Step
|
State
|
Pathway
|
Q-junction
|
Comment - Events (E) and Marks (M)
|
mt
|
REN
|
|
|
mt
- Respiration in the REN state is due to the presence of residual endogenous substrates.
- In the absence of endogenous substrates, mt can be used for calibration of fully oxidized NAD.
|
1D.1
|
|
|
|
mt;1D.1
- In the presence of endogenous substrates, the addition of a low concentration of ADP (0.1 μM) is a crucial step to detect the fully oxidized NAD for the calculation of the NAD redox state. ADP stimulates the consumption of endogenous substrates, which can be observed as a decrease in respiration and the NADH fluorescence signal.
|
2PGM
|
PGM
|
N
|
CI
|
mt;1D.1;2PGM
- NADH-linked substrates (type N-pathway to Q).
- The addition of fuel substrates may trigger a transient peak increase in mitochondrial respiration due to the presence of low ADP concentrations, and reduction of the NAD pool. Once ADP is phosphorylated to ATP the respiration decreases and a further reduction of NAD can be observed.
- In the presence of ATPases in the sample, ATP is recycled to ADP, making it not possible to measure LEAK state.
|
3D2.5
|
PGMP
|
N
|
CI
|
mt;1D.1;2PGM;3D2.5
|
4Anox
|
|
N
|
CI
|
mt;1D.1;2PGM;3D2.5;4Anox
- Anoxia, after the biological sample has consumed the O2 in the O2k-chamber, is used to detect the fully reduced NAD for calculation of the NAD redox states.
|
5Myx
|
|
N
|
CI
|
mt;1D.1;2PGM;3D2.5;4Anox;5Myx
- The addition of myxothiazol after anoxia is a crucial step to detect the fully reduced NAD for calculation of the NAD redox state. Myxothiazol can induce a further reduction of NAD under anoxia.
|
6Reox
|
ROX
|
|
|
mt;1D.1;2PGM;3D2.5;4Anox;5Myx;6Reox
- The reoxygenation by opening the chamber in the presence of Myx allows for Rox-correction of the O2 fluxes.
|