SUIT-009 O2 mt D015

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SUIT-009 O2 mt D015

Description

Abbreviation: NS(P)

Reference: A: SUIT-009 short protocol for respiration in mitochondrial preparations (isolated mitochondria and tissue homogenate).

SUIT number: D015_1S;2D;3P;4Rot;5Ama

SUIT-category: NS(P)

SUIT protocol pattern: orthogonal 1S;2D;3P;4Rot

SUIT 9 is a short protocol for simultaneous determination of O₂ flux and the rate of H₂O₂ production (H₂O₂ flux) on isolated mitochondria,tissue homogenate (except of liver) and permeabilized cells. Succinate (S) supports the reverse electron transfer (RET) initiated H₂O₂ production in the LEAK state. Addition of ADP decreases H₂O₂ flux due to depolarization of the mitochondrial membrane. Pyruvate (P) supports NADH-pathway (N), and usually in the presence of ADP it does not increase further the H₂O₂ flux. Antimycin A (Ama) inhibits Complex III (CIII) and results in the elevation of the H₂O₂ flux. The sensitivity of the Amplex UltraRed® assay (for determining H₂O₂ production) is changing over the experimental time and upon addition of chemicals. To correct H₂O₂ flux for the sensitivity changes several H₂O₂ calibration steps are done during the experiment. The measurement is carried out in MiR05-Kit at 37 °C.

For O2-Application, choose SUIT-009 O2 mt D015; for O2 and H₂O₂ measurements (AmR), choose: D021_1S;2D;3P;4Rot;5Ama.

Communicated by Iglesias-Gonzalez J,  Komlodi T and Gnaiger E (last update 2019-01-22)

DLP applications

References

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).
2D	S_P	S	CII	1S;2D Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3P	NS_P	NS	CI&II	1S;2D;3P Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4Rot	S_P	S	CII	1S;2D;3P;4Rot Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state. Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway.
5Ama	ROX			1S;2D;3P;4Rot;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).

Step	Respiratory state	Pathway control	ET-Complex	Comment
## AsTm	AsTm_E	CIV	CIV
## Azd	CHB

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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

For H₂O₂-Application, choose SUIT-009 AmR mt D021; for O₂ and H₂O₂ measurements (AmR), choose: SUIT-009 AmR mt D021.
Cytochrome c test can be performed in the S-OXPHOS state.

+Reasonable duration of the experiment.

+This protocol can be extended with the Complex IV module.

Compare SUIT protocols

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Respirometry