Optimised measurement of mitochondrial respiratory capacity in frozen rat liver and heart

High-resolution respirometry is widely used to assess mitochondrial respiratory capacity and oxidative phosphorylation coupling in preparations of fresh tissues, but is significantly limited in freeze-thawed preparations owing to mitochondrial membrane disruption. Reconstitution of the electron transfer system (ETS) by adding exogenous cytochrome c and using NADH as a complex I electron donor can partially circumvent these limitations, allowing assessment of ETS capacity uncoupled from ADP phosphorylation. We sought to optimise this approach for rat liver and heart homogenates but found that use of NADH resulted in high residual O 2 consumption, independent of canonical electron flow to complex IV. This could be minimised in liver homogenates through the inclusion of superoxide dismutase (SOD) and catalase to reconstitute O 2 from superoxide formed, and in heart homogenates by the further inclusion of S1QEL1.1, an I Q site-specific inhibitor of superoxide formation at complex I. We validated this modified approach by measuring ETS capacities in frozen liver and heart samples from rats exposed to inhalation hypoxia, replicating findings in fresh preparations from the same rats. • Measurement of electron transfer system (ETS) capacity, uncoupled from ADP-phosphorylation is feasible in homogenates of previously-frozen liver and heart • Residual O 2 consumption supported by NADH can be minimised by superoxide dismutase/catalase, and if needed, site-specific inhibition of superoxide formation • This approach can reveal biologically-relevant differences in ETS capacity, reflecting findings in fresh tissue