*2.4. Reactive Oxygen Species Analysis*

ROS levels were determined by evaluating the oxidation of the 2 , 7 -dichlorodihydrofluorescein diacetate fluorescent probe (H2DCFDA). A total of 0.5 mg/mL of homogenate from each muscle was placed in test tubes and incubated at 4 ◦C with constant shaking for 20 min in a buffer with 10 mM HEPES, 100 mM KCl, 3 mM MgCl2, 3 mM KH2PO4 (pH 7.4), and 1.25 mM of H2DCFDA in a total volume of 2 mL. This suspension was placed in a quartz cell, and the basal fluorescence was determined. One minute later, 10 mM glutamate/malate was added as substrate, and the changes in fluorescence were determined for an additional 20 min [26].

Fluorescence changes were measured on a Shimadzu RF-5301PC spectrofluorometer (λex 485 nm; λem 520 nm). The data were expressed as the difference in fluorescence obtained by subtracting the fluorescence units obtained at the end of the 20 min with the substrate minus the fluorescence obtained before the substrate's addition. The result was expressed as arbitrary fluorescence units/min.
