SUIT-025 O2 mt D057

From Bioblast
Jump to: navigation, search
Bioblasts - Richard Altmann and MiPArt by Odra Noel
MitoPedia         Terms and abbreviations         Concepts and methods         Oroboros O2k         MiP and biochemistry         Preprints and history



MitoPedia

SUIT-025 O2 mt D057

Description

1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Rot;9Ama.png

Abbreviation: FNS(Oct,PGM)

Reference: - SUIT-025

SUIT number: D057_1D;2M;3Oct;3c;4M;5P;6G;7S;8Rot;9Ama

O2k-Application: O2

MitoPedia: SUIT -
SUIT-category: FNS(Oct,PGM)
SUIT protocol pattern: lineal 1D;2M;3Oct;3c;4M;5P;6G;7S;8Rot-

The SUIT-025 protocol is based on SUIT-002 specially designed to give information on F-pathway in OXPHOS state avoiding 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)
MitoPedia: SUIT

Steps and respiratory states

1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Rot;9Ama.png

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. 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 (P, OXPHOS-capacity with saturating [ADP]).
4M2 OctMP F(N) FAO 1D;2M.1;3Oct;4M2
5P OctPMP FN FAO&CI 1D;2M.1;3Oct;4M2;5P
  • Respiratory stimulation by simultaneous action of fatty acid (F) and type N substrates (N) with convergent electron flow in the FN-pathway for reconstitution of TCA cycle function and 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 fatty acid (F) and type N substrates (N) with convergent electron flow in the FN-pathway for reconstitution of TCA cycle function and 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 fatty acid (F), type N substrates (N), and succinate (S), 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).


Questions.jpg


Click to expand or collaps

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.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry