Difference between revisions of "SUIT-008 O2 ce-pce D025"
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::: '''[[SUIT protocol pattern]]:''' diametral ce1;1Dig;1PM;2D;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd | ::: '''[[SUIT protocol pattern]]:''' diametral ce1;1Dig;1PM;2D;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd | ||
The SUIT-008 O2 ce-pce D025 protocol is designed to assess the additivity between the [[NADH_Electron_transfer-pathway_state| N-]] and [[Succinate pathway control state| 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 ce-pce D025 can be easily extended with the CIV assay module. | The SUIT-008 O2 ce-pce D025 protocol is designed to assess the additivity between the [[NADH_Electron_transfer-pathway_state| N-]] and [[Succinate pathway control state| S-pathway]] in the [[Q-junction]], providing a physiologically relevant estimate of the 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 ce-pce D025 can be easily extended with the CIV assay module. | ||
In this protocol, non-permeabilized cells are added in the chamber, and [[ROUTINE]] respiration is measured. The cells are further permeabilized with [[digitonin]] inside the O2k chamber. | In this protocol, non-permeabilized cells are added in the chamber, and [[ROUTINE]] respiration is measured. The cells are further permeabilized with [[digitonin]] inside the O2k chamber. | ||
__TOC__ | __TOC__ | ||
Communicated by [[Huete-Ortega M]], [[Iglesias-Gonzalez J]] and [[Gnaiger E]] (last update 2019- | Communicated by [[Huete-Ortega M]], [[Iglesias-Gonzalez J]] and [[Gnaiger E]] (last update 2019-04-03) | ||
{{Template:SUIT-008 pce}} | {{Template:SUIT-008 pce}} | ||
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:::+ The presence of PGM and S establishes fully operative TCA cycle activity. | :::+ 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). | :::+ This protocol allows to analyse the convergence of pathways at the Q-junction (N, NS, S). | ||
:::+ Mitochondrial | :::+ Mitochondrial outer 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. | :::+ Reasonable duration of the experiment. | ||
:::+ Complex IV activity can be measured. | :::+ Complex IV activity can be measured. |
Revision as of 17:05, 3 April 2019
Description
Abbreviation: NS(PGM)
Reference: A: for cells-permeabilized cells Lemieux 2017 Sci Rep - SUIT-008
SUIT number: D025_ce1;1Dig;1PM;2D;2c;3G;4S;4D;5U;6Rot;7Ama;8AsTm;9Azd
O2k-Application: O2
- MitoPedia: SUIT
- SUIT-category: NS(PGM)
- SUIT protocol pattern: diametral ce1;1Dig;1PM;2D;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd
The SUIT-008 O2 ce-pce D025 protocol is designed to assess the additivity between the N- and S-pathway in the Q-junction, providing a physiologically relevant estimate of the 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 ce-pce D025 can be easily extended with the CIV assay module.
In this protocol, non-permeabilized cells are added in the chamber, and ROUTINE respiration is measured. The cells are further permeabilized with digitonin inside the O2k chamber.
Communicated by Huete-Ortega M, Iglesias-Gonzalez J and Gnaiger E (last update 2019-04-03)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
1Dig | REN | ce1;1Dig
|
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 |
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » 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).
- + Mitochondrial outer 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 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.
- - 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.
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
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 concepts:
SUIT protocol,
SUIT A,
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MitoPedia methods:
Respirometry