SUIT-005

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SUIT-005

Description

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

Abbreviation: RP2-short

Reference: A: when malate-anaplerotic activity is zero

SUIT protocol pattern: 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 and Gnaiger E (last update 2019-06-05)

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
3P OctPMP FN F&CI 1OctM;2D;2c;3P
  • 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.
4S OctPMSP FNS F&CI&II 1OctM;2D;2c;3P;4S
  • 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.
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
## AsTm AsTmE CIV CIV
## Azd ROX


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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% (Lemieux et al 2011).
- SRotE may be underestimated if S is not saturating.


Compare SUIT protocols

  • SUIT-002 or SUIT-025 to measure F-OXPHOS capacity when malate anaplerotic activity is present;
  • SUIT-027 to check for malate-linked anaplerotic activity
  • SUIT-017

References

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry