SUIT-018 AmR mt D040

high-resolution terminology - matching measurements at high-resolution

SUIT-018 AmR mt D040

Description

Abbreviation: NS(GM)

Reference: A: simultaneous determination of O₂ and H₂O₂ flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) at high oxygen concentration (tissue hyperoxia)-SUIT-018

SUIT number: D040_1GMS;2D;3Ama

O2k-Application: AmR

MitoPedia: SUIT

SUIT-category: NS(GM)

SUIT protocol pattern: lineal 1GMS;2D-

SUIT-018 AmR mt D040 is a short protocol to study simultaneously the oxygen dependence of O₂ flux and H₂O₂ production in mitochondrial preparations. Is it possible to run the protocol under different oxygen concentration conditions: 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 altered injecting nitrogen with a syringe. We recommend to run SUIT-018 AmR mt D031 simultaneously at low oxygen concentration as a control.

Communicated by Iglesias-Gonzalez J, 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)
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

SUIT-018 AmR mt D031 should be runned in parallel (tissue normoxia condition).

+ Reasonable duration of the experiment.

+ Simple protocol to evaluate the oxygen dependence of H₂O₂ production.

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

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

- CIV activity and cytochrome c test cannot be performed together with the fluorescence.

Compare SUIT protocols

SUIT-018 AmR mt D041 for simultaneous determination of O₂ and H₂O₂ flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) changing the oxygen concentration in the same protocol.
SUIT-006 AmR mt D048 to investigate the dependence of H₂O₂ flux on the mt-membrane potential on the N-control state in mitochondrial preparations.
SUIT-009 to investigate H₂O₂ production driven by the reverse electron transfer (RET) in isolated mitochondria, tissue homogenate and permeabilized cells.

References

	Year	Reference	Organism	Tissue;cell
MiPNet24.10 H2O2 flux analysis	2021-10-22	Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4
Komlodi 2021 BEC AmR-O2	2021	Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi.org/10.26124/bec:2021-0004	Saccharomyces cerevisiae	Other cell lines
MiPNet20.14 AmplexRed H2O2-production	2019-06-24	O2k-FluoRespirometry: HRR and simultaneous determination of H₂O₂ production with Amplex UltraRed.	Mouse	Heart
Komlodi 2018 Methods Mol Biol	2018	Komlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55.	Human Mouse	Skeletal muscle HEK
Makrecka-Kuka 2015 Biomolecules	2015	Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. https://doi.org/10.3390/biom5031319	Human Mouse	Heart Nervous system HEK
Krumschnabel 2015 Methods Mol Biol	2015	Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61.	Mouse	Nervous system

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