SUIT-039 O2 mt D092

high-resolution terminology - matching measurements at high-resolution

SUIT-039 O2 mt D092

Description

Reference: A - SUIT-039

SUIT number: D092_1D;2M.1;3Pal;3c;4M2;5P;6G;7S;8U;9Rot;10Ama

O2k-Application: O2

The SUIT-039 O2 mt D092 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-039 O2 mt D092 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. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS pathways) and in ET state (FNS and S) can be evaluated by using this SUIT protocol.

Communicated by Grings M, Cecatto C and Cardoso LHD (last update 2023-04-13)

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.
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]).
4M2	PalMP	F(N)	FAO	1D;2M.1;3Pal;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	PalPMP	FN	FAO&CI	1D;2M.1;3Pal;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;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.
7S	PalPGMSP	FNS	FAO&CI&II	1D;2M.1;3Pal;4M2;5P;6G;7S 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.
8U	PalPGMSE	FNS	FAO&CI&II	1D;2M.1;3Pal;4M2;5P;6G;7S;8U 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. Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency.
10Rot	SE	S	CII	1D;2M.1;3Pal;4M2;5P;6G;7S;8U;9Rot Succinate, S ( type S-pathway to Q). Noncoupled electron transfer state, ET state, with ET capacity E.
11Ama	ROX			1D;2M.1;3Pal;4M2;5P;6G;7S;8U;9Rot;10Ama 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).

Strengths and limitations

• SUIT-039 allows assessing FAO-linked respiration avoiding overestimation by endogenous substrates or malate anaplerosis. Other electron transfer pathways are also analyzed in OXPHOS (FN, FNS) and ET-state (FNS, S).

+ SUIT-039 allows the depletion of endogenous substrates with ADP (1D).

+ The protocol provides information on F-pathway in OXPHOS state with palmitoylcarnitine, a long-chain acylcarnitine commonly found in vivo.

+ 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) and in ET state (FNS and S) can be observed.

+ Multiple pathways converging into Q (FNS) are assessed in OXPHOS and ET states. Therefore, P/E (7S/8U) at high ET capacity can be calculated.

- LEAK state is not investigated.

- Glycerophosphate (Gp) pathway is not investigated.

Compare SUIT protocols

• SUIT-036_O2_mt_D089: Similar protocol, including malate kinetics to assess mitochondrial malic enzyme and anaplerosis.

• 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).

• SUIT-041 O2 mt D096: A protocol to determine the optimum concentration of acylcarnitine.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1D	ADP (D)
2M.1	Malate (M)
3Pal	Palmitoylcarnitine (Pal)
3c	Cytochrome c (c)
4M2	Malate (M)
5P	Pyruvate (P)
6G	Glutamate (G)
7S	Succinate (S)
8U	Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)	Can be substituted for other uncoupler
9Rot	Rotenone (Rot)
10Ama	Antimycin A (Ama)

Suggested stock concentrations are shown in the specific DL-Protocol.

References

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