Mitochondrial Respiration Assay

Mitochondrial respiratory function was assessed in isolated mitochondria polarographically at 25°C, by measuring O₂ consumption using an Oroboros Oxygraph (Oroboros Instruments, Innsbruck, Austria). In a 2-ml, closed, thermostatic, magnetically stirred glass chamber, respiration medium (225 mmol/L mannitol, 70 mmol/L sucrose, 1 mmol/L EDTANa₂, 20 mmol/L KH₂PO₄, and 1 mg/ml BSA, pH 7.4) was saturated with ambient oxygen to reach a concentration of 258 μmol/L. After standardized instrument and chemical calibrations, 300 μg of isolated mitochondria were added to the reaction system. State 2 respiration was assessed with the addition of a 15-μl mixture of 0.8 mol/L malic acid and 1 mol/L glutamic acid, and then, state 3 respiration was assessed by the addition of ADP (0.5 mM). After all of the ADP had been phosphorylated to ATP, the respiratory rate turned to state 4. The respiratory control ratio (RCR) was calculated as the respiratory rate in state 3 divided by that in state 4.