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 2023-11-30) 

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.
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

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.