A system is in a steady state if the levels of a specific variable (e.g. O2 flux, H2O2 flux, mitochondrial membrane potential) in the system do not change over time. High-resolution respirometry allows for the measurement of different parameters (e.g. O2 flux, H2O2 flux, mitochondrial membrane potential) at a steady state. Combination with the Titration-Injection microPump (TIP2k) allows operation with programmable titration regimes and at quasi steady-states, yielding an expanded flexibility in experimental design by combining the technical advantages of closed and open systems approaches.
Reference: MiPNet06.01_O2k-Overview, Harrison 2015 J Appl Physiol
MitoFit Quality Control System
MitoPedia O2k and high-resolution respirometry:
Communicated by Doerrier C, 2020-04-20, updated 2020-04-23, 2020-05-28
DatLab oxygen flux: performance and data analysis
- Steady state in oxygen fluxes should be reached before the next chemical addition is performed.
- The following DatLab traces illustrate examples of steady and non-steady states in respirometry:
. Oxygen flux per volume (right axis, red trace) is not stable in OXPHOS-state
(2D). Consequently, OXPHOS-capacity is underestimated. The addition of cytochrome c
(2c) if the steady state has not yet been reached in OXPHOS may lead to an overestimation of the cytochrome c effect
, since the increase in the oxygen flux per volume is not due to the cytochrome c
addition, but instead because the steady state had not yet been reached before its titration (see Figure 2 for more detail).
. Illustration of 1) the underestimation of OXPHOS-capacity (2D) and 2) the overestimation of cytochrome c effect
. The increase in the oxygen flux per volume (from 2D to 2c) is not due to the cytochrome c
addition, but instead because the steady state had not yet been reached before its titration.
. Oxygen flux is stable in 2D (steady state has been reached), therefore we measured OXPHOS-state
. In this case, the addition of cytochrome c
(2c) will not lead to an overestimation of the cytochrome c effect
. Oxygen flux may need time to reach steady state (see 2D). It is important to wait until a steady state is reached to avoid underestimation of respiratory fluxes. Artefacts by reoxygenations may lead to an overestimation of respiratory fluxes. In this case, after a reoxygenation it is also important to wait until the steady state is reached again.
- After the addition of inhibitors:
(Complex I inhibitor) addition in the presence of NS-linked substrates
(PGMS) in ET-state
) allows for measurement of the S-pathway
in ET-state (SE
, 6Rot). Frequently, rotenone addition leads to a quick steady state.
. Rotenone (or other inhibitors, e.g. Antimycin A
) addition may require time to reach steady state (which frequently happens with permeabilized muscle fibers - but can also happen with other sample preparations
). It is important to wait until steady state is reached to avoid overestimation of respiratory fluxes.