Comparison of the effect of chemically and biologically induced inflammation on the volatile metabolite production of lung epithelial cells by GC×GC-TOFMS

Abstract

Exhaled breath analysis has a high potential for early non-invasive diagnosis of lung inflammatory diseases, such as asthma. The characterization and understanding of the inflammatory metabolic pathways involved into volatile organic compounds (VOCs) production could bring exhaled breath analysis into clinical practice and thus open new therapeutic routes for inflammatory diseases. In this study, lung inflammation was simulated in vitro using A549 epithelial cells. We compared the VOC production from A549 epithelial cells after a chemically induced oxidative stress in vitro, exposing the cells to H₂O₂, and a biological stress, exposing the cells to an inflammatory pool of sputum supernatants. Special attention was devoted to define proper negative and positive controls (8 different types) for our in vitro models, including healthy sputum co-culture. Sputum from 25 asthmatic and 8 healthy patients were collected to create each pool of supernatants. Each sample type was analyzed in 4 replicates using solid-phase microextraction (SPME) comprehensive two-dimensional gas chromatography hyphenated to time-of-flight mass spectrometry (GC×GC-TOFMS). This approach offers high resolving power for complex VOC mixtures. According to the type of inflammation induced, significantly different VOCs were produced by the epithelial cells compared to all controls. For both chemical and biological challenges, an increase of carbonyl compounds (54%) and hydrocarbons (31%) was observed. Interestingly, only the biological inflammation model showed a significant cell proliferation together with an increased VOC production linked to asthma airway inflammation. This study presents a complete GC×GC-TOFMS workflow for in vitro VOC analysis, and its potential to characterize complex lung inflammatory mechanisms.