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SUIT-017 O2 mt D046

From Bioblast


high-resolution terminology - matching measurements at high-resolution


SUIT-017 O2 mt D046

Description

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

Abbreviation: FNS(Oct,GM)

Reference: A: SUIT-017

SUIT number: D046_1OctM;2D;2c;3G;3c;4S;5U;6Rot;7Ama

O2k-Application: O2


MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry 




SUIT-category: FNS(Oct,GM)
SUIT protocol pattern: diametral 1OctM;2D;3G;4S;5U;6Rot-

SUIT-017 O2 mt D046 gives information on F-pathway in LEAK state and OXPHOS state. In addition, the pathway control of FN and FNS in OXPHOS state and of FNS and S in ET state is evaluated.

Communicated by Cardoso LHD, Gnaiger E (last update 2019-07-31)

Representative traces

D046 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

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

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1OctM OctML(n) F(N) FAO 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) FAO 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.
2c OctMcP FN FAO 1OctM;2D;2c
  • NADH-linked substrates (type N-pathway to Q). 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. 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]).
3G OctGMP FN F&CI 1OctM;2D;2c;3G;
4S OctGMSP FNS F&CI&II 1OctM;2D;2c;3G;4S
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. 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.
5U OctGMSE FNS F&CI&II 1OctM;2D;2c;3G;4S;5U
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. 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. Noncoupled electron transfer state, ET state, with ET capacity E.
6Rot SE S CII 1OctM;2D;2c;3G;4S;5U;6Rot
7Ama ROX 1OctM;2D;2c;3G;4S;5U;6Rot;7Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.
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 F-OXPHOS capacity in the absence of a significant OXPHOS capacity in the malate-anaplerotic pathway control state.
+ Glutamate is easier to prepare compared to pyruvate.
+ Reasonable duration of the experiment.
+ This protocol can be extended with the Complex IV module, which can prolong the experimental time by ~30 min.
- This protocol should not be used to analyze FAO in samples that express mitochondrial malic enzyme and thus present a significant OXPHOS capacity in the malate-anaplerotic pathway control state.
- This protocol analyses only GM as NADH-linked substrates. Comparison of GM- with PM-capacity yields important information on N-pathway respiratory control upstream of CI (Lemieux et al 2017; Votion et al 2012).
- F-OXPHOS capacity may be underestimated with Oct. In human heart muscle addition of Oct to palmitoylcarnitine (Pal) & malate increased OXPHOS capacity by 26% (Lemieux et al 2011).

Compare SUIT protocols

  • SUIT-002 or SUIT-025 to measure F-OXPHOS capacity when malate anaplerotic activity is present; are also more comprehensive protocols including additional substrate states.
  • SUIT-005: A comparable protocol using pyruvate (P) instead of glutamate (G).


Chemicals and syringes

Step Chemical(s) and link(s) Comments
1OctM Octanoylcarnitine (Oct) and Malate (M)
2D ADP (D)
2c Cytochrome c (c)
3G Glutamate (G)
4S Succinate (S)
5U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler
6Rot Rotenone (Rot)
7Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.

References