A fluorescence-based technique for screening compounds that protect against damage to brain mitochondria

Mitochondrial failure to generate ATP can be due to damage to their membranes, which leads to release of solutes, e.g., pyridine nucleotides, from the mitochondrial matrix. We developed a highly sensitive fluorescence assay for detecting a pathologic increase in mitochondrial membrane permeability. The assay is based on coupled enzymatic reactions that produce hydrogen peroxide in the presence of the reduced or oxidized form of nicotinamide adenine dinucleotide (NADH/NAD). The hydrogen peroxide is a substrate for horseradish peroxidase that converts Amplex Red into highly fluorescent Resorufin. The assay is able to detect nanomolar levels of pyridine nucleotides in the medium. Calcium additions to isolated rat brain or liver mitochondria incubated in a potassium-based medium with added enzymes caused osmotic swelling, as detected with light scattering, and production of Resorufin, due to release of NADH/NAD. These events were blocked by cyclosporin A (CsA) or Bongkrekic acid (BKA), inhibitors of the mitochondrial permeability transition (MPT). These results indicate that the NADH/NAD release assay is a simple, reliable, and sensitive method for detecting mitochondrial damage and for screening of compounds that protect mitochondria from injury.