Reference: A SUIT-019
SUIT number: D045_1PalM;2D;2c;3Oct;4P;5G;6U;7S;8Rot;9Ama
SUIT-019 O2 pfi D045 gives information on F-pathway in LEAK and OXPHOS states in permeabilized fibers. Moreover, this SUIT protocol allows the pathway control evaluation of FN in OXPHOS state and FN, FNS and S in ET state. 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, Doerrier C and Gnaiger E (last update 2019-03-06)
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
- SUIT-019 provides an estimation of F OXPHOS capacity, physiologically relevant estimate of maximal Complex I capacity as well as of FNS ET capacity.
- + The protocol provides information on FAO capacity in the absence of other, potentially interfering pathways, both in the LEAK state and in OXPHOS.
- + F OXPHOS capacity is estimated in presence of both Oct and Pal therefore it is more accurate than an estimate in the presence of only one of the substrates. In human heart muscle addition of Oct to palmitoylcarnitine (Pal) + malate increased OXPHOS by 26% (Lemuieux et al 2011).
- + FN OXPHOS capacity comprises the most important Complex I-linked pathways in many cell types and thus provides a physiologically relevant estimate of maximum CI capacity.
- + FNS ET capacity is a good estimate of overall ET capacity in many cell types
- + The presence of PMG and S establishes fully operative TCA cycle activity.
- + Reasonable duration of the experiment.
- - Oct and Pal can have an uncoupling effect during F OXPHOS and FN OXPHOS states if their concentration is not optimized.
- - SRot(E) may be underestimated if S is not saturating.
- - CIV activity is not measured, to save experimental time.
Compare SUIT protocols
- SUIT-002 (RP2): SUIT protocol specially designed to give information on F-pathway in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be evaluated by using this SUIT protocol.
Chemicals and syringes
|Step||Chemical(s) and link(s)||Comments|
|1PalM||Palmitoylcarnitine (Pal) and Malate (M)|
|2c||Cytochrome c (c)|
|6U||Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)||Can be substituted for other uncoupler|
|9Ama||Antimycin A (Ama)|
- Suggested stock concentrations are shown in the specific DL-Protocol.
|Lemieux 2011 Int J Biochem Cell Biol||2011||Lemieux H, Semsroth S, Antretter H, Höfer D, Gnaiger E (2011) Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int J Biochem Cell Biol 43:1729–38.||Human||Heart|
MitoPedia methods: Respirometry