SUIT-014 O2 pfi D042: Difference between revisions
No edit summary |
Timon Alba (talk | contribs) No edit summary ย |
||
(One intermediate revision by one other user not shown) | |||
Line 4: | Line 4: | ||
|info='''A''' '''[[SUIT-014]]''' | |info='''A''' '''[[SUIT-014]]''' | ||
|application=O2 | |application=O2 | ||
|SUIT number=D042_1GM;2D;3P;4S;5U;6Rot;7Ama | |SUIT number=D042_1GM;2D;2c;3P;4S;5U;6Rot;7Ama | ||
}} | }}ย ย | ||
::: '''[[Categories of SUIT protocols |SUIT-category]]:''' NS(PGM) | ::: '''[[Categories of SUIT protocols |SUIT-category]]:''' NS(PGM) | ||
::: '''[[SUIT protocol pattern]]:''' diametral 1GM;2D;3P;4S;5U;6Rot- | ::: '''[[SUIT protocol pattern]]:''' diametral 1GM;2D;3P;4S;5U;6Rot- | ||
Line 13: | Line 12: | ||
__TOC__ | __TOC__ | ||
== Representative traces == | == Representative traces == | ||
Line 36: | Line 34: | ||
::::* [[SUIT-011]]: 1GM;2D;2c;3S;4U;5Rot-; similar version omitting P. | ::::* [[SUIT-011]]: 1GM;2D;2c;3S;4U;5Rot-; similar version omitting P. | ||
::::* [[SUIT-012]]: 1PM;2D;2c;3G;4U-; shorter version with PM preceding PGM and omitting S. | ::::* [[SUIT-012]]: 1PM;2D;2c;3G;4U-; shorter version with PM preceding PGM and omitting S. | ||
== Chemicals and syringes == | |||
{{Template:Chemicals SUIT-014}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | == References == |
Latest revision as of 11:39, 29 February 2024
Description
Abbreviation: NS(PGM)
Reference: A SUIT-014
SUIT number: D042_1GM;2D;2c;3P;4S;5U;6Rot;7Ama
O2k-Application: O2
- SUIT-category: NS(PGM)
- SUIT protocol pattern: diametral 1GM;2D;3P;4S;5U;6Rot-
The SUIT-014 O2 pfi D042 protocol is designed for tissue fibers that display a preference for glutamate & malate (GM) over pyruvate & malate (PM) to support NADH-linked respiration. Adding pyruvate in the OXPHOS state enables to check if NADH-linked respiration can be further stimulated to determine N-pathway P capacity. SUIT-014 protocols provide an assessment of the coupling control L- P with NADH linked-substrates (GM) and P- E with N- and S-pathways combined (PGMS). The pathway control in OXPHOS state (N, NS) and ET state (NS, S) is also evaluated, covering the contribution of two pathways which are most important in the mitochondria of many species, tissues and cell types. 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.
Representative traces
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1GM | GML(n) | N | CI | 1GM
|
2D | GMP | N | CI | 1GM;2D
|
2c | GMcP | N | CI | 1GM;2D;2c
|
3P | PGMP | N | CI | 1GM;2D;2c;3P
|
4S | PGMSP | NS | CI&II | 1GM;2D;2c;3P;4S
|
5U | PGMSE | NS | CI&II | 1GM;2D;2c;3P;4S;5U
|
6Rot | SE | S | CII | 1GM;2D;2c;3P;4S;5U;6Rot
|
7Ama | ROX | 1GM;2D;2c;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
- + 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 oter 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.
- - It was observed that addition of P (5 mM) after GM inhibited respiration in horse skeletal muscle fibres (Votion et al 2012), which was not the case when P was titrated in steps of 1 mM.
- - 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). Otherwise, 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.
- - Rox may be lower in substrate states earlier in the SUIT protocol. Therefore, this Rox measurement is frequently taken as a methodological control rather than as the final basis of Rox correction of mitochondrial respiration (mt).
- - Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
- + This protocol can be extended with the Complex IV module, which can prolong the experimental time with ~30 min.
Compare SUIT protocols
- SUIT-008: 1PM;2D;2c;3G;4S;5U-; similar version starting with PM, and then adding G. Used in combination with SUIT-014 in Lemieux 2017 for pfi.
- SUIT-011: 1GM;2D;2c;3S;4U;5Rot-; similar version omitting P.
- SUIT-012: 1PM;2D;2c;3G;4U-; shorter version with PM preceding PGM and omitting S.
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1GM | Glutamate (G) 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) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry