Metabolomic profiling of extracellular vesicles reveals enhanced oxidative stress and energy metabolism during intense military training; an exploratory study

Extracellular vesicles (EVs) are small, membrane-bound vesicles that transfer biological content through the extracellular environment. The role of EVs in energy metabolism have primarily focused on EV proteins and microRNAs, with less attention on the metabolic content of EVs. Purpose: This exploratory study assessed changes in the EV metabolome in response to an arduous, 16-day military training exercise. Methods: Forty male Soldiers (21±2 years, 24.8±2.7 kg/m 2 ) provided blood from which circulating EVs were isolated, and completed assessments of body composition, and lower body power on days 1 (PRE) and 16 (POST) of a mountain training exercise (MTX). Results: Total daily energy expenditure (TDEE) during the MTX was 4,187±519 kcal·d -1 . Fat mass (POST-PRE [95%CI]; −0.9[−1.3,−0.6] kg), lean body mass (−1.6[−2.0,−1.2] kg), body fat percentage (−0.7[−1.1,−0.3]%), and lower body power (−133[−204,−63] Watts) decreased from PRE to POST ( p<0.05). Global metabolite profiling identified 81 metabolites from lipid (81%), energy (5%), cofactor and vitamin (5%), xenobiotic (4%), carbohydrate (2%), amino acid (1%) and nucleotide (1%) pathways in serum-derived EVs. After adjusting for EV concentration, 11 metabolites were different PRE to POST ( p<0.05, Q<0.20), with the largest increases in the oxidative stress-associated metabolites 5-oxoproline and benzoate. Changes in lean body mass were positively associated with changes the energy metabolites citrate (ρ=0.361, p=0.022) and phosphate (ρ=0.369, p=0.019). Conclusion: Findings suggest that EV metabolites change in response to physiological stress and reflect increased oxidative stress, energy metabolism, and fatty acid metabolism, which may provide early indicators of stress adaptations relevant for optimizing training and sustaining military performance.