SUIT-012: Difference between revisions
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:::+ Mitochondrial external membrane integrity 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. | :::+ Mitochondrial external membrane integrity 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. | :::+ Reasonable duration of the experiment. | ||
:::+ This protocol can be extended with the Complex IV module, which can prolong the experimental time withΒ ~30 min. | |||
:::+ GM and PM yield typically identical fluxes in human skeletal muscle fibres. However, PM is the superior alternative to GM: the fraction of the [[NADH Electron transfer-pathway state| N-pathway]] is lower and of the [[Succinate pathway control state| 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. | :::+ GM and PM yield typically identical fluxes in human skeletal muscle fibres. However, PM is the superior alternative to GM: the fraction of the [[NADH Electron transfer-pathway state| N-pathway]] is lower and of the [[Succinate pathway control state| 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. | ||
:::- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler. | :::- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler. |
Revision as of 11:35, 7 March 2019
Description
Abbreviation: N(PGM)
Reference: A: Linear coupling control (L- P- E) with NADH-linked substrates (PM) Β»Versions
- SUIT-category: N(PGM)
- SUIT protocol pattern: diametral 1PM;2D;2c;3G;4U-
The SUIT-012 protocols specifically focus on assessing the linear coupling control (L- P- E) with NADH-linked substrates (PM). Addition of G in NADH-supported OXPHOS enables evaluating the glutamate anaplerotic pathway control state. SUIT-012 can be extended with the CIV assay module.
Communicated by Cardoso LH, Doerrier C, Huete-Ortega M, Iglesias-Gonzalez J and Gnaiger E (last update 2019-01-28)
Specific SUIT protocols
- SUIT-012 O2 mt D027 for isolated mitochondria
- SUIT-012 O2 pce D052 for permeabilized cells
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
|
4U | PGME | N | CI | 1PM;2D;2c;3G;4U
|
5Ama | ROX | 1PM;2D;2c;3G;4U;5Ama
|
Step | Respiratory state | Pathway control | ET-Complex | Comment |
---|---|---|---|---|
## AsTm | AsTmE | CIV | CIV | |
## Azd | CHB |
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- 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
- + This protocol allows to evaluate the function of the TCA cycle without the involvement of the complex II ( S-pathway). It is thus useful to understand the contribution and the activity of the dehydrogenases providing NADH.
- + Mitochondrial external membrane integrity 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.
- + This protocol can be extended with the Complex IV module, which can prolong the experimental time with ~30 min.
- + 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.
- - Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
Compare SUIT protocols
References
MitoPedia concepts: MiP concept, SUIT protocol, Recommended
MitoPedia methods:
Respirometry