Emilia Viklund

Characterization of exhaled breath particles collected by an electret filter technique.

Aerosol particles that are present in exhaled breath carry nonvolatile components and have gained interest as a specimen for potential biomarkers. Nonvolatile compounds detected in exhaled breath include both endogenous and exogenous compounds. The aim of this study was to study particles collected with a new, simple and convenient filter technique. Samples of breath were collected from healthy volunteers from approximately 30 l of exhaled air. Particles were counted with an optical particle counter and two phosphatidylcholines were measured by liquid chromatography-tandem mass spectrometry. In addition, phosphatidylcholines and methadone was analysed in breath from patients in treatment with methadone and oral fluid was collected with the Quantisal device. The results demonstrated that the majority of particles are  <1 μm in size and that the fraction of larger particle contributes most to the total mass. The phosphatidylcholine PC(16 : 0/16 : 0) dominated over PC(16 : 0/18 : 1) and represented a major constituent of the particles. The concentration of the PC(16 : 0/16 : 0) homolog was significantly correlated (p  <  0.001) with total mass. From the low concentration of the two phosphatidylcholines and their relative abundance in oral fluid a major contribution from the oral cavity could be ruled out. The concentration of PC(16 : 0/16 : 0) in breath was positively correlated with age (p  <  0.01). An attempt to use PC(16 : 0/16 : 0) as a sample size indicator for methadone was not successful, as the large intra-individual variability between samplings even increased after normalization. In conclusion, it was demonstrated that exhaled breath sampled with the filter device represents a specimen corresponding to surfactant. The possible use of PC(16 : 0/16 : 0) as a sample size indicator was supported and deserves further investigations. We propose that the direct and selective collection of the breath aerosol particles is a promising strategy for measurement of nonvolatiles in breath.

Low Levels of Exhaled Surfactant Protein A Associated With BOS After Lung Transplantation

Background There is no clinically available marker for early detection or monitoring of chronic rejection in the form of bronchiolitis obliterans syndrome (BOS), the main long-term complication after lung transplantation. Sampling and analysis of particles in exhaled air is a valid, noninvasive method for monitoring surfactant protein A (SP-A) and albumin in the distal airways. Methods We asked whether differences in composition of exhaled particles can be detected when comparing stable lung transplant recipients (LTRs) (n = 26) with LTRs who develop BOS (n = 7). A comparison between LTRs and a matching group of healthy controls (n = 33) was also conducted. Using a system developed in-house, particles were collected from exhaled air by the principal of inertial impaction before chemical analysis by immunoassays. Results Surfactant protein A in exhaled particles and the SP-A/albumin ratio were lower (P = 0.002 and P = 0.0001 respectively) in the BOS group compared to the BOS-free group. LTRs exhaled higher amount of particles (P < 0.0001) and had lower albumin content (P < 0.0001) than healthy controls. Conclusions We conclude that low levels of SP-A in exhaled particles are associated with increased risk of BOS in LTRs. The possibility that this noninvasive method can be used to predict BOS onset deserves further study with prospective and longitudinal approaches.

Particles in exhaled air (PExA): non-invasive phenotyping of small airways disease in adult asthma

RATIONALE Asthma is often characterised by inflammation, damage and dysfunction of the small airways, but no standardised biomarkers are available. OBJECTIVES Using a novel approach-particles in exhaled air (PExA)-we sought to (a) sample and analyse abundant protein biomarkers: surfactant protein A (SPA) and albumin in adult asthmatic and healthy patients and (b) relate protein concentrations with physiological markers using phenotyping. METHODS 83 adult asthmatics and 21 healthy volunteers were recruited from a discovery cohort in Leicester, UK, and 32 adult asthmatics as replication cohort from Sweden. Markers of airways closure/small airways dysfunction were evaluated using forced vital capacity, impulse oscillometry and multiple breath washout. SPA/albumin from PEx (PExA sample) were analysed using ELISA and corrected for acquired particle mass. Topological data analysis (TDA) was applied to small airway physiology and PExA protein data to identify phenotypes. RESULTS PExA manoeuvres were feasible, including severe asthmatic subjects. TDA identified a clinically important phenotype of asthmatic patients with multiple physiological markers of peripheral airway dysfunction, and significantly lower levels of both SPA and albumin. CONCLUSION We report that the PExA method is feasible across the spectrum of asthma severity and could be used to identify small airway disease phenotypes.

Utility of single versus multiple breath washout in adult asthma

Nitrogen multiple breath washout (N2 MBW) is a sensitive method to identify peripheral airway involvement in asthma, but is a time‐consuming test. The N2 vital capacity single breath (VC SBW) test offers greater time efficiency, but concordance with N2 MBW is poorly understood. The prevalence of peripheral airway abnormality was determined by N2 MBW and N2 SBW tests in 194 asthmatic subjects aged 18–1 years. N2 MBW data were related to findings in 400 healthy controls, aged 17–71 years, while N2 SBW data were compared to findings in 224 healthy controls, aged 15–65 years, to derive equipment‐specific reference values. Amongst asthmatic subjects, relationships between N2 SBW and N2 MBW outcomes were studied. N2 SBW relationship with clinical history, spirometry, blood eosinophils and fraction exhaled nitric oxide (FENO) data was also explored. The prevalence of peripheral airway involvement (i.e. abnormal ventilation distribution) determined by N2 SBW‐derived phase III slope (N2 SIII) was 24·7%, compared to 44% determined by N2 MBW‐derived lung clearance index (LCI) (P<0·001). Predictors of abnormal N2 SIII were older age, smoking history and lower FEV1. N2 SBW offers lower sensitivity than N2 MBW to detect small airway dysfunction in adult asthma, but may be a marker of more severe disease.

Two techniques to sample non-volatiles in breath—exemplified by methadone

The particles in exhaled breath provide a promising matrix for the monitoring of pathological processes in the airways, and also allow exposure to exogenous compounds to be to assessed. The collection is easy to perform and is non-invasive. The aim of the present study is to assess if an exogenous compound-methadone-is distributed in the lining fluid of small airways, and to compare two methods for collecting methadone in particles in exhaled breath. Exhaled particles were collected from 13 subjects receiving methadone maintenance treatment. Two different sampling methods were applied: one based on electret filtration, potentially collecting exhaled particles of all sizes, and one based on impaction, collecting particles in the size range of 0.5-7 μm, known to reflect the respiratory tract lining fluid from the small airways. The collected samples were analyzed by liquid chromatography mass spectrometry, and the impact of different breathing patterns was also investigated. The potential contribution from the oral cavity was investigated by rinsing the mouth with a codeine solution, followed by codeine analysis of the collected exhaled particles by both sampling methods. The results showed that methadone was present in all samples using both methods, but when using the method based on impaction, the concentration of methadone in exhaled breath was less than 1% of the concentration collected by the method based on filtration. Optimizing the breathing pattern to retrieve particles from small airways did not increase the amount of exhaled methadone collected by the filtration method. The contamination from codeine present in the oral cavity was only detected in samples collected by the impaction method. We conclude that methadone is distributed in the respiratory tract lining fluid of small airways. The samples collected by the filtration method most likely contained a contribution from the upper airways/oral fluid in contrast to the impaction method.

Lipid composition of particles in exhaled air (PEx) from workers exposed to welding fumes

More than two million workers are exposed to pneumotoxic welding aerosols and there is a need for biomarkers of effects on the respiratory system. The lipid composition of the respiratory tract lining fluid (RTLF) is such a potential marker. The most abundant pulmonary surfactant phospholipid is dipalmitoylphosphocholine (DPPC). It is specific for the airways, while palmitoyloleoylphosphatidylcholine (POPC) is a common lipid in tissues and body fluids. We hypothesize that the amounts of or ratio between DPPC and DOPC are changed due to short term and/or long term exposure to welding fumes. We have developed a method for the collection of PEx, based on counting of the exhaled particles and subsequent collection by impaction on a teflon membrane. We have also developed a method for analysis of lipids in PEx based on LC/MS. We measured the exposure to iron, manganese, chromium and nickel of 18 stainless steel welders and also analyzed DPPC and DOPC in PEx samples taken at the end of the exposure measurement day. The welders working history was also recoded and summarized as welding years. Spirometry and nitrogen multiple breath wash out were also measured but the results are not yet evaluated. There were no significant correlations between the short term exposure to either iron, manganese, chromium or nickel and the fraction of DPPC in PEx or the ratio DPPC/DOPC. However, there was a tendency of correlation (Spearman correlation coefficient= 0.407 with p-value 0.09) between welding years and the DPPC/DOPC ratio. In this pilot study we could not establish short term effects of welding exposure on the RTFL lipid composition but a tendency of change due the long time exposure.

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.