2.1.4. Mitochondrial respiration

Mitochondrial respiration was assessed in both IFM and SSM as described previously [21], [23]. Isolated mitochondria (0.5 mg/mL) were respired in respiration buffer containing 100 mM KCl, 50 mM MOPS, 5 mM KH₂PO₄, 1 mM EGTA and 1 mg/mL BSA/Fraction V, using a polarographic oxygen sensing systems for measurements of dissolved oxygen consumption in liquid suspension (Qubit System, Kingston, ON, Canada). States 3 and 4 were measured with glutamate + malate (10 and 5 mM, respectively), palmitoylcarnitine (40 μM) and Succinate (20 mM) with rotenone (7.5 μM) was used to assess respiration through Complex II of the ETC exclusively. State 3 respiration (ADP-stimulated) was measured in the presence of 200 μM ADP. State 4 respiration (ADP-limited) was assessed after ADP consumption. Respiratory Control Ratio, the ratio of State 3 to State 4 was calculated to assess the control of oxygen consumption by phosphorylation. The ratio of ADP added in the chamber to the total amount of oxygen consumed in state 3 (ADP:O ratio) was calculated as an index of the efficiency of oxidative phosphorylation.