SUIT-032 O2 mt D109

high-resolution terminology - matching measurements at high-resolution

SUIT-032 O2 mt D109

Description

Reference: A: protocol for simultaneous determination of O₂ flux and NADH autofluorescence in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- SUIT-006

SUIT number: D109_mt;1PGM;2D;3Myx.png

O2k-Application: NADH

The coupling-control protocol SUIT-032 O2 mt D109 is used as a control protocol for SUIT-032 NADH mt D078, allowing for cytochrome c test and the evaluation of mitochondrial respiration in two coupling control states: LEAK, and OXPHOS in the N-pathway

After the addition of mitochondria in the absence of fuel substrates and ADP, Ren, respiration due to oxidation of endogenous substrates remaining after mitochondrial isolation is measured.

The addition of the complex III inhibitor myxothiazol allows the measurement of Rox.

Communicated by  Grings M, Cardoso Luiza HD (last update 2023-12-21) 

Representative traces

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Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
mt	REN			mt Respiration in the REN state is due to the presence of residual endogenous substrates.
1PGM	PGML(n)	N	CI	1PGM 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	PGMP	N	CI	1PGM;2D NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3Myx	ROX			1PGM;2D;3Myx Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of myxothiazol (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).

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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

• SUIT-032 NADH mt D078 in combination with SUIT-033 NADH mt D081 provides NAD redox ratios in LEAK and OXPHOS states, measured simultaneously with respiration.

+ Reasonable duration of the experiment.

+ H₂ gas from Oxia or N₂/argon can be used to decrease O₂ concentration to obtain anoxia faster.

- Fully oxidized NAD can only be obtained with the combination with SUIT-033 NADH mt D081 or with samples in which endogenous substrates are absent.

- Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors. The addition of liver homogenate is recommended in the washing protocol to bind strong inhibitors.

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

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

- Antimycin A and CCCP cannot be used due to the high chemical background effect on fluorescence.

- Cytochrome c test cannot be performed during the protocol as it affects fluorescence. Cytochrome c test can be performed in the following protocol: SUIT-032 O2 mt D109.

• After myxothyazol titration, this protocol can be extended with the Complex IV assay.

Compare SUIT protocols

• SUIT-006 NADH mt D084: Protocol for simultaneous determination of O2 flux and NADH autofluorescence in isolated mitochondria. Similar protocol with uncoupler titrations and ET state evaluation.
• SUIT-033 NADH mt D081: Protocol for simultaneous determination of O₂ flux and NADH autofluorescence in isolated mitochondria, allowing for calibration of the NAD redox ratios with samples that contain residual endogenous substrates. Additional titration of low concentration of ADP (0.1 μM) for depletion of endogenous substrates and calibration of fully reduced NAD, allowing for cross-calibration with SUIT-032 NADH mt D078.
• SUIT-032 O2 mt D109: Control protocol for respiration only, allowing for cytochrome c test.

Chemicals and syringes

If the experiment is performed as a control for D078, we recommend using the same chemicals.

Step	Chemical(s) and link(s)	Comments
1PGM	Pyruvate (P), Glutamate (G), and Malate (M)
2D	ADP (D)
3Myx	Myxothiazol

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