SUIT-033 O2 mt D110

high-resolution terminology - matching measurements at high-resolution

SUIT-033 O2 mt D110

Description

Reference: A: protocol for determination of O₂ flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- SUIT-033

SUIT number: D110_mt;1D.1;2PGM;3D2.5;4Myx

O2k-Application: O2

SUIT-033 O2 mt D110 is used as a control protocol for SUIT-033 NADH mt D081, allowing for cytochrome c test and the evaluation of mitochondrial respiration in OXPHOS in the N-pathway. In the absence of ATPases in the sample, LEAK state can also be evaluated. In order to measure LEAK in the presence of ATPases, this protocol should be performed in parallel to SUIT-032 O2 mt D109.

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 titration of a small concentration of ADP stimulates the depletion of the endogenous substrates, leading to a decrease in mitochondria respiration.

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

Communicated by  Grings M, Cardoso Luiza HD (last update 2024-01-12) 

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.
1D.1				mt;1D.1 The addition of a low concentration of ADP (0.1 μM) stimulates the consumption of endogenous substrates, which can be observed as a decrease in respiration.
2PGM	PGM	N	CI	mt;1D.1;2PGM NADH-linked substrates (type N-pathway to Q). The addition of fuel substrates may trigger a transient peak increase in mitochondrial respiration due to the presence of low ADP concentrations. Once ADP is phosphorylated to ATP the respiration decreases. In the presence of ATPases in the sample, ATP is recycled to ADP, making it not possible to measure LEAK state.
3D2.5	PGMP	N	CI	mt;1D.1;2PGM;3D2.5 NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4Myx	ROX			mt;1D.1;2PGM;3D2.5;4Myx 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

• Control protocol of SUIT-033 NADH mt D081 for respiration only, allowing for cytochrome c test.

+ Reasonable duration of the experiment.

- When ATPases are present in the sample, it is not possible to reach the LEAK state.

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

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

Compare SUIT protocols

• SUIT-033 NADH mt D081: Similar protocol to simultaneously determine O₂ flux and NADH autofluorescence in isolated mitochondria.

Chemicals and syringes

If the experiment is performed as a control for D081, we recommend using the same chemicals.

Step	Chemical(s) and link(s)	Comments
1D.1	ADP (D)	40 mM stock solution.
2PGM	Pyruvate (P), Glutamate (G), and Malate (M)
3D2.5	ADP (D)	500 mM stock solution.
5Myx	Myxothiazol

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