Reference: A: Additivity between the N- and S-pathway in the Q-junction
- SUIT protocol pattern: 1PM;2D;3G;4S;5U;6Rot-
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
- SUIT-008 O2 pfi D014 for permeabilized fibers
- SUIT-008 O2 ce-pce D025 for cells-permeabilized cells (ce-pce)
- SUIT-008 O2 mt D026 for isolated mitochondria and tissue homogenate
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
|Step||Respiratory state||Pathway control||ET-Complex entry into Q-junction||Comment|
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- Pathway control
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-014: 1GM;2D;3P;4S;5U;6Rot-; similar version starting with GM, and then adding P. Used in combination with SUIT-008 in Lemieux 2017.
- 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.
|Lemieux 2017 Sci Rep||2017||Lemieux 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||Mouse||Heart|
MitoPedia methods: Respirometry