SUIT-011

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SUIT-011

Description

Abbreviation: NS(GM)

Reference: A  »Versions

MitoPedia concepts: SUIT A 

MitoPedia methods: Respirometry 

SUIT-category: NS(GM)

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

References

	Year	Reference	Organism	Tissue;cell
Votion 2012 PLoS One	2012	Votion DM, Gnaiger E, Lemieux H, Mouithys-Mickalad A, Serteyn D (2012) Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle. PLoS One 7:e34890. https://doi.org/10.1371/journal.pone.0034890	Horse	Skeletal muscle
Boushel 2007 Diabetologia	2007	Boushel RC, Gnaiger E, Schjerling P, Skovbro M, Kraunsoee R, Dela F (2007) Patients with Type 2 diabetes have normal mitochondrial function in skeletal muscle. Diabetologia 50:790-6.	Human	Skeletal muscle

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

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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

• 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-

1GM;2D;3S;3c;4U;5Rot;6Ama