SUIT-037 O2 mt D090

high-resolution terminology - matching measurements at high-resolution

SUIT-037 O2 mt D090

Description

Abbreviation: FAO(Oct) & M kinetics

Reference: A - SUIT-037 - F-pathway and malate anaplerosis

SUIT number: D090_1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U;10Rot;11Ama

O2k-Application: O2

The SUIT-037 O2 mt D090 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-037 O2 mt D090 is specially designed to give information on F-pathway with octanoylcarnitine 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

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.
3Oct	OctMP	F	FAO	1D;2M.1;3Oct 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.
3c	OctMcP	F	FAO	1D;2M.1;3Oct;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	OctMP	F(N)	FAO	1D;2M.1;3Oct;3c;4M.2 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. Malate kinetics in the presence of saturating [ADP] allows the evaluation of malate anaplerotic pathways. High concentration of malate, typically 2 mM, saturates the N-pathway. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4M.5	OctMP	F(N)	FAO	1D;2M.1;3Oct;3c;4M.2;4M.5 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. Malate kinetics in the presence of saturating [ADP] allows the evaluation of malate anaplerotic pathways. High concentration of malate, typically 2 mM, saturates the N-pathway. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4M1	OctMP	F(N)	FAO	1D;2M.1;3Oct;3c;4M.2;4M.5;4M1 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. Malate kinetics in the presence of saturating [ADP] allows the evaluation of malate anaplerotic pathways. High concentration of malate, typically 2 mM, saturates the N-pathway. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4M2	OctMP	F(N)	FAO	1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;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. Malate kinetics in the presence of saturating [ADP] allows the evaluation of malate anaplerotic pathways. High concentration of malate, typically 2 mM, saturates the N-pathway. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5P	OctPMP	FN	FAO&CI	1D;2M.1;3Oct;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	OctPGMP	FN	FAO&CI	1D;2M.1;3Oct;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	OctPGMSP	FNS	FAO&CI&II	1D;2M.1;3Oct;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	OctPGMSP	FNS	FAO&CI&II	1D;2M.1;3Oct;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	OctPGMSGpP	FNSGp	FAO&CI&II&GpDH	1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp Respiratory stimulation by simultaneous action of fatty acid (F), type N substrates (N), succinate (S), and glycerophosphate with convergent electron flow in the FNSGp-pathway to the Q-junction. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
9U	OctPGMSGpE	FNSGp	FAO&CI&II&GpDH	1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U Respiratory stimulation by simultaneous action of fatty acid (F), type N substrates (N), succinate (S), and glycerophosphate with convergent electron flow in the FNSGp-pathway to the Q-junction. 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	SGpE	SGp	CII&GpDH	1D;2M.1;3Oct;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;3Oct;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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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-037 in combination with SUIT-036 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 S_E capacity.

+ SUIT-037 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-036_O2_mt_D089: Similar protocol, using palmitoylcarnitine instead of octanoylcarnitine.

• SUIT-038 O2 mt D091: Control protocol, without acylcarnitine.

• SUIT-002 O2 mt D005: Reference protocol SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized). Also allows evaluation of FAO 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: Also allows evaluation of FAO without overestimation by using low malate concentration, besides this, provides information on pathway control in OXPHOS state (F, F(N), FN, FNS pathways).

• SUIT-017_O2_mt_D046: For samples without mitochondrial malic enzyme activity, allows to assess FAO with octanoylcarnitine. Besides this, it is possible to analyse FN and FNS-linked respiration in OXPHOS state, and FNS- and S-linked respiration in and ET-state.

• 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)
3Oct	Octanoylcarnitine (Oct)
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

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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