SUIT-005 O2 pfi D011: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr= | |abbr=RP2-short pfi | ||
|description=[[File:1OctM;2D;2c;3P;4S;5U;6Rot;7Ama;8AsTm;9Azd.png|450px | |description=[[File:1OctM;2D;2c;3P;4S;5U;6Rot;7Ama;8AsTm;9Azd.png|450px]] | ||
|info='''A''': '''[[SUIT-005]]''' short protocol linked to [[SUIT-002 O2 pfi D006]] - '''SUIT RP2''' (human skeletal muscle) | |info='''A''': - '''[[SUIT-005]]''' short protocol linked to [[SUIT-002 O2 pfi D006]] - '''SUIT RP2''' (human skeletal muscle) ย | ||
|application=O2 | |application=O2 | ||
|SUIT number=D011_1OctM;2D;3P;4S;5U;6Rot;7Ama;8AsTm;9Azd | |SUIT number=D011_1OctM;2D;2c;3P;4S;5U;6Rot;7Ama;8AsTm;9Azd | ||
}} | }} | ||
: | {{Template:SUIT text D011}} {{Template:Setting oxygen pfi}} | ||
__TOC__ | |||
Communicated by [[Doerrier C]], [[Gnaiger E]] (last update 2019-06-05) | |||
== Representative traces == | |||
[[File:SUIT-005 O2 pfi D11.png|600px]] | |||
{{Template:SUIT-005}} | {{Template:SUIT-005}} | ||
== Strengths and limitations == | == Strengths and limitations == | ||
:::+ The protocol provides information on FAO capacity in the absence of other, potentially interfering pathways, both in the [[LEAK | :::+ The protocol provides information on FAO capacity in the absence of other, potentially interfering pathways, both in the [[LEAK respiration| LEAK state]] and in [[Oxidative phosphorylation| OXPHOS]]. | ||
:::+ [[FNS |FNS]] [[OXPHOS | :::+ [[FNS |FNS]] [[OXPHOS capacity| OXPHOS capacity]] comprises the most important pathways in many cell types and thus provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity. | ||
:::+ [[FNS |FNS]] [[ET | :::+ [[FNS |FNS]] [[ET capacity| ET capacity]] is a good estimate of overall ET capacity in many cell types. | ||
:::+ The presence of PM and S establishes a fully operative TCA cycle activity ([[PMS]]) if the 2-oxoglutarate carrier does not outcompete the sources of 2-oxoglutarate | :::+ The presence of PM and S establishes a fully operative TCA cycle activity ([[PMS]]) if the 2-oxoglutarate carrier does not outcompete the sources of 2-oxoglutarate | ||
:::+ Application of the [[cytochrome c]] test early in the protocol ensures comparability of all states in case of any effect of 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 (1.5 to 2 h); 2-3 h when the CIV module is added. | :::+ Reasonable duration of the experiment (1.5 to 2 h); 2-3 h when the CIV module is added. | ||
:::- F-[[OXPHOS | :::- F-[[OXPHOS capacity| OXPHOS capacity]] may be underestimated. In human heart muscle addition of Oct to palmitoylcarnitine (Pal) + malate increased OXPHOS by 26% (Lemuieux et al 2011). | ||
:::- SRot<sub>''E''</sub> may be underestimated if S is not saturating. | :::- SRot<sub>''E''</sub> may be underestimated if S is not saturating. | ||
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== Compare SUIT protocols == | == Compare SUIT protocols == | ||
::::* [[SUIT-002]] | ::::* [[SUIT-002]]: A comparable but more comprehensive protocol comprising additional substrate states. | ||
::::* [[SUIT-002 O2 pfi D006]] | ::::* [[SUIT-002 O2 pfi D006]]: A comparable but more comprehensive pfi-specific protocol comprising additional substrate states. | ||
::::* [[Isolated mitochondria |imt]], ROX: 1OctM;2D;2c;3P;4S;5U;6Rot;7Ama | ::::* [[Isolated mitochondria |imt]], ROX: 1OctM;2D;2c;3P;4S;5U;6Rot;7Ama | ||
::::* [[Permeabilized muscle | ::::* [[Permeabilized muscle fibers |pfi]], high O<sub>2</sub>, no ROX: FNS(PM)01_pfiO2,1OctM,2D(c),3P,4S,5U,6Rot | ||
ย | |||
== Chemicals and syringes == | |||
{{Template:Chemicals SUIT-005}} | |||
{{Template:Chemicals CIV}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | == References == |
Latest revision as of 11:37, 29 February 2024
Description
Abbreviation: RP2-short pfi
Reference: A: - SUIT-005 short protocol linked to SUIT-002 O2 pfi D006 - SUIT RP2 (human skeletal muscle)
SUIT number: D011_1OctM;2D;2c;3P;4S;5U;6Rot;7Ama;8AsTm;9Azd
O2k-Application: O2
SUIT-005 O2 pfi D011 protocol provides information on the F-pathway, the combined FN pathway, and the convergence FNS pathways in the OXPHOS state. FNS comprises the most important pathways in many cell types and thus provides a physiologically relevant estimate of maximum OXPHOS- and ET capacity. SUIT-005 can be extended with the CIV assay module. This protocol is linked to SUIT-002 O2 pfi D006 - SUIT RP2, specifically for human skeletal muscle mitochondria. SUIT-005 O2 pfi D011 is harmonized with SUIT-004 O2 pfi D010. The F-pathway has to be tested previously. 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 ยตM O2) must be employed. To set the optimal oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.
Communicated by Doerrier C, Gnaiger E (last update 2019-06-05)
Representative traces
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1OctM | OctML(n) | F(N) | FAO | 1OctM
|
2D | OctMP | F(N) | FAO | 1OCtM;2D
|
2c | OctMcP | F(N) | FAO | 1OCtM;2D;2c
|
3P | OctPMP | FN | F&CI | 1OctM;2D;2c;3P
|
4S | OctPMSP | FNS | F&CI&II | 1OctM;2D;2c;3P;4S
|
5U | OctPMSE | FNS | F&CI&II | 1OctM;2D;2c;3P;4S;5U
|
6Rot | SE | S | CII | 1OctM;2D;3P;4S;5U;6Rot
|
7Ama | ROX | 1OctM;2D;3P;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
- + The protocol provides information on FAO capacity in the absence of other, potentially interfering pathways, both in the LEAK state and in OXPHOS.
- + FNS OXPHOS capacity comprises the most important pathways in many cell types and thus provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
- + FNS ET capacity is a good estimate of overall ET capacity in many cell types.
- + The presence of PM and S establishes a fully operative TCA cycle activity (PMS) if the 2-oxoglutarate carrier does not outcompete the sources of 2-oxoglutarate
- + 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 (1.5 to 2 h); 2-3 h when the CIV module is added.
- - F- OXPHOS capacity may be underestimated. In human heart muscle addition of Oct to palmitoylcarnitine (Pal) + malate increased OXPHOS by 26% (Lemuieux et al 2011).
- - SRotE may be underestimated if S is not saturating.
Compare SUIT protocols
- SUIT-002: A comparable but more comprehensive protocol comprising additional substrate states.
- SUIT-002 O2 pfi D006: A comparable but more comprehensive pfi-specific protocol comprising additional substrate states.
- imt, ROX: 1OctM;2D;2c;3P;4S;5U;6Rot;7Ama
- pfi, high O2, no ROX: FNS(PM)01_pfiO2,1OctM,2D(c),3P,4S,5U,6Rot
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1OctM | Octanoylcarnitine (Oct) and Malate (M) | |
2D | ADP (D) | |
2c | Cytochrome c (c) | |
3P | Pyruvate (P) | |
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
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry