SUIT-032 NADH mt D078

high-resolution terminology - matching measurements at high-resolution

SUIT-032 NADH mt D078

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: D078_File:1mt;1PGM;2D;3Anox;4Myx;5Reox.png

O2k-Application: NADH

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. In the absence of endogenous substrates, mt can be used for calibration of fully oxidized NAD.
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.
3Anox		N	CI	1PGM;2D;3Anox 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.
4Myx		N	CI	1PGM;2D;3Anox;4Myx 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.
5Reox	ROX			1PGM;2D;3Anox;4Myx;5Reox 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-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-034 NADH mt D082: 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

Step	Chemical(s) and link(s)	Comments
1PGM	Pyruvate (P), Glutamate (G), and Malate (M)
2D	ADP (D)
3Anox		The O2 concentration in the O2k-chamber can be decreased by N2 or H2 injection to reach faster anoxia, see: Setting the oxygen concentration.
4Myx	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.
5Reox		Reoxygenation can be performed by opening the chamber, see: Open chamber.

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