Discussion
PEx
DISEASE, INFLAMMATORY RESPONSE, LEAKAGE
Our data demonstrates that, despite an overall similarity, the PL composition of PEx is more closely related to BAL than to ISP, which is in line with the proposed peripheral origin of PEx. PEx analysis was capable to show the inter-individual differences in the level of some lipids that were observed in BAL and ISP. Repetitive PEx sampling demonstrated that PL analysis in PEx yielded very reproducible data. LPS affected most targeted lipids in the challenged lobe but only the increase of SM(d34:1) could be detected in PEx, which are sampled from all lobes. The inhalation challenge showed a similar, but an overall weaker response. In addition, it most likely acted predominantly in the central airways and was therefore not detectable in PEx. The lipid composition of PEx provide further circumstantial support for the suggested mechanism for particle formation during re-opening of small peripheral airways. The peripheral lung origin of PEx could be an advantage for the study of PL composition compared to BAL, which naturally also contains portions of larger airways.We standardized our LC/MS analysis of lipids, by always using the same amount of BAL, ISP and PEx collection filters extraction fluid. Internal standard was added to these samples prior to the processing, however, this cannot account for variable PL recovery and dilution during collection of BAL and ISP or potential dilution effects due to LPS-induced leakage of fluid into the airways. Therefore, normalization of lipid data was performed. There are two major approaches. First, reference to a major or abundant lipid, e.g. PC(32:0) can be made with computation of the ratio of each lipid relative to this reference. This bears the risk, that the selected reference lipid itself is affected by the LPS challenge (or disease). Therefore, we normalized each lipid to the sum of all analysed lipids. The resulting mol% have the additional benefit, that the relative levels between BAL, ISP and PEx can be better compared. Due to the lack of normalization not all changes shown in Table S1 are comparable to those shown in Table The OPLS-DA analysis was performed to identify differences in the lipid profile between sampling methods. For this analysis we selected the BAL baseline visit, and the ISP and PEx baseline visits which were performed on the same day. There is no visit, for which baseline visits from all three methods are available and we are aware that this approach favors a closer similarity between PEx and ISP Nevertheless, our analysis showed that there was a greater similarity between BAL samples and PEx samples. With induced sputum being derived from the more central airways [PC(32:0) or dipalmitoyl-phosphatidylcholine (DPPC) is an essential PL for maintaining low surface tension of airway lining fluid. It displayed the lowest inter-individual variability, indicating that its function requires a well-defined concentration. It is known that inter-individual differences between lipid levels exist [Both segmental and inhalation LPS challenge resulted in a clearly detectable neutrophilic influx [Overall, the changes in PL composition in PEx were small. It could be argued, that PEx analysis is therefore not sensitive enough to detect LPS induced inflammatory changes. However, it needs to be kept in mind, that the segmental LPS challenge is limited to one lobe of the lung, while PEx are sampled from the small airways of all five lobes.The inhalation LPS challenge elicited only minimal PL changes in PEx and a smaller, but basically similar inflammatory response in ISP as compared to BAL from the challenged segment. It could be argued again, that PEx analysis was not sensitive enough to detect these small LPS induced changes. But it needs to be considered, that based on the inhalation maneuver and particle size of the inhaled LPS, the majority of LPS-induced changes in PL composition are probably limited to the larger airways. The small increments in PEx SM(d34:1) would then indicate that inflammation-related changes to proximal lung PL composition have to be substantial before they can be detected in samples from the distal lung. Based on our data it is tempting to conclude that the PExA analysis could even be a better tool than BAL to investigate PL composition in the peripheral lung. Not only because it is non-invasive, which will enable new study designs with more frequent sampling, but also because it samples ELF undiluted and with potentially less contaminations from larger airways as compared to BAL.