Reference: A pfi: permeabilized fibers- SUIT-016
SUIT number: D044_1OctM;2D;3G;4S;5Rot;6Omy;7U;8Ama
SUIT-016 O2 pfi D044 gives information on F-pathway in LEAK state and OXPHOS state in permeabized fibers, avoiding FAO overestimation in the presence of anaplerotic pathways. In addition, the pathway control of FN and FNS in OXPHOS state and of S in LEAK state and ET state is evaluated. 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 µM O2) must be employed. To set the optimal oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.
Communicated by Cardoso LHD and Gnaiger E (last update 2019-02-14)
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
|Step||Respiratory state||Pathway control||ET-Complex entry into Q-junction||Comment|
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- Pathway control
Strengths and limitations
- + This protocol provides information on FAO capacity in the absence of other potentially interfering pathways both in LEAK and OXPHOS coupling control states.
- + 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.
- + This is a good protocol to analyse coupling control at S-pathway, which is an advantage compared to SUIT-015 and SUIT-017.
- + Glutamate is easier to prepare compared to pyruvate.
- - In some tissues GM is not sufficient to fully support N-pathway capacity
- - SRot(E) may be underestimated if S is not saturating.
- - SRot(E) may be underestimated in the presence of Omy in some tissues and cell types
- - CIV activity is not measured, to save experimental time.
- It is possible to add cytochrome c in different steps:
- (2c), when cytochrome c is added after 2D, it allows to compare all steps in OXPHOS even if there is damage of the mitochondrial outer membrane. This approach can be chosen when the cytochrome c effect is not an exclusion criterion.
- (3c) when cytochrome c is added after 3G, due to the higher flux, it is easier to evaluate whether there is an increase in respiration indicating damages in the mitochondrial outer membrane. However, if there is a strong cytochrome c effect, it is not possible to compare OctMP with the next steps in OXPHOS.
- (7c) when cytochrome c is added after 7U: if there is no cytochrome c effect, it indicates that even after the whole lenght of the protocol, there were no damages to the mitochondrial outer membrane. This approach can be used as an exclusion criterion for samples that normally do not present mitochondrial outer membrane damage after the sample preparation, e.g. human permeabilized muscle fibers.
Compare SUIT protocols
|Gnaiger 2015 Scand J Med Sci Sports||2015||Gnaiger E, Boushel R, Søndergaard H, Munch-Andersen T, Damsgaard R, Hagen C, Díez-Sánchez C, Ara I, Wright-Paradis C, Schrauwen P, Hesselink M, Calbet JAL, Christiansen M, Helge JW, Saltin B (2015) Mitochondrial coupling and capacity of oxidative phosphorylation in skeletal muscle of Inuit and caucasians in the arctic winter. Scand J Med Sci Sports 25 (Suppl 4):126–34.||Human||Skeletal muscle|
MitoPedia methods: Respirometry