SUIT-009

high-resolution terminology - matching measurements at high-resolution

SUIT-009

Description

Abbreviation: NS(P)

Reference: A  »Versions

SUIT-category: NS(P)

SUIT protocol pattern: orthogonal 1S;2D;3P;4Rot;5Ama

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 2019-01-23)

DLP applications

• SUIT-009 AmR mt D021
• SUIT-009 AmR pce D019
• SUIT-009 O2 mt D015
• SUIT-009 O2 pce D016

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

Steps and respiratory states

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).

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

• A succinate concentration > 10 mM might be required to reach saturating S-OXPHOS capacity.
• In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway.

+ Contribution of N pathway to the NS can be checked after the addition of Rot.

+ Reasonable duration of the experiment.

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

- Rox may be lower in substrate states earlier in the SUIT protocol. Therefore, this Rox measurement is frequently taken as a methodological control rather than as the final basis of Rox correction of mitochondrial respiration (mt).

- Careful washing is required after the experiment to avoid carry-over of inhibitors.

Compare SUIT protocols

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 

MitoPedia methods: Respirometry, Fluorometry