Biomarkers of Redox Balance Adjusted to Exercise Intensity as a Useful Tool to Identify Patients at Risk of Muscle Disease through Exercise Test

Abstract

The screening of skeletal muscle diseases constitutes an unresolved challenge. Currently, exercise tests or plasmatic tests alone have shown limited performance in the screening of subjects with an increased risk of muscle oxidative metabolism impairment. Intensity-adjusted energy substrate levels of lactate (La), pyruvate (Pyr), β-hydroxybutyrate (BOH) and acetoacetate (AA) during a cardiopulmonary exercise test (CPET) could constitute alternative valid biomarkers to select "at-risk" patients, requiring the gold-standard diagnosis procedure through muscle biopsy. Thus, we aimed to test: (1) the validity of the V'O₂-adjusted La, Pyr, BOH and AA during a CPET for the assessment of the muscle oxidative metabolism (exercise and mitochondrial respiration parameters); and (2) the discriminative value of the V'O₂-adjusted energy and redox markers, as well as five other V'O₂-adjusted TCA cycle-related metabolites, between healthy subjects, subjects with muscle complaints and muscle disease patients. Two hundred and thirty subjects with muscle complaints without diagnosis, nine patients with a diagnosed muscle disease and ten healthy subjects performed a CPET with blood assessments at rest, at the estimated 1st ventilatory threshold and at the maximal intensity. Twelve subjects with muscle complaints presenting a severe alteration of their profile underwent a muscle biopsy. The V'O₂-adjusted plasma levels of La, Pyr, BOH and AA, and their respective ratios showed significant correlations with functional and muscle fiber mitochondrial respiration parameters. Differences in exercise V'O₂-adjusted La/Pyr, BOH, AA and BOH/AA were observed between healthy subjects, subjects with muscle complaints without diagnosis and muscle disease patients. The energy substrate and redox blood profile of complaining subjects with severe exercise intolerance matched the blood profile of muscle disease patients. Adding five tricarboxylic acid cycle intermediates did not improve the discriminative value of the intensity-adjusted energy and redox markers. The V'O₂-adjusted La, Pyr, BOH, AA and their respective ratios constitute valid muscle biomarkers that reveal similar blunted adaptations in muscle disease patients and in subjects with muscle complaints and severe exercise intolerance. A targeted metabolomic approach to improve the screening of "at-risk" patients is discussed.

Keywords: Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS); cardiopulmonary exercise test (CPET); exercise intolerance; maximal oxygen uptake; metabolomics; myopathy; tricarboxylic citric acid cycle.

Figure 1

Flow-chart diagram of the studied subjects and assessments. AA: Acetoacetate; ßOH: beta-hydroxybutyrate; CPET: cardiopulmonary exercise test; Lac: lactate; LC-MS/MS: chromatography liquid tandem mass spectrometry; Pyr: pyruvate.

Figure 2

V’O₂-adjusted biological parameters measured by routine automate in healthy subjects, muscle complaint subjects and muscle disease patients during CPET: (A) lactate; (B) pyruvate; (C) lactate/pyruvate ratio; (D) beta-hydroxybutyrate; (E) acetoacetate and (F) beta-hydroxybutyrate/acetoacetate ratio. Each patient was measured at three timepoints: rest, 50% of predicted maximal intensity and at maximal intensity. Data are presented as mean ± SEM at each time point. Significant p-value for post-hoc test $: healthy vs. muscle complaint subjects. AA: Acetoacetate; BOH: beta-hydroxybutyrate; CPET: Cardiopulmonary Exercise Test; La: lactate; Pyr: pyruvate; SEM: Standard Error of the Mean.

Figure 3

V’O₂-adjusted TCA cycle intermediates measured by LC-MS/MS in healthy subjects, muscle complaint subjects with no (V’O_2max > 85% pred.), mild (60% pred. < V’O_2max < 85% pred.), and severe (V’O_2max < 60% pred.) exercise intolerance during CPET: (A) lactate; (B) pyruvate; (C) lactate/pyruvate ratio; (D) malate; (E) succinate; (F) fumarate; (G) citrate; (H) beta-hydroxybutyrate and (I) alpha-ketoglutarate. Each patient was measured at three timepoints: rest, 50% of predicted maximal intensity and at maximal intensity. Data are presented as mean ± SEM at each time point. BOH: beta-hydroxybutyrate; CPET: Cardiopulmonary Exercise Test; La: lactate; Pyr: pyruvate; SEM: Standard Error of the Mean; TCA: Tricarboxylic Acid.