SUIT-026 AmR mt D064

high-resolution terminology - matching measurements at high-resolution

SUIT-026 AmR mt D064

Description

Abbreviation: RET

Reference: A - SUIT-026 - Protocol for the evaluation of ROS production by RET in mtprep

SUIT number: D064_mt;1S;2Rot;3D;4Ama

O2k-Application: AmR

SUIT-026 AmR mt D064 has been designed to study the RET-initiated ROS production 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 leading to decreased ROS production. If rotenone would have been added before S titration, it had inhibited the RET. 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. Addition of Ama leads to increased ROS production via inhibition of Complex III at the Qi site.

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. In this protocol, we suggest not to go under ~100 µM O₂. SUIT-018 AmR mt D041 has been designed to study O₂ dependence of H₂O₂ flux in mitochondrial preparations.

Communicated by Komlodi T, Gnaiger E (last update 2024-09-19)

Representative traces

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)
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.
4Ama	ROX			1S;2Rot;3D;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.
This protocol must be run in parallel with SUIT-026 O2 mt D063 to have a respiratory control, since many fluorescence dyes can inhibit components of the ET system, thus affecting the respiration.

+ Short duration of the experiment.

+ Rotenone provides an excellent control step for RET.

- 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 additing an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state.

- The addition of cytochrome c cannot be done in this SUIT protocol (SUIT-026 O2 mt D063 must be run in parallel to assess the mt outer membrane integrity as a quality control of the sample).

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

Compare SUIT protocols

SUIT-026 O2 mt D063: Respiratory control protocol.
SUIT-006 O2 mt D022: CCP with mtprep for S-pathway.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
H₂O₂	Hydrogen Peroxide (H₂O₂)

Step	Chemical(s) and link(s)	Comments
0DTPA	DTPA	This step can be skipped
0SOD	Superoxide Dismutase (SOD)
0HRP	Horseradish peroxidase (HRP)
0AmR	Amplex UltraRed (AmR)

Step	Chemical(s) and link(s)	Comments
1S	Succinate (S)
2Rot	Rotenone (Rot)
3D	ADP (D)
4Ama	Antimycin A (Ama)

Suggested stock concentrations are shown in the specific DL-Protocol.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Fluorometry