Abbreviation: RP2-short pfi
Reference: A: - SUIT-005
SUIT number: D011_1OctM;2D;3P;4S;5U;6Rot;7Ama;8AsTm;9Azd
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 µM O2) must be employed. To set the optimal oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.
Communicated by Doerrier C, Iglesias-Gonzalez J and Gnaiger E (last update 2019-06-05)
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
- + 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
MitoPedia methods: Respirometry