SUIT-016

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SUIT-016

Description

1OctM;2D;3G;4S;5Rot;6Omy;7U-.png

Abbreviation: F+G+S+Rot_OXPHOS+Omy

Reference: A: F-pathway in LEAK state and OXPHOS state

SUIT protocol pattern: 1OctM;2D;3G;4S;5Rot;6Omy;7U-

SUIT-016 gives information on F-pathway in LEAK state and OXPHOS state 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.

Communicated by Doerrier C, Huete-Ortega M, Cardoso LHD and Gnaiger E (last update 2019-06-05)

Specific SUIT protocols

1OctM;2D;3G;4S;5Rot;6Omy;7U;7c;8Ama.pngSUIT-016 O2 pfi D044 traces.png

MitoPedia: SUIT

Steps and respiratory states

1OctM;2D;3G;4S;5Rot;6Omy;7U;8Ama.png


Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1OctM OctML(n) F(N) CETF 1OctM
  • Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration.
  • Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(n), in the absence of ADP, ATP, AMP (no adenylates).
2D OctMP F(N) CETF 1OctM;2D
  • Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration.
  • Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
3G OctGMP FN CETF&I 1OctM;2D;3G
  • Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. & NADH-linked substrates (type N-pathway to Q).
  • Respiratory stimulation by simultaneous action of fatty acid (F) and type N substrates (N) with convergent electron flow in the FN-pathway for reconstitution of TCA cycle function and additive or inhibitory effect of F.
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
4S OctGMSP FNS CETF&CI&II 1OctM;2D;3G;4S
5Rot SP S CII 1OctM;2D;3G;4S;5Rot
6Omy SL(Omy) S CII 1OctM;2D;3G;4S;5Rot;6Omy
  • Succinate, S ( type S-pathway to Q).
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
7U SE S CII 1OctM;2D;3G;4S;5Rot;6Omy;7U
8Ama ROX 1OctM;2D;3G;4S;5Rot;6Omy;7U;8Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of Antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).
Step Respiratory state Pathway control ET-Complex entry into Q-junction Comment
## AsTm AsTmE CIV CIV
## Azd ROX


Questions.jpg


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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 criteria.
  • (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 criteria for samples that normally do not present mitochondrial outer membrane damage after the sample preparation, e.g. human permeabilized muscle fibers.

Compare SUIT protocols

References

 YearReferenceOrganismTissue;cell
Gnaiger 2015 Scand J Med Sci Sports2015Gnaiger 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.HumanSkeletal muscle


MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry