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 Doerrier C and Gnaiger E (last update 2019-09-24)

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 consumption of endogenous fuel-substrates. 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.
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.
9Rot	SE	S	CII	1D;2M.1;3Oct;4M2;5P;6G;7S;8U;9Rot Succinate, S ( type S-pathway to Q). Noncoupled electron transfer state, ET state, with ET capacity E.
10Ama	ROX			1D;2M.1;3Oct;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-002 in combination with SUIT-001 provides a common reference for comparison of respiratory control in a large variety of species, tissues and cell types. Both SUIT protocols provide a mitochondrial mapping which allows:

1. to obtain reference values.

2. to evaluate mitochondrial physiological diversity, generating a mt-database on comparative mitochondrial physiology.

3. to screen specific defects.

• SUIT-001 and SUIT-002 are used in the MitoFit Proficiency test for inter-individual and inter-laboratory reproducibility quality control.
• A succinate concentration of >10 mM may be required for saturating S_E capacity.

+ SUIT-002 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 (3Oct-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.

+ Harmonization with SUIT-001 allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements.

+ Harmonization with many SUIT protocols (up to step 7S).

+ In SUIT-002, the full set of pathways converging into Q (FNSGp) is obtained in OXPHOS and ET states. Therefore, P/E (8Gp/9U) at high ET capacity can be calculated.

+ This protocol can be extended with the Complex IV module.

- S-pathway in ET state is not obtained (it is obtained in SUIT-001).

- Lengthy duration of the experiment.

- We do not generally recommended the addition of already permeabilized cells, but we recommend to perform the permeabilization of the cell plasma membrane in the chambers (see SUIT-002 O2 ce-pce D007).

Compare SUIT protocols

• SUIT-001 O2 mt D001 (RP1): Harmonized SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized).

• SUIT-002 O2 ce-pce D007: SUIT-002 for non-permeabilized cells. Permeabilization of the cell plasma membrane occurs in the chambers through digitonin addition. Therefore, SUIT-002 O2 ce-pce D007 protocol provides information of non-permeabilized cell respiration (ce) and permeabilized cell respiration (pce).

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.