Exhaled Breath Reflects Prolonged Exercise and Statin Use during a Field Campaign

Abstract

Volatile organic compounds (VOCs) in exhaled breath provide insights into various metabolic processes and can be used to monitor physiological response to exercise and medication. We integrated and validated in situ a sampling and analysis protocol using proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) for exhaled breath research. The approach was demonstrated on a participant cohort comprising users of the cholesterol-lowering drug statins and non-statin users during a field campaign of three days of prolonged and repeated exercise, with no restrictions on food or drink consumption. The effect of prolonged exercise was reflected in the exhaled breath of participants, and relevant VOCs were identified. Most of the VOCs, such as acetone, showed an increase in concentration after the first day of walking and subsequent decrease towards baseline levels prior to walking on the second day. A cluster of short-chain fatty acids including acetic acid, butanoic acid, and propionic acid were identified in exhaled breath as potential indicators of gut microbiota activity relating to exercise and drug use. We have provided novel information regarding the use of breathomics for non-invasive monitoring of changes in human metabolism and especially for the gut microbiome activity in relation to exercise and the use of medication, such as statins.

Keywords: PTR-ToF-MS; breath; prolonged exercise; short-chain fatty acids; statin.

Figure 1

Raw signals of m/z 59.05 (acetone) and m/z 69.07 (isoprene) from five consecutive participants duplicate breath samples (each participant is separated using the dashed boxes). The red line represents the trigger that was activated when the CO₂ concentration in exhaled breath reached 4%. On average, two minutes per participant are sufficient to collect the samples.

Figure 2

Multilevel partial least squares discriminant analysis (M-PLS-DA) model for the effect of walking on the breath volatile organic compound (VOC) profile for St0. The model includes all sample time points. A clear separation between Day 0 and the remaining time points (Day 1–Day 3) is revealed, indicating a significant change in the breath profile at the onset of exercise compared against Day 0.

Figure 3

M-PLS-DA model for St0 for all post walking measurements (Day 1, Day 2, and Day 3) with a difference in clustering along the first latent variable (LV1). There is high overlap between Day 2 and Day 3 and a degree of separation between Day 1 and the other time points on LV1, suggesting a possible adaptation to prolonged exercise after the 2nd and 3rd day of walking.

Figure 4

Relative change in the median concentrations of the significant VOCs in relation to the effect of exercise during the 4 days. The concentrations are relative to baseline measurements of before walking of non-statin users (St0) and range scaled by row. All the VOCs, including the short-chain fatty acids (SCFA), reflect a recovery type effect for all participants, by increasing following Day 1 of exercise and then subsequently decreasing after a rest period (Day 2 am), and increasing again after exercise (Day 2, Day 3). Isoprene is an exception, and it shows the opposite trend, i.e., decreasing in relation to exercise.

Figure 5

Changes in the concentration of the short-chain fatty acids (SCFAs) cluster over days for each group. Exercise (walking) induced an increase (p < 0.001) in SCFAs for all the groups (Day 1, Day 2, and Day 3) compared to (Day 0 and Day 2 am). The SCFAs levels were higher for the non-statin group (St0) compared to statin users (St1: p = 0.013 and St2: p = 0.005). No difference between St1 and St2 over the post-walking period (p = 0.2843). For St0, the SCFAs concentration reached baseline level (p = 0.08), whilst for St1 (p = 0.009) and St2 (p = 0.007), the SCFAs concentration was elevated after a period of rest compared to Day 0. Concentration values are presented as mean ± standard error.

Figure 6

Schematic diagram of the breath sampling setup. Participants exhaled through a bacterial mouth filter (A) connected to a CO₂ sensor (B) into a heated buffer pipe (C) at a constant flow of 50 ml/sec following a visual indicator on the screen of the breath sampler (D) for as long as was comfortable. The pipe is connected to the proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) via a heated sampling line (E). When the CO₂ concentration reached 4%, a CO₂-flow monitoring trigger signal (F) was sent to the PTR-ToF-MS to aid with data processing.