SUIT-014 O2 pfi D042: Difference between revisions
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The SUIT-014 O2 pfi D042 protocol is designed for tissue fibers that display a preference for [[GM_pathway_control_state |GM]] over [[PM_pathway_control_state |PM]] to support NADH-linked respiration. Adding [[Pyruvate|pyruvate]] in the OXPHOS state enables to check if NADH-linked respiration can be further stimulated to determine N-pathway [[Oxidative phosphorylation| ''P'']] capacity. | |||
SUIT-014 protocols provide an assessment of the linear coupling control ([[LEAK-respiration|''L'']]-[[Oxidative phosphorylation| ''P'']]-[[ET-capacity| ''E'']]) with NADH linked-substrates ([[PGM|PGM]]) and the control in ET state ([[NADH_Electron_transfer-pathway_state|N]], [[NS-pathway control state| NS]], [[Succinate pathway control state| S]]), covering the contribution of two pathways which are most important in the mitochondria of many species, tissues and cell types. | |||
::: '''[[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- |
Revision as of 09:47, 6 March 2019
Description
Abbreviation: NS(PGM)
Reference: A
SUIT number: D042_1GM;2D;3P;4S;5U;6Rot;7Ama
O2k-Application: O2
The SUIT-014 O2 pfi D042 protocol is designed for tissue fibers that display a preference for GM over 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 linear coupling control (L- P- E) with NADH linked-substrates (PGM) and the control in ET state (N, NS, S), covering the contribution of two pathways which are most important in the mitochondria of many species, tissues and cell types.
- SUIT-category: NS(PGM)
- SUIT protocol pattern: diametral 1GM;2D;3P;4S;5U;6Rot-
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 |
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- 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 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.
- - 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.
- - CIV activity is not measured, to save experimental time.
Compare SUIT protocols
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry