Measurement of Mitochondrial Respiratory Activity

Oxygen consumption was measured polarographically at 25 °C using 1.0 to 2.5 mg protein from the hippocampal mitochondrial fraction in respiration medium consisting of 20 mM KCl, 5 mM MgCl₂, 10 mM KH₂PO₄, 10 mM tris–HCl, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 225 mM sucrose, at pH 7.4, using a Clark-type electrode. Five mM glutamate and malate were added as the respiratory substrates, and the mitochondrial respiration was initiated by adding 50 nmol adenosine-5-diphospate (ADP). Oxygen consumption measured in the presence of added ADP was defined as state III respiration, while that measured following the consumption of ADP was defined as state IV respiration. The respiration control ratio (RCR) was calculated as the ratio of state III respiration to state IV respiration and used as a marker of mitochondrial respiratory activity. The ADP/O ratio was calculated as the ratio of the added ADP concentration to the consumption of oxygen during state III respiration. Mitochondrial respiration was calculated as nanomoles of O₂ per min per milligram of protein. Mitochondrial protein was determined by the Pierce BCA Protein Assay Kit using BSA as standard and 10-fold dilutions to prevent reaction inhibition.