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-032

SUIT number: D078_File:1mt;1PGM;2D;3Anox;4Myx;5Reox

O2k-Application: NADH

The coupling-control protocol SUIT-032 NADH mt D078 allows the study of mitochondrial respiration and NADH fluorescence 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. If these substrates are fully consumed by the mitochondria, this step can be used for an approximate calibration of oxidized NAD (NAD defined as the sum of the oxidized NAD⁺ and the reduced NADH). If this is not possible, this protocol should be used in combination with SUIT-033 NADH mt D081, where the titration of a small concentration of ADP leads to depletion of endogenous substrates, thus leading to accumulation of oxidized NAD, allowing to calibrate for the fully oxidized NAD.

Anoxia is reached by letting mitochondria fully consume the oxygen in the O2k-chambers. In the absence of O2, the ETS upstream of CIV is reduced and thus leads to an accumulation of reduced NAD. Under anoxia the complex III inhibitor myxothiazol is added and a further increase in the reduced NAD fraction can be observed. This step is then used for the calibration of the fully reduced NAD. At the end of the protocol, the reoxigenation of the chamber 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. In the absence of endogenous substrates, mt can be used for calibration of fully oxidized NAD.
1PGM	PGM_L(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	PGM_P	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 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-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

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.

References