SUIT-008

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SUIT-008

Description

1PM;2D;3G;4S;5U;6Rot.png

Abbreviation: PM+G+S_OXPHOS+Rot_ET

Reference: A: Additivity between the N- and S-pathway in the Q-junction

SUIT protocol pattern: 1PM;2D;3G;4S;5U-

The SUIT-008 protocols are designed to assess the additivity between the N- and S-pathway in the Q-junction, providing a physiologically relevant estimate of maximum mitochondrial respiratory capacity. SUIT-008 also serves as a diagnostic tool for the activity of the glutamate dehydrogenase and its linked pathways, which could be relevant in some pathologies. SUIT-008 can be easily extended with the CIV assay module.


Communicated by Cardoso LHD, Doerrier C, Huete-Ortega M, Gnaiger E (last update 2019-06-05)

Specific SUIT protocols

1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd.png D014 O2 traces.png

Ce1;1Dig;1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd.png D025 O2 traces.png

1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd.png D026 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

SUIT-008

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1PM PML(n) N CI 1PM
2D PMP N CI 1PM;2D
2c PMcP N CI 1PM;2D;2c
  • NADH-linked substrates (type N-pathway to Q).
  • 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.
3G PGMP N CI 1PM;2D;2c;3G
4S PGMSP NS CI&II 1PM;2D;2c;3G;4S
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
5U PGMSE NS CI&II 1PM;2D;2c;3G;4S;5U
6Rot SE S CII 1PM;2D;2c;3G;4S;5U;6Rot
7Ama ROX 1PM;2D;2c;3G;4S;5U;6Rot;7Ama
  • 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).
Step Respiratory state Pathway control ET-Complex entry into Q-junction Comment
## AsTm AsTmE CIV CIV
## Azd ROX


Questions.jpg


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Strengths and limitations

+ NS-OXPHOS capacity provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
+ The presence of PGM and S establishes fully operative TCA cycle activity.
+ This protocol allows to analyse the convergence of pathways at the Q-junction (N, NS, S).
+ Outer mitochondrial membrane integrity can be evaluated by the addition of cytochrome c (cytochrome c test). The early addition of cytochrome c in the protocol ensures comparability of all states in case of any effect of cytochrome c'.
+ GM and PM yield typically identical fluxes in human skeletal muscle fibres. However, PM is the superior alternative to GM: the fraction of the N-pathway is lower and S-pathway contribution is higher with GM compared to PM. PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since impairment of N-pathway capacity can be compensated partially by activation of the S-pathway. This is an advantage compared to SUIT-011 for the diagnosis of N-capacity.
+ Reasonable duration of the experiment.
+ This protocol can be extended with the Complex IV module.
- F-pathway is not analysed.
- For additive effect evaluation of N- and S-pathways, it has to be considered that NSP and NSE capacities can only be compared with NP and SE capacities. This is not a problem when NSP = NSE (Gnaiger 2009). In this case, it may be assumed that SP = SE (Votion et al 2012), such that NSP can be compared with NP + SP. SUIT-004 should be chosen for the additive effect in the ET-state.
- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

Compare SUIT protocols

  • SUIT-004 1PM;2D;3U;4S;5Rot- The SUIT-004 protocols provide a quick assessment of the linear coupling control (L- P- E) with NADH linked-substrates (PM) and the control in ET state (N, NS, S)
  • SUIT-011 1GM;2D;3S;4U;5Rot- The SUIT-011 protocols are designed to study physiologically relevant maximum mitochondrial respiratory capacity (OXPHOS with NS substrates) and coupling/pathway control.

References

 YearReferenceOrganismTissue;cell
Lemieux 2017 Sci Rep2017Lemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep 7:2840, DOI:10.1038/s41598-017-02789-8.MouseHeart
Votion 2012 PLoS One2012Votion DM, Gnaiger E, Lemieux H, Mouithys-Mickalad A, Serteyn D (2012) Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle. PLoS One 7:e34890.HorseSkeletal muscle

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry