SUIT-014

high-resolution terminology - matching measurements at high-resolution

SUIT-014

Description

Abbreviation: GM+P+S_OXPHOS+Rot_ET

Reference: A: Cells or tissue types that display a preference for GM over PM to support NADH-linked respiration

SUIT protocol pattern: 1GM;2D;3P;4S;5U;6Rot-

The SUIT-014 protocols are designed for cells or tissue types that display a preference for glutamate & malate (GM) over pyruvate & malate (PM) to support NADH-linked respiration. Adding pyruvate in the OXPHOS state enables to check if NADH-linked respiration can be further stimulated to determine N-pathway P capacity. SUIT-014 protocols provide an assessment of the coupling control L- P with NADH linked-substrates (GM) and P- E with N- and S-pathways combined (PGMS). The pathway control in OXPHOS state (N, NS) and ET state (NS, S) is also evaluated, covering the contribution of two pathways which are most important in the mitochondria of many species, tissues and cell types.

Communicated by Cardoso LHD, Doerrier C, Huete-Ortega M, Gnaiger E (last update 2024-09-19)

Specific SUIT protocols

SUIT-014 O2 pfi D042 for permeabilized muscle fibers

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1GM	GM_L(n)	N	CI	1GM 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	GM_P	N	CI	1GM;2D NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	GMc_P	N	CI	1GM;2D;2c NADH-linked substrates (type N-pathway to Q). 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]). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3P	PGM_P	N	CI	1GM;2D;2c;3P NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4S	PGMS_P	NS	CI&II	1GM;2D;2c;3P;4S 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.
5U	PGMS_E	NS	CI&II	1GM;2D;2c;3P;4S;5U Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. 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. Noncoupled electron transfer state, ET state, with ET capacity E.
6Rot	S_E	S	CII	1GM;2D;2c;3P;4S;5U;6Rot Succinate, S ( type S-pathway to Q). Noncoupled electron transfer state, ET state, with ET capacity E.
7Ama	ROX			1GM;2D;2c;3P;4S;5U;6Rot;7Ama 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

+ NS-OXPHOS capacity provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.

+ The presence of PGM and S establishes fully operative TCA cycle activity.

+ This protocol allows to analyze the convergence of pathways at the Q-junction (N, NS, S).

+ Mitochondrial external membrane integrity can be measured with the addition of cytochrome c. Application of the cytochrome c test early in the protocol ensures comparability of all states in case of any effect of c.

- It was observed that addition of P (5 mM) after GM inhibited respiration in horse skeletal muscle fibres (Votion et al 2012), which was not the case when P was titrated in steps of 1 mM.

- When evaluating the additive effect of the N- and S-pathway, it has to be considered that NS_P- and NS_E-capacities can only be compared with N_P- and S_E-capacities. This is not a problem when NS_P = NS_E (Gnaiger 2009). Otherwise, it may be assumed that S_P = S_E (Votion et al 2012), such that NS_P can be compared with N_P + S_P. SUIT-004 should be chosen for the additive effect in the ET-state.

- Rox may be lower in substrate states earlier in the SUIT protocol. Therefore, this Rox measurement is frequently taken as a methodological control rather than as the final basis of Rox correction of mitochondrial respiration (mt).

- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

+ This protocol can be extended with the Complex IV module, which can prolong the experimental time ~30 min.

Compare SUIT protocols

SUIT-008: 1PM;2D;3G;4S;5U-; similar version starting with PM, and then adding G. Used in combination with SUIT-014 in Lemieux 2017.
SUIT-011: 1GM;2D;2c;3S;4U;5Rot-; similar version, omitting P.
SUIT-012: 1PM;2D;2c;3G;4U-; shorter version with PM preceding PGM and omitting S.

References

	Year	Reference	Organism	Tissue;cell
Lemieux 2017 Sci Rep	2017	Lemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep 7:2840. doi:10.1038/s41598-017-02789-8	Mouse	Heart

MitoPedia concepts: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Respirometry