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SUIT-015

From Bioblast


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SUIT-015

Description

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

Abbreviation: F+G+P+S_OXPHOS+Rot_ET

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

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

SUIT-015 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 FNS and S in ET state can be evaluated. SUIT-015 can be extended with the CIV assay module.


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

Specific SUIT protocols

1OctM;2D;3G;3c;4P;5S;6U;7Rot;8Ama.pngD042 Traces.png

MitoPedia: SUIT

Steps and respiratory states

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

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1OctM OctML 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.
  • 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.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3G OctGMP FN CETF&CI 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 the F-pathway and N-pathway with convergent electron flow in the FN-pathway for evaluation of an additive or inhibitory effect of F.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3c OctGMcP FN CETF&CI 1OctM;2D;3G;3c
  • Respiratory stimulation by simultaneous action of the F-pathway and N-pathway with convergent electron flow in the FN-pathway for evaluation of an additive or inhibitory effect of F.
  • Addition of cytochrome c yields a test for integrity of the mtOM (cytochrome c control efficiency). Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (OXPHOS capacity P with saturating [ADP]).
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4P OctPGMP FN CETF&CI 1OctM;2D;3G;3c;4P
  • 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 the F-pathway and N-pathway with convergent electron flow in the FN-pathway for evaluation of an additive or inhibitory effect of F.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5S OctPGMSP FNS CETF&CI&II 1OctM;2D;3G;3c;4P;5S
6U OctPGMSE FNS CETF&CI&II 1OctM;2D;3G;3c;4P;5S;6U
7Rot SE S CII 1OctM;2D;3G;3c;4P;5S;6U;7Rot
8Ama ROX 1OctM;2D;3G;3c;4P;5S;6U;7Rot;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 Comment
## AsTm AsTmE CIV CIV
## Azd CHB


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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 PMG and S establishes a fully operative TCA cycle activity.
+ Reasonable duration of the experiment.
- Application of the cytochrome c test after 3G in the protocol does not ensure comparability of all states in case of any effect of c. However, it allows to analyse whether the outer mt-membrane integrity is compromised.
- F OXPHOS capacity may be underestimated. In human heart muscle addition of Oct to palmitoylcarnitine (Pal) + malate increased OXPHOS by 26% (Lemieux et al 2011).
- SRot(E) may be underestimated if S is not saturating.
- CIV activity is not measured, to save experimental time.

Compare SUIT protocols

References

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry