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SUIT-041 O2 mt D096

From Bioblast


high-resolution terminology - matching measurements at high-resolution


SUIT-041 O2 mt D096

Description

1D;2M.1;3AC;3c;4M2;5P;6S;7Rot;8Ama.png

Abbreviation: Optimum [acylcarnitine] test

Reference: A - SUIT-041 - F-pathway and determination of optimum acylcarnitine concentration

SUIT number: D096_1D;2M.1;3AC;3c;4M2;5P;6S;7Rot;8Ama

O2k-Application: O2


The SUIT-041 O2 mt D096 protocol provides a common reference for comparison of respiratory control of mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species, tissues and cell types. SUIT-041 O2 mt D096 is specially designed to give information on F-pathway with titration of acylcarnitine as a FAO substrate to determine the optimal concentration. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS pathways) can be evaluated by using this SUIT protocol.

Communicated by Grings M, Cecatto C and Cardoso LHD (last update 2023-04-12)

Representative traces

MitoPedia: SUIT

Steps and respiratory states

1D;2M.1;3AC;3c;4M2;5P;6S;7Rot;8Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1D ROX 1D
  • ADP is added to stimulate the consumption of endogenous fuel-substrates.


2M.1 1D;2M.1
3AC ACMP F FAO 1D;2M.1;3AC
  • 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.
3c ACMcP F FAO 1D;2M.1;3AC;3c
  • 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]).
4M2 ACMP F(N) FAO 1D;2M.1;3AC;3c;4M2
5P ACPMP FN FAO&CI 1D;2M.1;3AC;4M2;5P
  • 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.
6S ACPMSP FNS FAO&CI&II 1D;2M.1;3AC;4M2;5P;6S
  • Respiratory stimulation by simultaneous action of the F-pathway, N-pathway, and S-pathway, with convergent electron flow in the FNS-pathway for reconstitution of TCA cycle function and additive or inhibitory effect of F.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
7Rot SE SGp CII 1D;2M.1;3AC;4M2;5P;6S;7Rot
  • Respiratory stimulation by action of succinate and glycerophosphate, Gp, with convergent electron flow in the SGp-pathway (CII&GpDH-linked pathway to the Q-junction).
  • Noncoupled electron transfer state, ET state, with ET capacity E.
8Ama ROX 1D;2M.1;3AC;4M2;5P;6S;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).


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Strengths and limitations

  • SUIT-041 has been developed to test the optimum concentration of acylcarnitine for high-resolution respirometry experiments. High concentrations of fatty acids or acylcarnitines might inhibit respiration, therefore, it is recommended to test the optimum concentration for new samples or different acylcarnitines.
+ SUIT-041 allows the depletion of endogenous substrates with ADP (1D).
+ The protocol provides information on F-pathway in OXPHOS state with multiple titrations of acylcarnitines (e.g., octanoylcarnitine, palmitoylcarnitine) until the optimum concentration is reached (respiration reaches its maximum and does not increase further with more acylcarnitine).
+ The low concentration of malate used in this protocol to assess F-pathway, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
+ F-pathway (3Pal-2M.1) can be compared to FN-pathway (5P) in OXPHOS state.
+ Pathway control in OXPHOS (F, F(N), FN, FNS pathways) can be observed.
- Very long duration of the experiment.
- Glycerophosphate (Gp) pathway is not investigated.
- LEAK and ET states are not investigated.


Compare SUIT protocols

  • SUIT-002 O2 mt D005: Reference protocol SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized). Allows evaluation of FAO with octanoylcarnitine, without overestimation by using low malate concentration. Besides this, provides information on pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and ET-state (FNSGp, SGp pathways).
  • SUIT-025_O2_mt_D057: Allows evaluation of FAO with octanoylcarnitine, without overestimation by using low malate concentration. Besides this, provides information on pathway control in OXPHOS state (F, F(N), FN, FNS pathways).

Chemicals and syringes

Step Chemical(s) and link(s) Comments
1D ADP (D)
2M.1 Malate (M)
3AC Template:Acylcarnitine
3c Cytochrome c (c)
4M2 Malate (M)
5P Pyruvate (P)
6S Succinate (S)
7Rot Rotenone (Rot)
8Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.

References

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