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. Permeabilized muscle fibers are sensitive to oxygen supply due to limited diffusion of oxygen to the fiber bundle core. To counteract this limitation, hyperoxic conditions (400-250 µM O2) must be employed. To set the optimal oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.
Communicated by Huete-Ortega M, 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-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 for pfi.
- 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.
Chemicals and syringes
|Step||Chemical(s) and link(s)||Comments|
|1PM||Pyruvate (P) and Malate (M)|
|2c||Cytochrome c (c)|
|5U||Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)||Can be substituted for other uncoupler|
|7Ama||Antimycin A (Ama)|
|Step||Chemical(s) and link(s)||Comments|
|## AsTm||Ascorbate (As) and TMPD (Tm)|
|## Azd||Azide (Azd)|
- Suggested stock concentrations are shown in the specific DL-Protocol.
|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