- high-resolution terminology - matching measurements at high-resolution
SUIT-025 O2 mt D057
Description
Abbreviation: FNS(Oct,PGM)
Reference: - SUIT-025
SUIT number: D057_1D;2M;3Oct;3c;4M;5P;6G;7S;8Rot;9Ama
O2k-Application: O2
- SUIT-category: FNS(Oct,PGM)
- SUIT protocol pattern: lineal 1D;2M;3Oct;3c;4M;5P;6G;7S;8Rot-
The SUIT-025 O2 mt D057 protocol is based on SUIT-002 specially designed to give information on F-pathway in OXPHOS state avoiding fatty acid oxidation (FAO) overestimation in the presence of anaplerotic pathways. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS, pathways) can be evaluated by using this SUIT protocol.
Communicated by Doerrier C (last update 2019-05-21)
Representative traces
Steps and respiratory states
Step
|
State
|
Pathway
|
Q-junction
|
Comment - Events (E) and Marks (M)
|
1D
|
ROX
|
|
|
1D
- ADP is added to stimulate consumption of endogenous fuel-substrates.
- Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.
|
2M.1
|
|
|
|
|
3Oct
|
OctMP
|
F
|
FAO
|
1D;2M.1;3Oct
- 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
|
OctMcP
|
F
|
FAO
|
1D;2M.1;3Oct;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
|
OctMP
|
F(N)
|
FAO
|
1D;2M.1;3Oct;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
|
OctPMP
|
FN
|
FAO&CI
|
1D;2M.1;3Oct;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
|
OctPGMP
|
FN
|
FAO&CI
|
1D;2M.1;3Oct;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
|
OctPGMSP
|
FNS
|
FAO&CI&II
|
1D;2M.1;3Oct;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.
|
8Rot
|
SP
|
S
|
CII
|
1D;2M.1;3Oct;4M2;5P;6G;7S;8Rot
|
9Ama
|
ROX
|
|
|
1D;2M.1;3Oct;4M2;5P;6G;7S;8Rot;9Ama
- 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).
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- » Coupling control state
- » ET capacity
- » OXPHOS capacity
- » LEAK respiration
- Pathway control
- » Electron transfer pathway
- » Fatty acid oxidation pathway control state, F
- » NADH electron transfer-pathway state, N
- » Succinate pathway control state, S
- » NS-pathway control state, NS
- » Glycerophosphate pathway control state, Gp
- » Complex IV single step, CIV
- » Anaplerotic pathway control state
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Glossary
- » List of SUIT states
- » SUIT concept
Strengths and limitations
- + SUIT-025 allows the depletion of endogenous substrates with ADP (1D).
- + The protocol provides information on F-pathway in OXPHOS state. The low concentration of malate used in this protocol, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
- + F-pathway (3Oct-2M.1) can be compared to FN-pathway (5P) in OXPHOS state.
- + Pathway control in OXPHOS (F, F(N), FN, FNS and S pathways) can be evaluated.
- + Harmonization with many SUIT protocols (up to step 7S).
- + Short experimental time.
- - ET state is not evaluated (for obtaining ET capacity with FNSGp and SGp substrate combinations see SUIT-002). To evaluate limitation of OXPHOS capacity by the phosphorylation system is it suggested to performed in parallel the SUIT-006 O2 mt D022 or to titrate the uncoupler after rotenone.
Compare SUIT protocols
- SUIT-002 (the reference protocol 2) differs from SUIT-025 in the coupling and pathway control states. SUIT-002 allows the evaluation of pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) whereas in SUIT-025 only F, F(N), FN and FNS pathways in OXPHOS state can be evaluated.
Chemicals and syringes
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
MitoPedia concepts:
SUIT protocol,
SUIT A,
Find
MitoPedia methods:
Respirometry