SUIT-008 O2 ce-pce D025: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr= | |abbr=Q-junction ce-pce | ||
|description=[[File:1PM;2D;3G;4S;5U;6Rot | |description=[[File:ce1;1Dig;1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Az.png|600px]] | ||
|info=''' | |info='''A: for cells-permeabilized cells''' [[Lemieux 2017 Sci Rep]] - '''[[SUIT-008]]''' | ||
|application=O2 | |application=O2 | ||
|SUIT number= | |SUIT number=D025_ce1;1Dig;1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd | ||
}} | }} | ||
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{{Template:SUIT text D025}} | |||
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__TOC__ | __TOC__ | ||
Communicated by [[Huete-Ortega M]], [[Gnaiger E]] (last update 2019-05-02) | |||
== Representative traces == | |||
[[File:D025 O2 traces.png|600px]] | |||
[[File: | {{Template:SUIT-008 pce}} | ||
== 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. | |||
:::-ย 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_Sci_Rep|Lemieux 2017]] for [[Permeabilized muscle fibers|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-ย 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 == | |||
{{Template:Chemicals ce}} | |||
{{Template:Chemicals SUIT-008}} | |||
{{Template:Chemicals CIV}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | == References == | ||
{{#ask:[[Category:Publications]] [[Additional label:: | {{#ask:[[Category:Publications]] [[Additional label::SUIT-008 O2 pce D025]] | ||
|?Was published in year=Year | |?Was published in year=Year | ||
|?Has title=Reference | |?Has title=Reference | ||
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{{MitoPedia concepts | {{MitoPedia concepts | ||
|mitopedia concept=SUIT protocol | |mitopedia concept=SUIT protocol, SUIT A, Find | ||
}} | }} | ||
{{MitoPedia methods | {{MitoPedia methods | ||
|mitopedia method=Respirometry | |mitopedia method=Respirometry | ||
}} | }} | ||
{{MitoPedia O2k and high-resolution respirometry}} | |||
{{MitoPedia topics}} |
Latest revision as of 23:58, 12 April 2021
Description
Abbreviation: Q-junction ce-pce
Reference: A: for cells-permeabilized cells Lemieux 2017 Sci Rep - SUIT-008
SUIT number: D025_ce1;1Dig;1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd
O2k-Application: O2
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, Gnaiger E (last update 2019-05-02)
Representative traces
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
- ยป Electron transfer pathway
- ยป Fatty acid oxidation pathway control state, F
- ยป NADH electron transfer-pathway state, N
- ยป Succinate pathway control state, S
- ยป NS-pathway control state, NS
- ยป Glycerophosphate pathway control state, Gp
- ยป Complex IV single step, CIV
- ยป Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- ยป Glutamate, G
- ยป Glycerophosphate, Gp
- ยป Malate, M
- ยป Octanoylcarnitine, Oct
- ยป Pyruvate, P
- ยป 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-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 |
---|---|---|
1Dig | Digitonin (Dig) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1PM | Pyruvate (P) and Malate (M) | |
2D | ADP (D) | |
2c | Cytochrome c (c) | |
3G | Glutamate (G) | |
4S | Succinate (S) | |
5U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler |
6Rot | Rotenone (Rot) | |
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.
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,
Find
MitoPedia methods:
Respirometry