SUIT-001

high-resolution terminology - matching measurements at high-resolution

SUIT-001

Description

Abbreviation: RP1

Reference: Doerrier 2018 Methods Mol Biol

MitoPedia concepts: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Respirometry

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

The SUIT-001 protocol in combination with SUIT-002 is specially designed to provide a common reference for comparison of respiratory control of mitochondrial preparations in a wide variety of species, tissues and cell types. SUIT-001 gives information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS, FNS, S and SGp pathways) can be evaluated by using this SUIT protocol. SUIT-001 can be extended with the CIV assay module.

Communicated by Timon-Gomez A, Doerrier C, Gnaiger E (last update 2024-04-09)

Specific SUIT protocols

SUIT-001 O2 mt D001

SUIT-001 O2 mt D001 for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber)

SUIT-001 O2 pfi D002

SUIT-001 O2 pfi D002 for permeabilized muscle fibers

SUIT-001 O2 ce-pce D003

SUIT-001 O2 ce-pce D003 for living cells (ce) and permeabilized cells (pce; permeabilization of the plasma membrane occurs into the chambers through digitonin addition)

SUIT-001 O2 ce-pce D004

SUIT-001 O2 ce-pce D004 for living cells (ce) and permeabilized PBMC and PLT (pce; permeabilization of the plasma membrane occurs into the chambers through digitonin addition)(SUIT-001 O2 ce-pce D004 is performed in the absence of Oct).

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1PM	PM_L(n)	N	CI	1PM NADH-linked substrates (type N-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	PM_P	N	CI	1PM;2D NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	PMc_P	N	CI	1PM;2D;2c NADH-linked substrates (type N-pathway to Q). 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]).
3U	PM_E	N	CI	1PM;2D;3U NADH-linked substrates (type N-pathway to Q). 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.
4G	PGM_E	N	CI	1PM;2D;3U;4G NADH-linked substrates (type N-pathway to Q). Noncoupled electron transfer state, ET state, with ET capacity E.
5S	PGMS_E	NS	CI&II	1PM;2D;3U;4G;5S Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. Noncoupled electron transfer state, ET state, with ET capacity E.
6Oct	OctPGMS_E	FNS	FAO&CI&II	1PM;2D;3U;4G;5S;6Oct 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. Noncoupled electron transfer state, ET state, with ET capacity E.
7Rot	S_E	S	CII	1PM;2D;3U;4G;5S;6Oct;7Rot Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway. Noncoupled electron transfer state, ET state, with ET capacity E.
8Gp	SGp_E	SGp	CII&GpDH	1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp 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.
9Ama	ROX			1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp;9Ama 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

Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

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-001 in combination with SUIT-002 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.

+ Linear coupling control (L-P-E) with N substrates (PM).

+ Pathway control in ET state (N, NS, FNS, S and SGp pathways).

+ Harmonization with SUIT-002 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 5S).

+ Oct is added in ET state to avoid uncoupler effect with fatty acids.

+ The additivity index of N- and S-pathways in ET state can be evaluated.

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

- The substrate combination for maximum ET capacity (FNSGp) is not obtained in SUIT-001 in favour of measuring S_E.

- Oct is not added in PBMC and PLT (SUIT-001 O2 ce-pce D004).

- Lengthy duration of the experiment.

Compare SUIT protocols

SUIT-001 O2 ce-pce D004 (SUIT-001 for permeabilized PBMC and PLT) differs from SUIT-001 O2 ce-pce D003 (general SUIT-001 for permeabilized cells) in the absence and presence of octanoylcarnitine, respectively.

SUIT-002 (RP2): Harmonized SUIT protocol.

SUIT-004: Short version of RP1; without G, F and Gp.
SUIT-006: Very short initial coupling control module.

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