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

Communicated by  Grings M, Cardoso Luiza HD (last update 2023-12-14) 

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 is detected due to oxidation of endogenous substrates remaining after mitochondrial isolation and can be used for an approximate calibration of oxidized NAD (NAD defined as the sum of the oxidized NAD⁺ and the reduced NADH). In order to perform a calibration for the fully oxidized NAD this protocol should be used in combination with SUIT-034 NADH mt D082, where the titration of a small concentration of ADP depletes the endogenous substrates and leads to the oxidation of NAD.

The use of oligomycin is optional, however, it provides important information when residual and endogenous adenylates are present (which may happen if ATPases are active in the sample). This situation may lead to overestimated LEAK respiration measured in the absence of adenylates - L(n). Therefore, oligomycin can be used to verify whether this occurs and obtain the LEAK state appropriately. Since higher concentrations of Omy can decrease the ET state induced upon the addition of uncoupler, the required concentration of Omy has to be assessed by the Omy titration test.

Anoxia was reached when the mitochondria consumed 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.

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.
(3Omy)	PGML(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	PGME	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 a common reference for comparison of respiratory control in a large variety of species, tissues and cell types. Both SUIT protocols provide a mitochondrial mapping which allows:

1. to obtain reference values.

2. to evaluate mitochondrial physiological diversity, generating a mt-database on comparative mitochondrial physiology.

3. to screen specific defects.

+ Reasonable duration of the experiment.

- Fully oxidized NAD can only be obtained with the combination with SUIT-034 NADH mt D082 or in the absence of endogenous substrates

- 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.

- Ama and CCCP cannot be used due to the high chemical background effect on fluorescence

- CIV activity cannot be determined and cytochrome c test cannot be performed together with the NADH-Module.

• Cytochrome c test can be performed in the following protocol:[[]].
• This protocol can be extended with the Complex IV module in the following protocol: [[]].

Compare SUIT protocols

• SUIT-032 NADH mt D078: Coupling-control protocol for simultaneous determination of O2 flux and NADH autofluorescence in mt-preparations (mtprep). Similar protocol without uncoupler titrations and ET state evaluation.
• SUIT-034 NADH mt D082: Coupling-control protocol for simultaneous determination of O2 flux and NADH autofluorescence in mt-preparations (mtprep). Additional titration of low concentration of ADP (0.1 μM) for depletion of endogenous substrates and calibration of fully reduced NAD. Cross-calibration with SUIT-006 NADH mt D084.
• [[]]

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.