SUIT-026

high-resolution terminology - matching measurements at high-resolution

SUIT-026

Description

Abbreviation: RET

Reference: A Short protocol to study RET-related H₂O₂ production

SUIT protocol pattern: 1S;2Rot;3D-

SUIT-026 AmR mt D064 has been designed to study the reverse electron transfer (RET)-initiated ROS production, while SUIT-026 O2 mt D063 serves as a respiratory control protocol for SUIT-026 AmR mt D064 in mitochondrial preparations: isolated mitochondria, tissue homogenate and permeabilized cells (which are already permeabilized when they are added to the chamber). The protocol is focused on LEAK state for the S-pathway to provide the conditions of high membrane potential and redox power in the Q-junction. Under these conditions, in several samples has been described that a reverse flow of the electrons into the membrane arm and the quinone binding site of the Complex I occurs, promoting the production of ROS. The addition of rotenone provides excellent control to this mechanism because this compound blocks the point on which the electrons leak from the Complex I. The titration of ADP at saturating concentrations to reach OXPHOS, allows us to harmonize this protocol with SUIT-006 O2 mt D022 for quality control and a full assessment of the coupling control. According to our results, the H₂O₂ production is linearly dependent on the O₂ concentration in MiR05-Kit, therefore, during the measurements the O₂ concentration has to be well-defined.

Communicated by Timon-Gomez A, Komlodi T, Cardoso LHD and Gnaiger E (last update 2024-09-19)

Specific SUIT protocols

SUIT-026 AmR mt D064

SUIT-026 AmR mt D064 to study RET-initiated H₂O₂ production using mitochondrial preparations (isolated mtiochondria, tissue homogenate or permeabilized cells)

SUIT-026 O2 mt D063

SUIT-026 O2 mt D063 serves as a control for SUIT-026 AmR mt D064 in order to study the effect of the AmR assay on respiration

SUIT-026 AmR ce-pce D087

SUIT-026 AmR ce-pce D087 to study RET-initiated H₂O₂ production using cells, which are permeabilized in the chamber (ce-pce)

SUIT-026 AmR mt D077

SUIT-026 AmR mt D077 to study oxygen dependence of RET-initiated H₂O₂ production using mitochondrial preparations (isolated mitochondria, tissue homogenate or permeabilized cells)

SUIT-026 O2 ce-pce D088

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SUIT-026 O2 ce-pce D088 serves as a control for SUIT-026 AmR ce-pce D087 in order to study the effect of the AmR assay on respiration.

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1S	S_L(n)	S	CII	1S Succinate, S ( type S-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2Rot	S_L(n)	S	CII	1S;2Rot Succinate, S ( type S-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates). Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway.
3D	S_P	S	CII	1S;2Rot;3D Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3c	S_P	S	CII	1S;2Rot;3D;3c Addition of cytochrome c yields a test for integrity of the mtOM (cytochrome c control efficiency). Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (OXPHOS capacity P with saturating [ADP]). Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4Ama	ROX			1S;2Rot;3D;3c;4Ama 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

The conditions on which RET is observable in isolated mitochondria are not physiological but it has been suggested that corresponds to some pathological states.
Many fluorescence dyes can inhibit components of the ET system, thus affecting the respiration. Therefore, a control run of the protocol should be done in the absence of the fluorescence dye. Choose SUIT-026 O2 mt D063 as a control for SUIT-026 AmR mt D064.

+ Rotenone provides an excellent control step for RET owing to its inhibitory effect on RET leading to decreased ROS formation.

+ Short duration of the experiment.

- CIV activity and cytochrome c test cannot be performed together with the fluorescence. SUIT-026 O2 mt D063 can be used in parallel, with cytochrome c addition to assess the mt outer membrane integrity as a quality control of the sample.

- This protocol does not provide information about all the coupling control states (LEAK respiration, OXPHOS and ET). However, it is possible to create a DLPU by adding an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state.

- Measurements with Amplex UltraRed assay cannot be carried out with homogenate.

Compare SUIT protocols

SUIT-006 AmR mt D048 to investigate the dependence of H₂O₂ flux on the mt-membrane potential on the N-control state in isolated mitochondria and tissue homogenate.
SUIT-006 O2 mt D022: CCP mtprep for S-pathway.

References

MitoPedia concepts: SUIT protocol, SUIT protocol, Recommended

MitoPedia methods: Fluorometry