SUIT-041 O2 mt D096

high-resolution terminology - matching measurements at high-resolution

SUIT-041 O2 mt D096

Description

Reference: A - SUIT-041

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

Steps and respiratory states

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 Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
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 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. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) the F-pathway capacity is overestimated, if there is an added contribution of NADH-linked respiration, F(N) (see SUIT-002). The FA concentration has to be optimized to saturate the FAO-pathway, without inhibiting or uncoupling respiration. High concentration of malate, typically 2 mM, saturates the N-pathway. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
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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Coupling control

» Coupling control state

» ET capacity

» OXPHOS capacity

» LEAK respiration

Pathway control

» Electron transfer pathway

» Fatty acid oxidation pathway control state, F

» NADH electron transfer-pathway state, N

» Succinate pathway control state, S

» NS-pathway control state, NS

» Glycerophosphate pathway control state, Gp

» Complex IV single step, CIV

» Anaplerotic pathway control state

Main fuel substrates

» Glutamate, G

» Glycerophosphate, Gp

» Malate, M

» Octanoylcarnitine, Oct

» Pyruvate, P

» Succinate, S

Glossary

» List of SUIT states

» SUIT concept

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-036_O2_mt_D089, SUIT-037 O2 mt D090, SUIT-038 O2 mt D091: A combination of protocols to assess FAO-linked respiration using palmitoylcarnitine and octanoylcarnitine, and investigating the presence of malic enzyme with malate kinetics.

• 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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