Encapsulation and perfusion of mitochondria in agarose beads for functional studies with 31P-NMR spectroscopy

An NMR method to study on-line mitochondrial function was developed, Mitochondria were maintained in a stable physiologic state in agarose beads that were continuously superfused with oxygenated buffer at 28 degrees C. Oxidative function of both heart and liver mitochondria was evaluated with P-31 NMR at 9.4 T using pyruvate plus malate as substrate, This method allows clear resolution of adenosine triphosphate-gamma (ATP gamma) and adenosine diphosphate-beta (ADP beta) phosphate signals, whereas alpha signals of ATP and ADP overlap. ATP production by mitochondria was documented to be very sensitive to different interventions (hypoxia, ischemia, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP)) and depended on the ADP concentration in superfusion medium, These data demonstrate that the new application of NMR to study mitochondrial function can discriminate, on-line, between several physiologic and biochemical processes in intact physiologically stable mitochondria.