Supplemental material captions
Supplemental Figure 1 – Comparison between MCW and boiling water as extraction
methods for (polar) metabolites. (A) Recoveries for non-endogenous carbon labeled
organic acids, amines and glucose. (B) For endogenous nucleotides and coenzymes the
relative abundance, expressed as the response ratio endogenous/ISTD, is shown. Error bars =
standard deviation (n=3). Results were obtained using 50 mg of pig muscle tissue (wet mass).
Supplemental Figure 2 – Effect of pH for metabolite extraction using MCW. (A)
Relative abundance of endogenous nucleotides and coenzymes using MCW extraction at pH
2, 6 and 9. (B) Recovery and partition of deuterium labeled fatty acids between aqueous and
organic phases of MCW extraction at the different pHs. Error bars = standard deviation
(n=3). Results were obtained using 50 mg of pig muscle tissue (wet mass).
Supplemental Figure 3 – Metabolite extraction efficiency using consecutive rounds of
homogenization and extraction. Endogenous organic acids, amines and glucose were
measured by GC-MS after 3 consecutive rounds of homogenization and extraction with
MCW. Efficiencies for each round are relative to the overall metabolite response
(endogenous/ISTD) obtained from the 3 rounds. Error bars = standard deviation (n=3).
Results were obtained using 50 mg of pig muscle tissue (wet mass).
Supplemental Figure 4 – Correlation between LC-MS and GC-MS measurements for
aspartic acid and glutamic acid.
Supplemental Figure 5 – Subject and intervention classification based on the metabolic
profile data using PCA analysis. Organic acids (15), amines (49), nucleotides (4),
acylcarnitines (22) and oxylipins (12) were used to classify muscle biopsy profiles at baseline
– before exercise – and in the right and left legs after 1h of one-legged cycling exercise (A).
Data obtained for 5 individuals is shown with each color representing one individual at
baseline and after exercise with the exercised and non-exercised leg. Similar analysis are
depicted in (B) comparing only baseline and exercised leg. Green or 1 = baseline and Red or
0 = exercised leg. Highlighted PCA plots (PC1 vs. PC2 and PC1 vs. PC5) are shown in more
detail in Figure 4.
Supplemental Figure 6 – Anthropometric and biochemical characteristics of the
subjects metabolically profiled in this study. (A) Biochemical parameters measured in
plasma before exercise (baseline) and right after 1h of one-legged cycling exercise. (B) Age,
height, weight and body mass index (BMI), hart frequency (HF), maximal aerobic capacity
(VO2max) and maximum work load (Wmax) were assessed for the subjects enrolled in this
study (n=5). In panel (C) the maximum HF and the VO2max are shown in detail for the five
subjects. *p<0.05 when comparing S1/S2 with S4/S5.
Supplemental Table 1 – Concentrations spiked into pig muscle extracts for the construction
of calibration lines. Data was obtained using approximately 10 mg of pig muscle tissue (wet
mass).
Supplemental Table 2 – Analytical validation results: recovery, matrix effects and ion
suppression data. Values were determined based on C13 or deuterium labeled ISTDs using
pig muscle extracts. Data was obtained using approximately 10 mg of pig muscle tissue (wet
mass).
Supplemental Table 3 – Analytical validation results: linearity (R2), sensitivity (LOD/LOQ)
and precision at C4 and C2 levels for the detected metabolites in pig tissue. Data was
obtained using approximately 10 mg of pig muscle tissue (wet mass).
Supplemental Table 4 – Biological validation results: intra- and inter-individual variation
(% RSD) of metabolites detected in pig and human tissue biopsies. Only metabolites
consistently detected in all human or pig samples are shown. Data was obtained using
approximately 10 mg of pig or human muscle tissue (wet mass).