Steady state

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Bioblasts - Richard Altmann and MiPArt by Odra Noel
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MitoPedia

Steady state

Description

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


MitoPedia concepts: MitoFit Quality Control System 


MitoPedia methods: Respirometry, Fluorometry, Spectrophotometry 


MitoPedia O2k and high-resolution respirometry: DatLab 



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:
  • OXPHOS state:
Figure 1. 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).
Figure 2. 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.
Figure 3. 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.
Figure 4. 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:
Figure 5. Rotenone (Complex I inhibitor) addition in the presence of NS-linked substrates (PGMS) in ET-state (NSE) allows for measurement of the S-pathway in ET-state (SE, 6Rot). Frequently, rotenone addition leads to a quick steady state.
Figure 6. 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.