SUIT-011

high-resolution terminology - matching measurements at high-resolution

SUIT-011

Description

Abbreviation: NS(GM)

Reference: A  »Versions

SUIT-category: NS(GM)

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

The SUIT-011 protocols are designed for cells or tissue types that either display a preference for GM over PM to support NADH-linked respiration or is used in comparison with SUIT-004 to evaluate whether such a preference exists. SUIT-011 protocols provide a quick assessment of the linear coupling control (L- P- E) with NADH linked-substrates (GM) and the control in ET state (N, NS, S), covering the contribution of two pathways which are most important in the mitochondria of many species, tissues and cell types. SUIT-011 can be extended with the CIV assay module.

Communicated by Doerrier C and Gnaiger E (last update 2019-01-31)

Specific SUIT protocols

• SUIT-011 O2 pfi D024 for permeabilized fibers

References

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1GM	GML(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	GMP	N	CI	1GM;2D NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	GMcP	N	CI	1GM;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]).
3S	GMSP	NS	CI&II	1GM;2D;2c;3S 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. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4U	GMSE	NS	CI&II	1GM;2D;2c;3S;4U 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.
5Rot	SE	S	CII	1GM;2D;2c;3S;4U;5Rot 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.
6Ama	ROX			1GM;2D;2c;3S;4U;5Rot;6Ama 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

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

• Comparison of GM- with PM-capacity yields important information on N-pathway respiratory control upstream of CI (Lemeux et al 2017; Votion et al 2012).
• A succinate concentration of >10 mM may be required for saturating SE capacity.
• Rox might be inhibited slightly further by inhibition of CIV by cyanide (KCN; 1 μM). But cyanide inhibits not only CIV, but also catalase and other oxygenases involved in ROX.

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

+ Glutamate is easier to prepare compared to pyruvate.

+ Application of the cytochrome c test early in the protocol ensures comparability of all states in case of any effect of c.

+ Reasonable duration of the experiment.

- 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 of the S-pathway is higher with GM compared to PM (GM_P is inhibited by the CII inhibitor malonic acid to a larger extent than PM_P). PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since an impairment of N-pathway capacity can be compensated partially by activation of the S-pathway. This is a disadvantage compared to SUIT-004 and SUIT-008 for diagnosis of N-capacity.

- To detect an additive effect of P after GM_P, pyruvate would have to be added as step 3 (before S). However, inhibition of respiration was observed after titration of P (5 mM) 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.

- CIV activity is not measured, to save experimental time.

Compare SUIT protocols

• GM and PM yield typically identical fluxes in human skeletal muscle fibres.
• SUIT-004 1PM;2D;3U;4S;5Rot-
• SUIT-008 1PM;2D;3G;4S;5U;6Rot-
• 1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp-
• 1PGM;2D;3S;4U;5Rot-

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 

MitoPedia methods: Respirometry