Untargeted LC-HRMS metabolomic analysis reveals exercise-induced biochemical alterations in endurance Arabian horses

Endurance exercise induces extensive metabolic remodeling in equine athletes, yet the global metabolic responses in purebred Arabian horses remain insufficiently characterized. This study aimed to investigate plasma metabolomic alterations associated with prolonged aerobic exercise using untargeted liquid chromatography–high-resolution mass spectrometry (LC-HRMS). Plasma samples from twelve trained Arabian horses were collected before and after 80-km endurance rides and analyzed using both hydrophilic interaction (HILIC) and reversed-phase (C18) chromatographic platforms. Multivariate analysis (PLS-DA) revealed a distinct separation between pre- and post-exercise metabolic profiles. Forty-seven metabolites exhibited significant concentration changes, primarily involving acylcarnitines, organic acids, and amino acid derivatives. Marked increases in medium- and long-chain acylcarnitines, 3-hydroxybutyric acid, pyruvate, and tricarboxylic acid (TCA) intermediates indicated a pronounced metabolic shift toward enhanced fatty-acid β-oxidation and oxidative phosphorylation. Concurrent decreases in tryptophan and small peptides reflected intensified amino acid catabolism and potential mechanisms of central fatigue. Classical exercise biomarkers—creatinine, urea, and lactic acid—were significantly elevated and strongly correlated with LC-HRMS results (r > 0.99, p < 0.05). Additional upregulated metabolites, including HpODE, allantoin, and homovanillic acid, suggested activation of oxidative stress and dopaminergic signaling pathways. This study provides comprehensive metabolomic evidence of systemic metabolic adaptation to prolonged endurance exercise in Arabian horses. The findings highlight key biochemical pathways involved in energy metabolism, oxidative stress, and neuromodulation, and identify potential plasma biomarkers for monitoring fatigue, metabolic stress, and performance in equine endurance athletes.