The calculation of the mitochondrial free [NAD+]/[NADH][H+] ratio in brain: effect of electroconvulsive seizure.

This study is an investigation into the validity of calculating the mitochondrial redox state in brain in vivo using models of seizure and anoxia in rats. At six intervals following electroconvulsive seizure (0.5-10 min) and after 5 min of complete anoxia, multiple metabolites were measured in freeze-blown or freeze-clamped brain. From substrate ratios, the apparent changes in the mitochondrial free [NAD+]/[NADH] [H+] ratio were calculated from the L-glutamate dehydrogenase reaction [EC 1.4.1.3] and compared with shifts in the oxidized to reduced ratio of total ubiquinone (a component of the mitochondrial phosphorylation chain). During complete anoxia the calculated mitochondrial free [NAD+]/[NADH] [H+] ratio and the ubiquinone redox ratio both became more reduced by a factor of approximately 7. In contrast, following seizure the two indicators of the mitochondrial redox state moved in opposite directions. Mainly because of a large increase in tissue NH4+, the calculated mitochondrial free [NAD+]/[NADH] [H+] ratio paradoxically became more oxidized, plateauing between 2 and 10 min post seizure at a value approximately double that of the control. At the same time, however, the ubiquinone redox state fell to one-half the control value at two min and moved back towards normal between 5 and 10 min after the onset of the seizure. The results have been taken to be evidence against the applicability of the calculation of the mitochondrial free [NAD+]/[NADH] [H+] ratio from the L-glutamate dehydrogenase reaction in brain at least under conditions of rapid change. The results also suggest the possibility that the NH4+ produced during seizure is extra-mitochondrial and has relatively little tendency to diffuse into the matrix.