SUIT-031 O2 mt D075

high-resolution terminology - matching measurements at high-resolution

SUIT-031 O2 mt D075

Description

400px

Abbreviation: PM+S+Rot

Reference: A to analyse O₂ flux in the NS- pathway control state on isolated mitochondria- SUIT-031

SUIT number: D0745_1mt;1PM;2D;3S;4Rot;5U;6Ama

O2k-Application: Q

This is a protocol to investigate oxygen flux N- and NS-pathway and S-pathway control state in the OXPHOS state and S-pathway control state in the ET state in isolated mitochondria, tissue homogenate and cells permeabilized before addition in the O2k-chamber. SUIT-031 Q mt D072 and SUIT-031 Q ce-pce D074 are designed to measure simultaneously O₂ flux and the Q-redox state in isolated mitochondria and permeabilized cells.

Communicated by Komlodi T (last update 2021-12-02) 

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). 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	PMSP	NS	CI&II	1PM;2D;2c;3S 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.
4Rot	SP	S	CII	1PM;2D;2c;3S;4Rot Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5U	SE	N	CII	1PM;2D;2c;3S;4Rot;5U Succinate, S ( type S-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.
6Ama	ROX			1PM;2D;2c;3S;4Rot;5U;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).

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

• SUIT-031 Q ce-pce D074 in combination with SUIT-006 Q ce-pce D073 provides a common reference for comparison of respiratory control in a large variety of species, tissues and cell types. Both SUIT protocols provide a mitochondrial mapping which allows:

1. to obtain reference values.

2. to evaluate mitochondrial physiological diversity, generating a mt-database on comparative mitochondrial physiology.

3. to screen specific defects.

• Cytochrome c test can be performed in the following protocol: [[ ]].
• This protocol can be extended with the Complex IV module in the following protocol: [[ ]].
• To study the effect of CoQ₂ on mitochondrial respiration, the following protocol can be used in a parallel experiment: [[ ]].

+ Reasonable duration of the experiment.

- The fully oxidized CoQ₂ cannot be detected with rotenone in this protocol. This protocol can be run simultaneously with SUIT-006 Q ce-pce D073 to determine the fully oxidized CoQ₂. If it is not applicable, the effect of rotenone can be tested on the Q-signal in a separate experiment using the same sample under the same experimental conditions (same respiration medium, same sample concentration). If rotenone does not have a further effect on the Q signal in the presence of sample and coenzyme Q2, this test can be omitted.

- Omy concentration has to be determined if used. Higher concentrations of Omy may inhibit the ET state.

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

- The concentration of the oxidized and reduced Q fraction cannot be determined.

- CIV activity cannot be determined and cytochrome c test cannot be performed together with the Q-Sensor.

Compare SUIT protocols

• SUIT-006 O2 mt D047 is a coupling-control protocol in the N-pathway for isolated mitochondria, tissue homogenate and cells permeabilized before addition to the O2k- chamber.
• SUIT-004 is designed to provide a quick assessment of the linear coupling control (L- P- E) with NADH-linked substrates (PM) and the contribution of the S-pathway to the ET state (N, NS, S).
• SUIT-008 is 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.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1PM	Pyruvate (P) and Malate (M)
2D	ADP (D)
3S	Succinate (S)
4Rot	Rotenone (Rot)
5U	Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)	Can be substituted for other uncoupler.
6Anox		The O2 concentration in the O2k-chamber can be decreased by N2 or H2 injection to reach faster anoxia, see: Setting the oxygen concentration.
7Ama	Antimycin A (Ama)	This step can be omitted.

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

References

	Year	Reference	Organism	Tissue;cell
MiPNet24.12 NextGen-O2k: Q-Module	2021-10-29	NextGen-O2k: Q-Module manual
Komlodi 2021 BEC Q	2021	Komlódi T, Cardoso LHD, Doerrier C, Moore AL, Rich PR, Gnaiger E (2021) Coupling and pathway control of coenzyme Q redox state and respiration in isolated mitochondria. Bioenerg Commun 2021.3. https://doi.org/10.26124/bec:2021-0003	Mouse	Heart Nervous system

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry