Indirect Calorimetry-Based Novel Approach for Evaluating Metabolic Flexibility and Its Association with Circulating Metabolic Markers in Middle-Aged Subjects

: We propose a novel method for assessing metabolic flexibility (MF) through indirect calorimetry. A total of twenty healthy volunteers (10 females; 10 males) aged 45-65 were categorized into a Low-Intensity activity group (LI, 0-1 session of 1 h per week) and a High-Intensity activity group (HI, 5-6 sessions of 2 h per week). Volunteers underwent a stepwise exercise test on a cycle ergometer, connected to a calorimeter, to examine respiratory gas exchange to evaluate peak fatty acid Oxidation (PFO) and peak carbohydrate oxidation (PCO). Circulating peroxisome proliferator-activated receptor α (PPARα) biomarkers, docosahexaenoic acid/eicosapentaenoic acid (DHA/EPA) ratio and N-oleoylethanolamine (OEA), and the endocannabinoid- 2-arachidonoylglycerol (2-AG), were evaluated. We developed two MF parameters: the MF index (MFI), calculated by the product of PFO normalized per kg of fat-free mass (FFM) and the percentage of VO2max at PFO, and the peak energy substrates' oxidation (PESO), computed by summing the kilocalories from the PFO and PCO, normalized per kg FFM. The MFI and PESO were significantly different between the HI and LI groups, showing strong correlations with the circulating bioactive substances. Higher DHA/EPA ratio (p ≤ 0.05) and OEA (p ≤ 0.01), but lower 2-AG levels (p ≤ 0.01) were found in the HI group. These new parameters successfully established a functional link between MF and the balance of PPARα/endocannabinoid systems.