SUIT-018 O2 mt D054

high-resolution terminology - matching measurements at high-resolution

SUIT-018 O2 mt D054

Description

Reference: B: SUIT-018

SUIT number: D054_mt;1GMS;2D;2c;3Ama

O2k-Application: O2

SUIT-018 O2 mt D054 is a short protocol to study the influence of a fluorescence dye on the O₂ flux. During this protocol the [O₂] can be modified by injecting nitrogen in the chamber.

Communicated by Komlodi T and Gnaiger E  (last update 2019-09-24)

Representative traces

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1GMS	GMSL(n)	NS	CI&CII	1GMS Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	GMSP	NS	CI&CII	1GMS;2D 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.
2c	GMScP	NS	CI&CII	1GMS;2D;2c 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. 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]).
3Ama	ROX			1GMS;2D;2c;3Ama 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	AsTmE	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

• This protocol can be used as a control for SUIT-018 AmR mt D031, SUIT-018 AmR mt D040 and SUIT-018 AmR mt D041. The oxygen concentration should be adjusted to the matching protocol with the Amplex UltraRed assay.

+ Reasonable duration of the experiment.

- CIV activity and cytochrome c test can be performed.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1GMS	Glutamate (G), Malate (M) and Succinate (S)
2D	ADP (D)
2c	Cytochrome c (c)
3Ama	Antimycin A (Ama)

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

MitoPedia concepts: SUIT protocol, SUIT B, Find 

MitoPedia methods: Respirometry