Muscles to molecules via mass spectrometry

Understanding exercise metabolism and the relationship with volatile organic compounds (VOCs) holds potential in health care and sports performance. The combination of approaches both in vitro and in vivo, linked together with analytical analysis has provided a platform to investigate exercise metabolism on two fronts. This research has investigated novel headspace (HS) analysis of skeletal muscle and exercise breath analysis aiming to find comparative markers of exercise metabolism.

An in vitro system was built to investigate the effect of electrical pulse stimulation (EPS) on the volatile constituents in the HS above contracting skeletal muscle. Determination of the cell culture protocol, EPS parameters and VOC sampling techniques provided a system capable of skeletal muscle stimulation and HS analysis. The C2C12 immortalised cell line was chosen based on its reliability and ease of use for proof-of-concept research into VOC characterisation of contracting skeletal muscle. The time to begin EPS and HS analysis was determined experimentally as seven days post terminal differentiation based on the contractility of differentiated myotubes. Further research should focus on improvements to the EPS system including the use of more relevant cell lines, improvements to the EPS system and quantification of myotube contraction.

The in vitro model was applied to the analysis of VOCs using three sampling techniques: solid phase micro-extraction (SPME), thermal desorption (TD) and direct atmospheric pressure chemical ionisation mass spectrometry (APCI-MS). Samples were collected under four conditions: environmental samples (enviro), acellular media HS samples (blank), skeletal muscle myotubes without stimulation HS samples (baseline) and stimulated skeletal muscle myotube HS (stim). TD sampling proved the most successful sampling technique with a four-fold increase in compounds included in the dataset compared with SPME sampling. Direct APCI-MS analysis rendered incompatible with cell culture HS VOC analysis. TD sampling was combined with gaschromatography mass spectrometry (GC-MS) and detected two compounds that, after multivariate and univariate statistical analysis, were identified as changing because of EPS. These compounds were tentatively assigned as 1,4-Dioxane-2,5-dione, 3,6-dimethyl- and 1-pentene. The former is a known lactid and the latter has been reported as a marker of oxidative stress. Both compounds imply metabolic relevance from previous literature and the trends exhibited in the current research. In addition to the improvements to the in vitro model discussed above, further research applying targeted analysis, metabolic assays and media analysis, has been recommended.

The application of direct APCI-MS analysis of single breath samples during an exercise study was investigated. This investigation aimed to collect comparative single breath samples that represented changes in exercise metabolism whilst testing the hypothesis that breath acetone increased with increased exercise intensity. Twentytwo participants (mean +/- standard deviation: age 22 ± 2.6yrs, height 181.5 ± 5.9cm, body mass 74.7 ± 10.7kg) provided single breath samples before exercise, after submaximal exercise and after maximal exercise. Mean acetone increased by 29% ± 46% from rest after the sub-maximal exercise, remained level after the maximal exercise and decreased 20% ± 15% after twenty minutes of post exercise seated rest. High levels of standard deviation and variability in individual response requires additional research into the capability to analyse acetone in single breath samples. Additionally, it has been recommended that the inclusion of TD-GC-MS would prove more fruitful in the analysis of exercise mediated breath VOCs.

Finally, TD-GC-MS analysis of single breath samples before, during and after two different forms of exercise (endurance and resistance) was investigated. Eleven participants (mean ± standard deviation: age 24 ± 5 yrs., height 182.9 ± 3.8cm, body mass 81.7 ± 11.4kg) completed endurance and resistance exercise visits, providing one-hundred and fifty-one breath samples. Two compounds, tentatively assigned as isoprene and benzaldehyde, were detected and through multivariate and univariate statistical analysis were concluded to change as a result of exercise. Isoprene changed during both endurance and resistance exercise whilst benzaldehyde only changed during the resistance exercise. A third compound, tentatively assigned acetophenone, was identified initially as changing significantly but further investigation rendered the changes non-significant. Correlations were investigated between each compound and the physiological markers of metabolism that were measured throughout the study. The potential of breath isoprene as a marker of blood lactate has been discussed. TD-GC-MS analysis of single breath samples rendered interesting results that warrant further analysis in a large cohort that includes trained individuals.

Both the in vitro and in vivo approaches collected resting measurements, subjected the skeletal muscle to a bout of stimuli (EPS and exercise) and collected samples during both the EPS and the exercise protocols. Samples were collected after the in vivo exercise but not after the EPS. It would be of interest to investigate any changes on VOC patterns following cessation of EPS. In both approaches, TD-GC-MS was the favoured technique for sample collection and analysis of VOCs. Of the two hundred compounds detected across the in-vitro and in-vivo approaches, thirty-seven compounds were detected in both methods. Benzaldehyde, identified as significantly changing in the in vivo analysis, was also detected in the in vitro analysis. However, it was not identified as significantly changing due to EPS. The same applied to acetophenone. Interestingly, isoprene was not detected in the in vitro model contrary to previous reports. The absence of isoprene was attributed to lower level in vitro compared to the concentrations detected in breath from in vivo whole body skeletal muscle contribution. Higher resolution mass spectrometry techniques would provide clarity on the in vitro presence of isoprene.