SUIT number: D014_1PM;2D;2c;3G;4S;4D;5U;6Rot;7Ama;8AsTm;9Azd
The SUIT-008 O2 pfi D014 protocol is 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 in permeabilized fibers. It 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 O2 pfi D014 can be easily extended with the CIV assay module.
Communicated by Huete-Ortega M, Iglesias-Gonzalez J and Gnaiger E (last update 2019-06-06)
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).
- + Mitochondrial external membrane can be measured with the addition of cytochrome c. Application of the cytochrome c test early in the protocol ensures comparability of all states in case of any effect of c.
- + Reasonable duration of the experiment.
- + Complex IV activity can be measured.
- + 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 of the S-pathway is higher with GM compared to PM (GMP is inhibited by the CII inhibitor malonic acid to a larger extent than PMP). PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since an impairment of N-pathway capacity can be compensated partially by activation of the S-pathway. This is an advantage compared to SUIT-011 for diagnosis of N-capacity.
- - Fatty acid oxidation is not analysed.
- - When evaluating the additive effect of the N- and S-pathway, 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.
|Votion 2012 PLoS One||2012||Votion 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.||Horse||Skeletal muscle|
MitoPedia methods: Respirometry