SUIT-006 NADH mt D084

high-resolution terminology - matching measurements at high-resolution

SUIT-006 NADH mt D084

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: D084_mt;1PGM;2D;3(Omy);4U;5Anox;6Myx;7Reox

O2k-Application: NADH

The coupling-control protocol SUIT-006 NADH mt D084 allows the study of mitochondrial respiration and NADH fluorescence in the three coupling control states LEAK, OXPHOS and ET 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-034 NADH mt D082, 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.
(3Omy)	PGM_L(Omy)	N	CI	1PGM;2D;(3Omy) Omy addition is skipped in SUIT-006 NADH mt D084 NADH-linked substrates (type N-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
4U	PGM_E	N	CI	1PGM;2D;(3Omy);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	1PGM;2D;(3Omy);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	1PGM;2D;(3Omy);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			1PGM;2D;(3Omy);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-006 NADH mt D084 in combination with SUIT-034 NADH mt D082 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 only be obtained with the combination with SUIT-034 NADH mt D082 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-006 O2 mt D108.

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

Compare SUIT protocols

SUIT-032 NADH mt D078: Protocol for simultaneous determination of O2 flux and NADH autofluorescence in isolated mitochondria. Similar protocol without uncoupler titrations and ET state evaluation.
SUIT-034 NADH mt D082: 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-006 NADH mt D084.
SUIT-006 O2 mt D108: 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)
(3Omy)	Oligomycin (Omy)	This step can be skipped.
4U	SF6847	We do not recommend the use of any other uncoupler, like 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 the use of any other inhibitor of complex III, like 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