Any useful assay of ROS production should not interfere with mitochondrial function. This can be checked by measuring mitochondrial respiration in the presence of the molecular probes constituting the chemical detection system. Addition of horseradish peroxidase and homo-vanillic acid, at the same final concentration used in the fluorometric H2O2 assay, does not change the rates of mitochondrial state 4 or state 3 respiration or the RCI. High purity horseradish peroxidase and superoxide dis-mutase should be used. Commercial solution of these two enzymes stabilized with agents capable of altering mitochondria should be avoided. The appropriateness of the particular commercial reactants used can be easily checked in pilot experiments measuring mitochondrial respiration in their absence and in their presence.
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