SUIT-012 O2 mt D027

high-resolution terminology - matching measurements at high-resolution

SUIT-012 O2 mt D027

Description

Abbreviation: N CCP mtprep

Reference: A Linear coupling control with NADH-linked substrates for isolated mitochondria, tissue homogenate and permeabilized cells- SUIT-012

SUIT number: D027_1PM;2D;2c;3G;4U;5Ama

O2k-Application: O2

The SUIT-012 O2 mt D027 protocol specifically focuses on assessing the coupling control (L- P- E) with NADH-linked substrates (PM and PGM). Addition of G enables evaluating the contribution of this substrate in NADH-supported OXPHOS respiration, which may be relevant in the presence of glutamate-anaplerotic pathway control state. SUIT-012 can be extended with the CIV assay module.

Communicated by Huete-Ortega M, Gnaiger E (last update 2019-06-06)

Representative traces

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.
4U	PGME	N	CI	1PM;2D;2c;3G;4U NADH-linked substrates (type N-pathway to Q). 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.
5Ama	ROX			1PM;2D;2c;3G;4U;5Ama 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

+ This protocol allows to evaluate the function of the TCA cycle without the involvement of the complex II ( S-pathway). It can be useful to understand the contribution and the activity of the dehydrogenases.

+ Mitochondrial outer 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 cytochrome c.

+ Reasonable duration of the experiment.

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

+ 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 (GMP is inhibited by the CII inhibitor malonic acid to a larger extent than PMP). 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 an advantage compared to SUIT-011 for diagnosis of N-capacity.

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

Compare SUIT protocols

• SUIT-008: 1PM;2D;3G;4S;5U-; longer version that also analyses S-pathway.
• SUIT-006: 1X;2D;3Omy;4U-; coupling control protocol with only one combination of substrates (e.g. 1PM;2D;3Omy;4U-).

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1PM	Pyruvate (P) and Malate (M)
2D	ADP (D)
2c	Cytochrome c (c)
3G	Glutamate (G)
4U	Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)	Can be substituted for other uncoupler
5Ama	Antimycin A (Ama)

Suggested stock concentrations are shown in the specific DL-Protocol.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry