Project description:Despite the attractiveness of breath analysis as a non-invasive means to retrieve relevant metabolic information, its introduction into routine clinical practice remains a challenge. Among all the different analytical techniques available to interrogate exhaled breath, secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) offers a number of advantages (e.g., real-time, yet wide, metabolome coverage) that makes it ideal for untargeted and targeted studies. However, so far, SESI-HRMS has relied mostly on lab-built prototypes, making it difficult to standardize breath sampling and subsequent analysis, hence preventing further developments such as multi-center clinical studies. To address this issue, we present here a number of new developments. In particular, we have characterized a new SESI interface featuring real-time readout of critical exhalation parameters such as CO2, exhalation flow rate, and exhaled volume. Four healthy subjects provided breath specimens over a period of 1 month to characterize the stability of the SESI-HRMS system. A first assessment of the repeatability of the system using a gas standard revealed a coefficient of variation (CV) of 2.9%. Three classes of aldehydes, namely 4-hydroxy-2-alkenals, 2-alkenals and 4-hydroxy-2,6-alkedienals―hypothesized to be markers of oxidative stress―were chosen as representative metabolites of interest to evaluate the repeatability and reproducibility of this breath analysis analytical platform. Median and interquartile ranges (IQRs) of CVs for CO2, exhalation flow rate, and exhaled volume were 3.2% (1.5%), 3.1% (1.9%), and 5.0% (4.6%), respectively. Despite the high repeatability observed for these parameters, we observed a systematic decay in the signal during repeated measurements for the shorter fatty aldehydes, which eventually reached a steady state after three/four repeated exhalations. In contrast, longer fatty aldehydes showed a steady behavior, independent of the number of repeated exhalation maneuvers. We hypothesize that this highly molecule-specific and individual-independent behavior may be explained by the fact that shorter aldehydes (with higher estimated blood-to-air partition coefficients; approaching 100) mainly get exchanged in the airways of the respiratory system, whereas the longer aldehydes (with smaller estimated blood-to-air partition coefficients; approaching 10) are thought to exchange mostly in the alveoli. Exclusion of the first three exhalations from the analysis led to a median CV (IQR) of 6.7 % (5.5 %) for the said classes of aldehydes. We found that such intra-subject variability is in general much lower than inter-subject variability (median relative differences between subjects 48.2%), suggesting that the system is suitable to capture such differences. No batch effect due to sampling date was observed, overall suggesting that the intra-subject variability measured for these series of aldehydes was biological rather than technical. High correlations found among the series of aldehydes support this notion. Finally, recommendations for breath sampling and analysis for SESI-HRMS users are provided with the aim of harmonizing procedures and improving future inter-laboratory comparisons.

Project description:Trihalomethanes (THM) are a class of disinfection by-products in chlorinated waters linked to deleterious health effects in humans although biological mechanisms are unclear. We aimed to study short-term changes in blood gene expression of adult recreational swimmers associated with physical activity and THM exposure. Adult volunteers (18-50 years, non-smokers, non-asthmatics) swam 40 minutes in an indoor chlorinated pool in Barcelona. Blood samples and THM measurements in exhaled breath were collected before and 5 min/1h after swimming, respectively. Physical activity intensity was calculated as metabolic equivalents (METs). Gene expression in whole blood RNA was evaluated using Illumina HumanHT-12v3 Expression-BeadChip. Linear mixed models, Gene Set Enrichment Analyses-GSEA and mediation analyses were used. The study population comprised 37 before-after pairs, with mean age 31 years (SD: 6.0), 60% female, and average changes before-after swimming of 1.75 METs (SD: 1.36) and 0.23 Âµg/m3 of exhaled bromoform (SD: 0.23). Among THM, bromoform yielded the strongest effect on gene expression changes. Eighty eight probes were associated with bromoform, 326 probes with MET and 77 probes overlapped. In mutually adjusted models, 15 probes remained significant for MET after False Discovery Rate (FDR). Although not FDR significant, in 23 nominally significant probes (p-value <0.05), fulfilling criteria for exploring mediation, 29.5 to 53.4% of MET effect was mediated by exhaled bromoform. Individual genes in this subset and the GSEA of the mutually adjusted gene lists of bromoform and MET were associated with pathways related to inflammatory/immune response and to several cancers. In this first study evaluating short-term gene expression changes associated with swimming in a chlorinated pool, changes in gene expression were observed in association with physical activity with part of this effect mediated through bromoform exposure. Identified genes were correlated with inflammatory, immune response and cancer pathways. These results need replication in larger studies. Expression profile differences were determined between total RNA extracted before and after exposure to trihalomethanes present in swimming pool water for 40 minute from whole blood samples from 33 healthy human individuals.

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