2.3.2. Mitochondrial High-Resolution Respirometry

Mitochondrial respiration was investigated using a high-resolution Oxygraph-2k (Oroboros Instruments, Innsbruck, Austria), which was composed of a two-chamber respirometer with a Peltier thermostat and electromagnetic stirrers [45]. The analysis was conducted in 2 mL of respiration medium that was previously equilibrated in each chamber with air at 30 °C and then stirred at 750 rpm until a stable air saturation signal was reached. All experiments were performed using a final concentration of 0.2–0.3 mg/mL fresh protein of isolated mitochondria in respiratory buffer. The isolated mitochondria were suspended in respiration buffer supplemented with 5 mM glutamate and 2.5 mM malate or with 5 mm succinate in the presence of rotenone as an energizing substrate. This was followed by measurement and recording of oxygen flux (J_O2) at 30 °C in a constantly stirred oxygraph vessel after the successive addition of 1 mm ADP (state 3_ADP or OXPHOS capacity) with 0.75 mm oligomycin, which acted as an ATPase inhibitor (state 4 or leak respiration). Expression of results was as pmol of oxygen consumed/min mg protein per respiratory state. Measurements were taken at 0.2 s intervals for 15–20 min, and a computer-driven data acquisition system (DatLab, Innsbruck, Austria) was used for recording these measurements. The respiratory control ratio (RCR), determined as the ratio of state 3 to state 4, was used as a general measure of mitochondrial function [5]. Determination of the phosphorylation coefficient (ADP/O ratio) was also performed and defined as the amount of ADP added to the respiratory medium divided by nanograms of oxygen consumed during state 3.