SUIT-008

high-resolution terminology - matching measurements at high-resolution

SUIT-008

Description

Abbreviation: PM+G+S_OXPHOS+Rot_ET

Reference: A: Additivity between the N- and S-pathway in the Q-junction

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

The SUIT-008 protocols are designed to assess the additivity between the N- and S-pathway in the Q-junction, providing a physiologically relevant estimate of maximum mitochondrial respiratory capacity. SUIT-008 also serves as a diagnostic tool for the activity of the glutamate dehydrogenase and its linked pathways, which could be relevant in some pathologies. SUIT-008 can be easily extended with the CIV assay module.

Communicated by Cardoso LHD, Doerrier C, Gnaiger E (last update 2019-06-05)

Specific SUIT protocols

• SUIT-008 O2 pfi D014 for permeabilized fibers

• SUIT-008 O2 ce-pce D025 for cells-permeabilized cells (ce-pce)

• SUIT-008 O2 mt D026 for isolated mitochondria and tissue homogenate

Steps and respiratory states

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). 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.
3G	PGMP	N	CI	1PM;2D;2c;3G NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4S	PGMSP	NS	CI&II	1PM;2D;2c;3G;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	PGMSE	NS	CI&II	1PM;2D;2c;3G;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	SE	S	CII	1PM;2D;2c;3G;4S;5U;6Rot Succinate, S ( type S-pathway to Q). 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.
7Ama	ROX			1PM;2D;2c;3G;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	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

+ 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 analyse the convergence of pathways at the Q-junction (N, NS, S).

+ Outer mitochondrial membrane integrity can be evaluated by the addition of cytochrome c (cytochrome c test). The early addition of cytochrome c in the protocol ensures comparability of all states in case of any effect of cytochrome c'.

+ GM and PM yield typically identical fluxes in human skeletal muscle fibres. However, PM is the superior alternative to GM: the fraction of the N-pathway is lower and S-pathway contribution is higher with GM compared to PM. PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since impairment of N-pathway capacity can be compensated partially by activation of the S-pathway. This is an advantage compared to SUIT-011 for the diagnosis of N-capacity.

+ Reasonable duration of the experiment.

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

- F-pathway is not analysed.

- For additive effect evaluation of N- and S-pathways, 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). In this case, 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.

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

Compare SUIT protocols

• SUIT-014: 1GM;2D;3P;4S;5U;6Rot-; similar version starting with GM, and then adding P. Used in combination with SUIT-008 in Lemieux 2017.
• SUIT-004: 1PM;2D;3U;4S;5Rot- The SUIT-004 protocols provide a quick assessment of linear coupling control (L- P- E) with NADH-linked substrates (PM) and pathway control in the ET state (N, NS, S)
• SUIT-011: 1GM;2D;3S;4U;5Rot- The SUIT-011 protocols are designed to study physiologically relevant maximum mitochondrial respiratory capacity (OXPHOS with NS substrates) and coupling/pathway control.

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