SUIT-033 NADH mt D081

high-resolution terminology - matching measurements at high-resolution

SUIT-033 NADH mt D081

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: D081_mt;1D.1;2PGM;3D2.5;4Anox;5Myx;6Reox

O2k-Application: NADH

Communicated by  Grings M, Cardoso Luiza HD (last update 2023-11-30) 

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. In the absence of endogenous substrates, mt can be used for calibration of fully oxidized NAD.
1D.1				mt;1D.1 In the presence of endogenous substrates, the addition of a low concentration of ADP (0.1 μM) is a crucial step to detect the fully oxidized NAD for the calculation of the NAD redox state. ADP stimulates the consumption of endogenous substrates, which can be observed as a decrease in respiration and the NADH fluorescence signal.
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, and reduction of the NAD pool. Once ADP is phosphorylated to ATP the respiration decreases and a further reduction of NAD can be observed. 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.
4Anox		N	CI	mt;1D.1;2PGM;3D2.5;4Anox Anoxia, after the biological sample has consumed the O2 in the O2k-chamber, is used to detect the fully reduced NAD for calculation of the NAD redox states.
5Myx		N	CI	mt;1D.1;2PGM;3D2.5;4Anox;5Myx The addition of myxothiazol after anoxia is a crucial step to detect the fully reduced NAD for calculation of the NAD redox state. Myxothiazol can induce a further reduction of NAD under anoxia.
6Reox	ROX			mt;1D.1;2PGM;3D2.5;4Anox;5Myx;6Reox The reoxygenation by opening the chamber in the presence of Myx allows for Rox-correction of the O2 fluxes.

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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-033 NADH mt D081 in combination with SUIT-032 NADH mt D078 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 be obtained due to low ADP (0.1 μM) titration before the addition of fuel substrates in contrast to SUIT-032 NADH mt D078 or SUIT-006 NADH mt D084.

- 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 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.

- 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-033 O2 mt D110.

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

Compare SUIT protocols

• SUIT-034 NADH mt D082: Protocol for simultaneous determination of O₂ flux and NADH autofluorescence in isolated mitochondria. Similar protocol with uncoupler titrations and ET state evaluation.
• SUIT-032 NADH mt D078: Protocol for simultaneous determination of O₂ flux and NADH autofluorescence in isolated mitochondria. Without titration of low concentration of ADP (0.1 μM) for depletion of endogenous substrates. Therefore, it is possible to measure LEAK respiration, harmonizing it with SUIT-033 NADH mt D081 in the presence of ATPases in the sample.
• SUIT-033 O2 mt D110: Control protocol for respiration only, allowing for cytochrome c test.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1D.1	ADP (D)
2PGM	Pyruvate (P), Glutamate (G), and Malate (M)
3D2.5	ADP (D)
4Anox		The O2 concentration in the O2k-chamber can be decreased by N2 or H2 injection to reach faster anoxia, see: Setting the oxygen concentration.
5Myx	Myxothiazol	We do not recommend the use of any other inhibitor of complex III, like Antimycin A (Ama), due to the chemical background effect on fluorescence.
6Reox		Reoxygenation can be performed by opening the chamber, see: Open chamber.

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