SUIT-029 O2 mt D066

high-resolution terminology - matching measurements at high-resolution

SUIT-029 O2 mt D066

Description

Abbreviation: QC_imt (PM)

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

SUIT number: D066_1PM;2T;2D;2c;3Omy;4U;5G;6S;6U;7Rot;8Ama

O2k-Application: O2

MitoPedia: SUIT

SUIT protocol pattern: 1mt;1PM;2T;2D;2c;3Omy;4U;5G;6S;6U;7Rot;-

The SUIT-029 O2 mt D066 is designed to evaluate the quality control of mitochondrial preparation. This protocol gives information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). SUIT-029 O2 mt D066 includes a control for contamination with other organelles containing ATPases (e.g., plasma membrane and endoplasmic reticulum.) In experiments with crude isolated mitochondria, tissue homogenates and permeabilized cells, it is expected to have active ATPases in the preparation. When ATPases are active, residual endogenous adenylates are phosphorylated to ATP in the LEAK state, which is recycled to ADP in isolated mitochondria contaminated by ATPases, which leads to an overestimation of LEAK respiration, when comparing L(n) and L(Omy). Therefore, it might be necessary to assess LEAK respiration in the presence of ATPase inhibitors, e.g. oligomycin. Additive effect of convergent electron flow through NADH- and NS-pathways is evaluated in the ET state.

The present SUIT-029 O2 mt D066 DLP file shows an application with imt, thom or pce in the pathway category NS. For using this protocol with other mitochondrial preparations or substrate/inhibitor combinations, a personalized DLPU can be created.

Communicated by  Antunes D, Komlodi T, Gnaiger E (last update 2020-02-28)

Representative traces

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Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1PM	PM_L(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	PM_L or PM_P	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	PM_P	N	CI	1PM;2T;2D NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	PMc_P	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	PM_LOmy	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	PM_E	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	PGM_E	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	PGMS_E	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	PGMS_E	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	S_E	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.

+ NADH-linked substrates (PM) make use of all the proton pumps in the ETS without the additivity effect of the S-pathway, which would decrease the coupling degree. Additionally, PM is the superior alternative to GM: the fraction of the N-pathway is lower and S-pathway contribution is higher with GM compared to PM. PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since impairment of N-pathway capacity can be compensated partially by activation of the S-pathway.

+OXPHOS capacity evaluation is included (2D).

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

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

+ LEAK respiration overestimation is prevented in the presence of Oligomycin, without the uncoupling promoted by the ATPase contamination and as a control with the L(n) previously obtained.

+ Internal ET-pathway control step is included as a test for limitation of OXPHOS-capacity by the phosphorylation system relative to ET-capacity. The evaluation of ET-state with NADH-linked substrates allows to calculate ET-coupling efficiency (1-L/E).

+ The additive effect of convergent flux through NADH-linked respiration in ET-state is evaluated (5G).

+ ET-state with NS-linked substrates is evaluated (5S) followed by additional U titration to ensure ET-capacity.

+ Assessment of ET-capacity not only in N- and NS-pathway but also in S-pathway (7Rot).

+ This protocol can be optionally harmonized with many other SUIT protocols (e.g., SUIT-001, SUIT-004, SUIT-008). Addition of G in NADH-supported OXPHOS enables evaluation of the glutamate anaplerotic pathway control state.

+ 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).

- F-pathway is not analysed.

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

Compare SUIT protocols

SUIT-001 O2 mt D001 (RP1): Harmonized SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized).
SUIT-002 O2 mt D005 (RP2): Harmonized SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized).

References

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Respirometry