SUIT-034 NADH mt D082

high-resolution terminology - matching measurements at high-resolution

SUIT-034 NADH mt D082

Description

Reference: A: protocol for simultaneous determination of O₂ flux and NADH autofluorescence in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- SUIT-034

SUIT number: D082_mt;1D.1;2PGM;3D2.5;4U;5Anox;6Myx;7Reox

O2k-Application: NADH

SUIT-034 NADH mt D082 allows the study of mitochondrial respiration and NADH fluorescence in two coupling control states, OXPHOS and ET 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-006 NADH mt D084.

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. For calibration of the fully oxidized NAD, this protocol contains a step with the titration of a small concentration of ADP, stimulating the depletion of the endogenous substrates and thus leading to accumulation of oxidized NAD. This protocol is used for cross-calibration of SUIT-006 NADH mt D084, where no low concentration of ADP is added before fuel substrates, allowing the measurement of LEAK respiration and NAD redox state.

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 2023-11-30)

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	PGM_P	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.
4U	PGM_E	N	CI	mt;1D.1;2PGM;3D2.5;4U NADH-linked substrates (type N-pathway to Q). Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency.
5Anox		N	CI	mt;1D.1;2PGM;3D2.5;4U;5Anox 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.
6Myx		N	CI	mt;1D.1;2PGM;3D2.5;4U;5Anox;6Myx 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.
7Reox	ROX			mt;1D.1;2PGM;3D2.5;4U;5Anox;6Myx;7Reox 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-034 NADH mt D082 in combination with SUIT-006 NADH mt D084 provides NAD redox ratios in LEAK, OXPHOS and ET 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 fuels substrates in contrast to SUIT-034 NADH mt D082.

- 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-034 O2 mt DXX.

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

Compare SUIT protocols

SUIT-033 NADH mt D081: Protocol for simultaneous determination of O2 flux and NADH autofluorescence in isolated mitochondria. Similar protocol without uncoupler titrations and ET state evaluation.
SUIT-006 NADH mt D084: 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-034_NADH_mt_D082 in the presence of ATPases in the sample.
SUIT-034 O2 mt D111: Control protocol for respiration only, allowing for cytochrome c test.

Chemicals and syringes

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.
4U	SF6847	We do not recommend using any other uncoupler, e.g. Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U), due to the chemical background effect on fluorescence.
5Anox		The O₂ concentration in the O2k-chamber can be decreased by N₂ or H₂ injection to reach faster anoxia, see: Setting the oxygen concentration.
6Myx	Myxothiazol	We do not recommend using any other inhibitor of complex III, e.g. Antimycin A (Ama), due to the chemical background effect on fluorescence.
7Reox		Reoxygenation can be performed by opening the chamber, see: Open chamber.

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

References