SUIT-006 AmR mt D048

high-resolution terminology - matching measurements at high-resolution

SUIT-006 AmR mt D048

Description

Abbreviation: N(PM)

Reference: A: SUIT-006 short protocol for simultaneous determination of O₂ flux and H₂O₂ in mitochondrial preparations (isolated mitochondria and tissue homogenate).

SUIT number: D048_1PM;2D;3Omy;4U;5Ama

O2k-Application: AmR

SUIT-category: N(PM)

SUIT protocol pattern: orthogonal 1PM;2D;3Omy;4U;5Ama

This is a protocol to investigate the N- control state in isolated mitochondria and tissue homogenate. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Since higher concentrations of Omy can decrease the ET state induced upon addition of uncoupler, the required concentration of Omy has to be determined. This is a short protocol to study simultaneously the O₂ flux and H₂O₂ flux, in particular the dependence of H₂O₂ flux on the mt-membrane potantial. Addition of ADP depolarises the mt-membrane which leads to the decrease of the H₂O₂ flux. Omy hyperpolarises the mt-membrane resulting in increased H₂O₂ production, while the addition of the uncoupler decreases the H₂O₂ generation owing to the depolarisation of the mt-membrane.

Communicated by  Komlodi T (last update 2019-02-20) 

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
0DTPA				DTPA is an iron chelator, which decreases the chemical fluorescence background created by the Amplex UltraRed assay. Administration of DTPA into the O2k-chamber is recommended before all other chemicals because the iron chelation capacity of the compound is time-dependent (approx. 10-15 min). However, the experiments can be carried out in the absence of DTPA.
0SOD				SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H2O2, making it accessible to the Amplex UltraRed assay.
0HRP				HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H2O2 towards the fluorescent resorufin.
0AmR				AmR or Amplex UltraRed forms resorufin with H2O2 catalysed by horseradish peroxidase HRP.

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1PM	PML(n)			1PM NADH-linked substrates (type N-pathway to Q).
2D	PMP			1PM;2D OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3Omy	PML(Omy)			1PM;2D;3Omy Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin. The addition of oligomycin is optional depending on the experimental question to resolve. If it is known that Ln has the same values for LOmy for a kind of sample and substrates, this step may be skipped. If the carrier of oligomycin (EtOH) is used instead, it is possible to evaluate whether it affects OXPHOS. In these cases, LEAK state is not reached again.
4U	PME			1PM;2D;3Omy;4U 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.
5Ama	ROX			1PM;2D;3Omy;4U;5Ama Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).

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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

• This protocol can be run under low oxygen concentration to test the oxygen dependency of mt-ROS production. For more technical details see: SUIT-018 AmR mt D031, SUIT-018 AmR mt D041.

+ Reasonable duration of the experiment.

+ This protocol can be extended with the Complex IV module in the following protocols: SUIT-006 O2 mt D047.

+ Cytochrome c test can be performed in the following protocols:SUIT-006 O2 mt D047.

- CIV activity cannot be determined and Cytochrome c test cannot be performed together with the fluorescence dyes.

- Omy concentration has to be determined. Higher concentrations of Omy may inhibit the ET state.

References

	Year	Reference	Organism	Tissue;cell
MiPNet20.13 Safranin mt-membranepotential	2019-06-24	O2k-FluoRespirometry: HRR and simultaneous determination of mt-membrane potential with safranin or TMRM.	Mouse	Nervous system
Krumschnabel 2014 Methods Enzymol	2014	Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. https://doi.org/10.1016/B978-0-12-416618-9.00009-1	Mouse	Nervous system

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