SUIT-009 AmR mt D021

high-resolution terminology - matching measurements at high-resolution

SUIT-009 AmR mt D021

Description

Abbreviation: H₂O₂ mtprep

Reference: B: short protocol for H₂O₂ (AmR) in mitochondrial preparations; mt: isolated mitochondria, tissue homogenate and permeabilized cells - SUIT-009short protocol for H₂O₂ (AmR) for isolated mitochondria, tissue homogenate and permeabilized cells

SUIT number: D021_1S;2D;3P;4Rot;5Ama

O2k-Application: AmR

SUIT-009 is a short protocol for simultaneous determination of O₂ flux and the rate of H₂O₂ production (H₂O₂ flux) in mitochondrial preparations such as isolated mitochondria, tissue homogenate (except of liver) and permeabilized cells (already permeabilized when they are added to the chamber). Succinate (S) supports the reverse electron transfer (RET)-related H₂O₂ production in the LEAK state. 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) at the Q_o level and could increase the H₂O₂ flux. The sensitivity of the Amplex UltraRed® assay (for determining H₂O₂ production) changes 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. Since the H₂O₂ flux measured in MiR05-Kit showed a linear correlation with the oxygen concentration, it is recommended to change the oxygen level during the experiment.

Communicated by Komlodi T, Gnaiger E (last update 2020-06-12)

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	SL(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	SP	S	CII	1S;2D Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3P	NSP	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	SP	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).

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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 is a protocol designed to analyze:

1. the S- and NS-pathway-linked respiration and H₂O₂ flux.

2. any Complex I defect, which would lead to a decrease in N-linked respiration and in reverse electron transfer-supported H₂O₂ flux (RET).

3. any Complex II defect, which would result in an altered S-linked respiration and H₂O₂ flux.

+ This protocol is designed to evaluate RET in the S-linked pathway in the LEAK state, which is decreased by ADP due to reduction of the mt-membrane potential.

+ Short protocol.

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

- We do not generally recommend the addition of already permeabilized cells into the chamber. Instead, we recommend performing the permeabilization of the plasma membrane in the chambers with titration of digitonin.

Compare SUIT protocols

• SUIT-006 AmR mt D048 to investigate the dependence of H₂O₂ flux on the mt-membrane potential on the N-control state in isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber).
• SUIT-018 are short protocols to study the oxygen dependence of O₂ flux and H₂O₂ production on isolated mitochondria or tissue homogenate.

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)
2D	ADP (D)
3P	Pyruvate (P)
4Rot	Rotenone (Rot)
5Ama	Antimycin A (Ama)

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

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

	Year	Reference	Organism	Tissue;cell
Chang 2018 Life Sciences Meeting 2018 Innsbruck AT	2018	pH dependence of mitochondrial respiration and H2O2 production in oral cancer cells – a pilot study.	Human	HEK Kidney

MitoPedia concepts: SUIT protocol, SUIT B, Find 

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