SUIT-038 O2 mt D091

high-resolution terminology - matching measurements at high-resolution

SUIT-038 O2 mt D091

Description

Abbreviation: FAO control & M kinetics

Reference: A - SUIT-038 - F-pathway and malate anaplerosis - control protocol

SUIT number: D091_1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp;8U;9Rot;10Ama

O2k-Application: O2

The SUIT-038 O2 mt D091 protocol is focused on the analysis of the malate anaplerotic pathway control state in 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. Malate alone does not support respiration if oxaloacetate is not metabolized further in the absence of acetyl-CoA. The careful titration of malate is required to analyze the activity of the mt-isoform of NADP- or NAD(P)-dependent malic enzyme (mtME). If these enzymes are present in the sample one should be able to reach with malate alone a high respiratory activity comparable to the NADH-linked pathway control states with more classical combinations of substrates (e.g. PM or GM). Moreover, the pathway control in OXPHOS state ((N), N, NS, NSGp pathways) and in ET state (NSGp 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. Malate kinetics in the presence of saturating [ADP] allows the evaluation of malate anaplerotic pathways.
2H2O				1D;2M.1;2H2O Titration of carrier (H2O) as a control, to be compared with protocols with titration of acylcarnitines to assess fatty acid oxidation-linked respiration.
3c				1D;2M.1;2H2O;2c 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]).
3M.2				1D;2M.1;2H2O;2c;3M.2 Malate kinetics in the presence of saturating [ADP] allows the evaluation of malate anaplerotic pathways. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3M.5				1D;2M.1;2H2O;2c;3M.2;3M.5 Malate kinetics in the presence of saturating [ADP] allows the evaluation of malate anaplerotic pathways. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3M1				1D;2M.1;2H2O;2c;3M.2;3M.5;3M1 Malate kinetics in the presence of saturating [ADP] allows the evaluation of malate anaplerotic pathways. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3M2				1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2 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.
4P	PM_P	N	CI	1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5G	PGM_P	N	CI	1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
6S10	PGMS_P	NS	CI&II	1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10 Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
6S50	PGMS_P	NS	CI&II	1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50 Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
7Gp	PGMSGp_P	NSGp	CI&II&GpDH	1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp Respiratory stimulation by simultaneous action of type N substrates (N), succinate (S), and glycerophosphate with convergent electron flow in the NSGp-pathway to the Q-junction. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
8U	PGMSGp_E	NSGp	CI&II&GpDH	1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp;8U Respiratory stimulation by simultaneous action of type N substrates (N), succinate (S), and glycerophosphate with convergent electron flow in the NSGp-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.
9Rot	SGp_E	SGp	CII&GpDH	1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp;8U;9Rot 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.
10Ama	ROX			1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp;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).

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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-038 provides a control protocol without fatty acids, to in combination with SUIT-036 and SUIT-037 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-038 allows the depletion of endogenous substrates with ADP (1D).

+ Pathway control in OXPHOS (N, NS, NSGp pathways) and in ET state (NSGp and SGp) can be observed.

+ Multiple pathways converging into Q (NSGp) are assessed in OXPHOS and ET states. Therefore, P/E (7Gp/8U) 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: Using palmitoylcarnitine to assess FAO - SUIT-038_O2_mt_D091 serves as a control protocol.

SUIT-037 O2 mt D090: Using octanoylcarnitine to assess FAO - SUIT-038_O2_mt_D091 serves as a control protocol.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1D	ADP (D)
2M.1	Malate (M)
2H2O
2c	Cytochrome c (c)
3M.2	Malate (M)
3M.5	Malate (M)
3M1	Malate (M)
3M2	Malate (M)
4P	Pyruvate (P)
5G	Glutamate (G)
6S10	Succinate (S)
6S50	Succinate (S)
7Gp	Glycerophosphate (Gp)
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

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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