SUIT-002

high-resolution terminology - matching measurements at high-resolution

SUIT-002

Description

Abbreviation: RP2

Reference: A: Doerrier 2018 Methods Mol Biol

SUIT protocol pattern: 1D;2M;3Oct;4M;5P;6G;7S;8Gp;9U;10Rot-

The SUIT-002 protocol in combination with SUIT-001 provides a common reference for comparison of respiratory control of mitochondrial preparations in a wide variety of species, tissues and cell types. SUIT-002 is specially designed to give information on F-pathway in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. 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. SUIT-002 can be extended with the CIV assay module.

Communicated by Doerrier C and Gnaiger E (last update 2024-04-09)

Specific SUIT protocols

SUIT-002 O2 mt D005

SUIT-002 O2 mt D005 for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber)

SUIT-002 O2 pfi D006

SUIT-002 O2 pfi D006 for permeabilized muscle fibers

SUIT-002 O2 ce-pce D007

SUIT-002 O2 ce-pce D007 for living cells (ce) and permeabilized cells (pce; permeabilization of the plasma membrane occurs into the chambers through digitonin addition)

SUIT-002 O2 ce-pce D007a

SUIT-002 O2 ce-pce D007a for living cells (ce) and permeabilized PBMC and PLT (pce; permeabilization of the plasma membrane occurs into the chambers through digitonin addition)

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1D	REN			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	OctM_P	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.
3c	OctMc_P	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]).
4M2	OctM_P	F(N)	FAO	1D;2M.1;3Oct;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	OctPM_P	FN	FAO&CI	1D;2M.1;3Oct;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	OctPGM_P	FN	FAO&CI	1D;2M.1;3Oct;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	OctPGMS_P	FNS	FAO&CI&II	1D;2M.1;3Oct;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.
8Gp	OctPGMSGp_P	FNSGp	FAO&CI&II&GpDH	1D;2M.1;3Oct;4M2;5P;6G;7S;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	OctPGMSGp_E	FNSGp	FAO&CI&II&GpDH	1D;2M.1;3Oct;4M2;5P;6G;7S;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	SGp_E	SGp	CII&GpDH	1D;2M.1;3Oct;4M2;5P;6G;7S;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;4M2;5P;6G;7S;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).

Step	Respiratory state	Pathway control	ET-Complex	Comment
## AsTm	AsTm_E	CIV	CIV
## Azd	CHB

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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-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.

Compare SUIT protocols

SUIT-002 O2 ce-pce D007 (general SUIT-002 for permeabilized cells) differs from SUIT-002 O2 ce-pce D007 (SUIT-002 for permeabilized PBMC and PLT) in the concentration and volume of the titrations.

SUIT-002 O2 ce-pce D007 for permeabilized cells (ROUTINE respiration in living cells is obtained before selectively permeabilizing the plasma membrane).
SUIT-002 O2 ce-pce D007a for permeabilized PBMC and PLT (ROUTINE respiration in living cells is obtained before selectively permeabilizing the plasma membrane).

SUIT-001 (RP1): Harmonized SUIT protocol.
SUIT-005
SUIT-025 SUIT-025 allows the evaluation of different ET-pathway states (F, F(N), FN, FNS and S) in OXPHOS state.
SUIT-027 SUIT-027 focuses on the analysis of the malate-anaplerotic pathway control state in mitochondrial preparations.

References

	Year	Reference	Organism	Tissue;cell
Doerrier 2018 Methods Mol Biol	2018	Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. https://doi.org/10.1007/978-1-4939-7831-1_3	Human Mouse Rat Saccharomyces cerevisiae	Heart Skeletal muscle Endothelial;epithelial;mesothelial cell Blood cells HEK Platelet

MitoPedia concepts: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Respirometry