Step
|
State
|
Pathway
|
Q-junction
|
Comment - Events (E) and Marks (M)
|
1D
|
ROX
|
|
|
1D
- ADP is added to stimulate the consumption of endogenous fuel-substrates.
|
2M.1
|
|
|
|
1D;2M.1
|
3Pal
|
PalMP
|
F
|
FAO
|
1D;2M.1;3Pal
- Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration.
|
3c
|
PalMcP
|
F
|
FAO
|
1D;2M.1;3Pal;3c
- Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration.
- OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
- Addition of cytochrome c yields a test for integrity of the mtOM (cytochrome c control efficiency). Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (OXPHOS capacity P with saturating [ADP]).
|
4M2
|
PalMP
|
F(N)
|
FAO
|
1D;2M.1;3Pal;3c;4M2
- Respiratory stimulation of the FAO-pathway, F, by fatty acid FA in the presence of malate M. Malate is a type N substrate (N), required for the F-pathway. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) the F-pathway capacity is overestimated, if there is an added contribution of NADH-linked respiration, F(N) (see SUIT-002). The FA concentration has to be optimized to saturate the FAO-pathway, without inhibiting or uncoupling respiration.
- High concentration of malate, typically 2 mM, saturates the N-pathway.
- OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
|
5P
|
PalPMP
|
FN
|
FAO&CI
|
1D;2M.1;3Pal;4M2;5P
- Respiratory stimulation by simultaneous action of the F-pathway and N-pathway with convergent electron flow in the FN-pathway for evaluation of an additive or inhibitory effect of F.
- OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
|
6G
|
PalPGMP
|
FN
|
FAO&CI
|
1D;2M.1;3Pal;4M2;5P;6G
- Respiratory stimulation by simultaneous action of the F-pathway and N-pathway with convergent electron flow in the FN-pathway for evaluation of an additive or inhibitory effect of F.
- OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
|
7S
|
PalPGMSP
|
FNS
|
FAO&CI&II
|
1D;2M.1;3Pal;4M2;5P;6G;7S
- Respiratory stimulation by simultaneous action of the F-pathway, N-pathway, and S-pathway, with convergent electron flow in the FNS-pathway for reconstitution of TCA cycle function and additive or inhibitory effect of F.
- OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
|
8U
|
PalPGMSE
|
FNS
|
FAO&CI&II
|
1D;2M.1;3Pal;4M2;5P;6G;7S;8U
|
10Rot
|
SE
|
S
|
CII
|
1D;2M.1;3Pal;4M2;5P;6G;7S;8U;9Rot
|
11Ama
|
ROX
|
|
|
1D;2M.1;3Pal;4M2;5P;6G;7S;8U;9Rot;10Ama
- Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).
|