Mitochondrial rates of oxidation and phosphorylation: measurement

Oxygen and magnesium green fluorescence signals were detected simultaneously, using respirometry chambers equipped with fluorescent sensors (as in Chinopoulos et al. 2014). Pure oxygen gas was added to the respirometry chambers to reach a concentration of 550 μmol/L. The titration protocol was as follows: respiration was stimulated by adding pyruvate (5 mmol/L) and malate (0.5 mmol/L). Magnesium green (2.1 μmol/L), EGTA (0.1 mmol/L) and EDTA (15 μmol/L) were subsequently added to the chamber. Then, stepwise additions of MgCl₂ were performed for calibration of the fluorescent signal into Mg²⁺. Succinate (10 mmol/L) was then added. State 3 was reached by adding a saturating concentration of ADP (2 mmol/L). In this condition, changes in oxygen and ATP concentrations in the chamber are representative of raw fluxes in oxygen and ATP (JO_2‐raw and JATP_raw, respectively). JO_2‐raw reflects the rate of oxygen consumption by the mitochondria but also by nonmitochondrial reactions; JATP_raw is the balance between the rate of mitochondrial ATP production and the rate of ATP disappearance due to the activity of the various ATPase enzymes (phosphorylases, phosphatases and kinases; for further details see Chinopoulos et al. 2014). Addition of cATR (4 μmol/L), an inhibitor of ATP‐ADP exchanger, allowed calculation of the rate of ATP disappearance due to ATPase activity under state 4 conditions (JATP_St4). Addition of complex I inhibitor, rotenone (0.5 μmol/L) and complex III inhibitor, antimycin A (2.5 μmol/L) determined nonmitochondrial, or residual, oxygen consumption (JO_2‐ROX). At each step the rates of changes in ATP and/or oxygen were allowed to stabilize. An identical trial was run on the homogenate obtained from the same fish 2 h later, in order to determine the repeatability of measurements and the stability of measurements in relation to time since sample preparation.