Description
Abbreviation: NS(PGM)
Reference: A: SUIT protocol for mt; mt: isolated mitochondria and tissue homogenate - SUIT-008
SUIT number: D026_1PM;2D;2c;3G;4S;4D;5U;6Rot;7Ama;8AsTm;9Azd
O2k-Application: O2
- MitoPedia: SUIT
- SUIT-category: NS(PGM)
- SUIT protocol pattern: diametral 1PM;2D;3G;4S;5U;6Rot-
The SUIT-008 O2 mt D026 protocol can be used with mitochondrial preparations (isolated mitochondria, tissue homogenate or cells permeabilized before addition to the O2k chamber). The 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. 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 mt D026 can be easily extended with the CIV assay module.
Communicated by Doerrier C and Gnaiger E (last update 2019-02-20)
Steps and respiratory states
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
|
3G | PGMP | N | CI | 1PM;2D;2c;3G
|
4S | PGMSP | NS | CI&II | 1PM;2D;2c;3G;4S
|
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
|
Step | Respiratory state | Pathway control | ET-Complex | Comment |
---|---|---|---|---|
## AsTm | AsTmE | CIV | CIV | |
## Azd | CHB |
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- Coupling control
- Pathway control
- Β» Electron transfer pathway
- Β» Fatty acid oxidation pathway control state, F
- Β» NADH electron transfer-pathway state, N
- Β» Succinate pathway control state, S
- Β» NS-pathway control state, NS
- Β» Glycerophosphate pathway control state, Gp
- Β» Complex IV single step, CIV
- Β» Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- Β» Glutamate, G
- Β» Glycerophosphate, Gp
- Β» Malate, M
- Β» Octanoylcarnitine, Oct
- Β» Pyruvate, P
- Β» Succinate, S
- Main fuel substrates
- Glossary
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.
- - Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
Compare SUIT protocols
- SUIT-004: SUIT-004 protocol provide a quick assessment of the linear coupling control (L-P-E) with NADH linked-substrates (PM) and the pathway control in ET state (N, NS, S pathways).
- SUIT-011: SUIT-011 protocol are designed to study physiologically relevant maximum mitochondrial respiratory capacity (OXPHOS capacity with GMS as NS-linked substrates).
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry