SUIT-001 O2 ce-pce D003

high-resolution terminology - matching measurements at high-resolution

SUIT-001 O2 ce-pce D003

Description

Abbreviation: FNSGp(Oct,PGM)

Reference: A Reference protocol RP1; ce: non-permeabilized cells, pce: permeabilized cells - SUIT-001

SUIT number: D003_ce1;1Dig;1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd

O2k-Application: O2

MitoPedia: SUIT - SUIT reference protocol RP1 for cells that are permeabilized in the chamber

SUIT-category: FNSGp(Oct,PGM)

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

The SUIT-001 O2 ce-pce D003 protocol in combination with SUIT-002 O2 ce-pce D007 is specially designed to provide a common reference for comparison of respiratory control of permeabilized cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol) in a wide variety of cell types. SUIT-001 O2 ce-pce D003 gives information of ROUTINE respiration (ce: non-permeabilized cells) and after the plasma membrane permeabilization (pce: permeabilized cells) of 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 O2 pce D003 can be extended with the CIV assay module.

Communicated by Doerrier C and Gnaiger E (last update 2019-03-27)

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
ce1	ROUTINE			ce1 ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
1Dig	REN			ce1;1Dig Optimum effective digitonin concentration for complete plasma membrane permeabilization.

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1PM	PML(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	PMP	N	CI	1PM;2D NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	PMcP	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	PME	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	PGME	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	PGMSE	NS	CI&II	1PM;2D;3U;4G;5S NADH-linked substrates (type N-pathway to Q). & Succinate, S ( type S-pathway to Q). 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	OctPGMSE	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	SE	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	SGpE	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			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	AsTmE	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-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.

+ In cells, SUIT-001 and SUIT-002 harmonize in: ROUTINE (ce1), SGp_E (8Gp in SUIT-001 and 10Rot in SUIT-002) and Complex IV.

+ Harmonization with many SUIT protocols (up to step 5S).

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

+ If NS- and FNS-pathways are similar (NS_E ~ FNS_E), 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 addition of non-permeabilized cells to the chambers provides additional information (ROUTINE respiration) which can not be obtained in SUIT-001 O2 mt D001 where the cells added to the chambers are already permeabilized.

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

- Lengthy duration of the experiment.

Compare SUIT protocols

• SUIT-001 O2 pce D004: SUIT-001 O2 pce D003 (general SUIT-001 for permeabilized cells) differs from SUIT-001 O2 pce D004 (SUIT-001 for permeabilized PBMCs and PLTs) in the presence and absence of octanoylcarnitine, respectively.

• SUIT-002 O2 pce D007 (RP2): Harmonized SUIT protocol for permeabilized cells.

• SUIT-001 O2 mt D001: SUIT-001 for permeabilized cells (cells added to the chamber have been permeabilized before their addition). This SUIT protocol provides no information on ROUTINE respiration of intact cells (ce).

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

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry