SUIT-005

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SUIT-005

Description

1OctM;2D;3P;4S;5U;6Rot-.png

Abbreviation: FNS(Oct,PM)

Reference: A: short RP2 when malate-anaplerotic activity is zero Bioblast pdf »Versions

SUIT-category: FNS(Oct,PM)
SUIT protocol pattern: orthogonal 1OctM;2D;3P;4S;5U;6Rot-

The SUIT-005 protocols provide information on the F-pathway, the combined FN pathway, and the convergence FNS pathways in the OXPHOS state. FNS comprises the most important pathways in many cell types and thus provides a physiologically relevant estimate of maximum OXPHOS- and ET-capacity. SUIT-005 can be extended with the CIV assay module.


Communicated by Iglesias-Gonzalez J, Krumschnabel G and Gnaiger E (last update 2019-01-26)

Specific SUIT protocols

SUIT-005 O2 pfi D11

1OctM;2D;2c;3P;4S;5U;6Rot;7Ama;8AsTm;9Azd.png SUIT-005 O2 pfi D11.png

MitoPedia: SUIT

Steps and respiratory states

1OctM;2D;2c;3P;4S;5U;6Rot;7Ama;8AsTm;9Azd.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1OctM OctML(n) F(N) FAO 1OctM
2D OctMP F(N) FAO 1OCtM;2D
2c OctMcP F(N) FAO 1OCtM;2D;2c
  • 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 FAO-pathway. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) FAO-pathway could be overestimated due to a 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.
  • Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
  • 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]).
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
3P OctPMP FN F&CI 1OctM;2D;2c;3P
  • 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.
4S OctPMSP FNS F&CI&II 1OctM;2D;2c;3P;4S
  • 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.
5U OctPMSE FNS F&CI&II 1OctM;2D;2c;3P;4S;5U
6Rot SE S CII 1OctM;2D;3P;4S;5U;6Rot
7Ama ROX 1OctM;2D;3P;4S;5U;6Rot;7Ama
  • 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.
Step Respiratory state Pathway control ET-Complex entry into Q-junction Comment
## Tm TmE CIV CIV
## Azd ROX


Questions.jpg


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Strengths and limitations

+ Reasonable duration of the experiment (1.5 to 2 h); 2-3 h when the CIV module is added.
+ The protocol provides information on F-OXPHOS capacity in the absence of a significant OXPHOS-capacity in the malate anaplerotic pathway control state.
+ FNS comprises the most important pathways in many cell types and thus provides a physiologically relevant estimate of maximum OXPHOS- and ET-capacity.
+ PM & S reconstitutes TCA cycle activity (PMS) if the 2-oxoglutarate carrier does not outcompete the sources of 2-oxoglutarate.
+ Application of the cytochrome c test early in the protocol ensures comparability of all states in case of any effect of c.
- F-OXPHOS capacity may be underestimated with Oct. In human heart muscle addition of Oct to palmitoylcarnitine (Pal) & malate increased OXPHOS-capacity by 26% (Lemuieux et al 2011).
- SRotE may be underestimated if S is not saturating.


Compare SUIT protocols

  • SUIT-002 check for malate-linked anaplerotic activity

References

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry