SUIT-014 O2 pfi D042

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SUIT-014 O2 pfi D042

Description

Abbreviation: NS(PGM)

Reference: A

SUIT number: D042_1GM;2D;3P;4S;5U;6Rot;7Ama

O2k-Application: O2

SUIT-category: NS(PGM)

SUIT protocol pattern: diametral 1GM;2D;3P;4S;5U;6Rot;7Ama

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). 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	PGMP	N	CI	1GM;2D;2c;3P NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4S	PGMSP	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	PGMSE	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	SE	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	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).

+ 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.

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

Compare SUIT protocols

• SUIT-011: 1GM;2D;2c;3S;4U;5Rot-; similar version of omitting P.
• SUIT-012: 1PM;2D;2c;3G;4U-; shorter version with PM preceding PGM and omitting S.

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry