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 31P NMR at 9.4 T using pyruvate plus malate as substrate. This method allows clear resolution of adenosine triphosphate-gamma (ATPgamma) and adenosine diphosphate-beta (ADPbeta) 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.