Talk:SUIT-029

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Talk:SUIT-029

Description

Reference: A: quality control evaluation of mitochondrial preparations - SUIT-029

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 

MitoPedia methods: Respirometry 

SUIT protocol pattern: 1PM;2T;3Omy;4U-

The SUIT-029 protocol is specially designed to evaluate the quality of mitochondrial preparations in a wide variety of species, tissues and cell types. SUIT-029 gives information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Using this protocol ATPase contamination and mitochondrial outer membrane integrity in different mitochondrial preparations with N substrates can be evaluated. In isolated mitochondria contaminated by ATPases, endogenous adenylates are phosphorylated to ATP in the LEAK state, which is recycled to ADP, which leads to an overestimation of LEAK respiration, when comparing L(Omy) and L(n). Therefore, it might be necessary to assess LEAK respiration in the presence of ATPases inhibitors, e.g. oligomycin. Optionally, additive effect of convergent electron flow through NADH- and NS-pathways can be evaluated in the ET state.

The present SUIT-029 DLP file shows an application in with imt, thom and pce in the pathway category in N or NS. For using this protocol with other sample preparations or substrate/inhibitor combinations, a personalized DLPU can be created.

Communicated by Antunes D, Komlodi T, Gnaiger E (last update 2020-05-12)

Specific SUIT protocols

SUIT-029 O2 mt D066

• SUIT-029 O2 mt D066 for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber)

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1PM	PML(n)	N	CI	1PM NADH-linked substrates (type N-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2T	PML or PMP	N	CI	1PM;2T NADH-linked substrates (type N-pathway to Q). In the absence of ATPase activity, the LEAK-state is maintained. However, if ATPases are active and thus generate ADP, respiration coupled to phosphorylation is stimulated. Stimulation is limited up to OXPHOS-capacity; therefore, higher ATPase activities cannot be determined.
2D	PMP	N	CI	1PM;2T;2D NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	PMcP	N	CI	1PM;2T;2D;2c NADH-linked substrates (type N-pathway to Q). 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]).
3Omy	PMLOmy	N	CI	1PM;2D;2c;3Omy NADH-linked substrates (type N-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
4U	PME	N	CI	1PM;2T;2D;2c;3Omy;4U NADH-linked substrates (type N-pathway to Q). Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency.
5G	PGME	N	CI	1PM;2T;2D;2c;3Omy;4U;5G NADH-linked substrates (type N-pathway to Q). Noncoupled electron transfer state, ET state, with ET capacity E.
6S	PGMSE	NS	CI+CII	1PM;2T;2D;2c;3Omy;4U;5G;6S Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. Noncoupled electron transfer state, ET state, with ET capacity E.
6U	PGMSE	NS	CI+CII	1PM;2T;2D;2c;3Omy;4U;5G;6S;6U Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency.
7Rot	SE	S	CI	1PM;2T;2D;2c;3Omy;4U;5G;6S;6U;7Rot Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway. Noncoupled electron transfer state, ET state, with ET capacity E.
8Ama	ROX			1PM;2T;2D;2c;3Omy;4U;5G;6S;6U;7Rot;8Ama 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 protocol allows to evaluate the quality of mitochondrial preparations taking into account the presence of ATPases.

+ PM is the superior alternative to GM: with GM the fraction of the N-pathway is lower and S-pathway contribution is higher compared to PM. PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since in the case of GM impairment N-pathway capacity can be compensated partially by activation of the S-pathway.

+ Evaluation of the ATPase contamination is included (2T).

+ Integrity of mitochondrial outer membrane is assessed ( 2c).

+ Overestimation of LEAK respiration is prevented in the presence of Oligomycin as a control with the L(n) previously obtained.

+ Evaluation of ET-state with NADH-linked substrates allows to calculate ET-coupling efficiency (1-L/E).

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

- Long duration of the experiment due to adjustment of Oligomycin concentration (in the case of a new isolation protocol). For a shorter protocol the titration of glutamate, succinate and rotenone could be omitted.

- The use of Oligomycin could affect the evaluation of the ET capacity (could inhibit ET-state).

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

Compare SUIT protocols

• SUIT-001 (RP1): Harmonized SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells.
• SUIT-006: Very short coupling protocol with PM as NADH-linked substrates.
• SUIT-008: A protocol to evaluate NS-linked pathway in the OXPHOS and ET states.
• SUIT-004: Short version of RP1; without G, F and Gp.
• SUIT-012: Coupling control with NADH-linked substrates (PM and PGM) without Omy addition.