SUIT-018 AmR mt D031

high-resolution terminology - matching measurements at high-resolution

SUIT-018 AmR mt D031

Description

[[Description::SUIT-018]]

Reference: [[Info::A: SUIT-018 »Versions to study the oxygen dependence of H₂O₂ (AmR) in mitochondrial preparations (isolated mitochondria and tissue homogenate).]]

SUIT number: Has SUIT number::D031_1GMS;2D;3Ama

O2k-Application: Has application::AmR

SUIT-category: NS(GM)

SUIT protocol pattern: linear 1GMS;2D;3Ama

This is a short protocol to study simultaneously the oxygen dependence of O₂ flux and H₂O₂ production on isolated mitochondria. You can run the protocol 1.)under tissue normoxia (50-60 µM = [O₂]), 2.) under tissue hyperoxia [> 120 µM = [O₂]]or 3.) you can change the oxygen concentration during the experiment. The oxygen concentration in the chamber can be changed by the injection of nitrogen with a syringe.

Communicated by Iglesias-Gonzalez J,  Komlodi T and Gnaiger E (last update 2019-02-12)

References

{{#ask: Additional label::SUIT-018 AmR mt D031 Instrument and method::O2k-Protocol | ?Was published in year=Year | ?Has title=Reference | ?Mammal and model=Organism | ?Tissue and cell=Tissue;cell | format=broadtable | limit=5000 | offset=0 | sort=Was published in year | order=descending }}

{{#ask: Additional label::SUIT-020 Fluo mt D033 | ?Was submitted in year=Year | ?Has title=Reference | ?Mammal and model=Organism | ?Tissue and cell=Tissue;cell | format=broadtable | limit=5000 | offset=0 | sort=Was published in year | order=descending }}

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)
1GMS	GMS_L(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	GMS_P	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.
3Ama	ROX			1GMS;2D;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).

Strengths and limitations

+ Reasonable duration of the experiment.

+ Simple protocol to evaluate the oxygen dependence of H₂O₂ production in LEAK and OXPHOS.

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

- H₂O₂ production can be studied in two different states, LEAK and OXPHOS.

- Reoxygenation and nitrogen injection could lead to bubble formation in the O2k-Chamber.

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