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SUIT-036 O2 mt D089

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SUIT-036 O2 mt D089

Description

1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U;10Rot;11Ama.png

Abbreviation: FAO(Pal) & M kinetics

Reference: A - SUIT-036 - F-pathway and malate anaplerosis

SUIT number: D089_1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U;10Rot;11Ama

O2k-Application: O2


The SUIT-036 O2 mt D089 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-036 O2 mt D089 is specially designed to give information on F-pathway with palmitoylcarnitine as a FAO substrate in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. The careful titration of malate is required to analyze the activity of the mt-isoform of NADP- or NAD(P)-dependent malic enzyme (mtME). 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.

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;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U;10Rot;11Ama.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
3Pal PalMP F FAO 1D;2M.1;3Pal
  • 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 PalMcP F FAO 1D;2M.1;3Pal;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]).
4M.2 PalMP F(N) FAO 1D;2M.1;3Pal;3c;4M.2
4M.5 PalMP F(N) FAO 1D;2M.1;3Pal;3c;4M.2;4M.5
4M1 PalMP F(N) FAO 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1
4M2 PalMP F(N) FAO 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2
5P PalPMP FN FAO&CI 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;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.
6G PalPGMP FN FAO&CI 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G
  • 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.
7S10 PalPGMSP FNS FAO&CI&II 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10
  • 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.
7S50 PalPGMSP FNS FAO&CI&II 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50
  • 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.
8Gp PalPGMSGpP FNSGp FAO&CI&II&GpDH 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp
9U PalPGMSGpE FNSGp FAO&CI&II&GpDH 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U
10Rot SGpE SGp CII&GpDH 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U;10Rot
  • 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.
11Ama ROX 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U;10Rot;11Ama
  • 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-036 in combination with SUIT-037 and SUIT-038 provides a basis to study fatty acid oxidation (FAO) in a large variety of species, tissues and cell types. These protocols in combination allow:
1. to assess FAO carefully avoiding overestimation by endogenous substrates or malate anaplerosis.
2. to compare FAO with palmitoylcarnitine or octanoylcarnitine.
3. to analyse malate anaplerosis with malate kinetics
4. to analyse OXPHOS respiration and ET-capacity with various pathways (F, N, S, Gp)
  • A succinate concentration of >10 mM may be required for saturating SE capacity.
+ SUIT-036 allows the depletion of endogenous substrates with ADP (1D).
+ The protocol provides information on F-pathway in OXPHOS state. The low concentration of malate used in this protocol, 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, FNSGp pathways) and in ET state (FNSGp and SGp) can be observed.
+ Multiple pathways converging into Q (FNSGp) are assessed in OXPHOS and ET states. Therefore, P/E (8Gp/9U) at high ET capacity can be calculated.
- Very long duration of the experiment.
- LEAK state is 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, also 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, also 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)
3Pal Palmitoylcarnitine (Pal)
3c Cytochrome c (c)
4M.2 Malate (M)
4M.5 Malate (M)
4M1 Malate (M)
4M2 Malate (M)
5P Pyruvate (P)
6G Glutamate (G)
7S10 Succinate (S)
7S50 Succinate (S)
8Gp Glycerophosphate (Gp)
9U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler
10Rot Rotenone (Rot)
11Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.

References



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FAT4BRAIN
The project FAT4BRAIN has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 857394

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