Evaluation of Respiration with Clark Type Electrode in Isolated Mitochondria and Permeabilized Animal Cells

In many studies, the evaluation of mitochondrial function is critical to understand how disease conditions or xenobiotics alter mitochondrial function. One of the classic end-points that can be assessed is oxygen consumption, which can be performed under controlled, yet artificial conditions. Oxygen is the terminal acceptor in the mitochondrial respiratory chain, namely at an enzyme called cytochrome oxidase, which produces water in the process and pumps protons from the matrix to the intermembrane space. Several techniques are available to measure oxygen consumption, including polarography with oxygen electrodes or fluorescent/luminescent probes. The present chapter will deal with the determination of mitochondrial oxygen consumption by means of the Clark-type electrode, which has been widely used in the literature and that still remains to be the most reliable technique. We focus our technical description in the measurement of oxygen consumption by isolated mitochondrial fractions and by permeabilized cells.