Talk:SUIT-029

high-resolution terminology - matching measurements at high-resolution

Talk:SUIT-029

Description

SUIT-029

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

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 

MitoPedia methods: Respirometry 

SUIT protocol pattern: 1PM;2D;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). With this protocol ATPase contamination and mitochondrial outer membrane integrity in different mitochondrial preparations with N substrates can be evaluated. Moreover, the LEAK state L(Omy) without the uncoupling promoted by the ATPase contamination and as a control with the L(n) previously obtained is also assessed. As a test for limitation of OXPHOS capacity by the phosphorylation system relative to ET-capacity the performance of uncoupler titrations might be carried out with N in order to calculate ET-coupling efficiency (1-L/E). The additive effect of convergent flux through NADH-linked respiration, NS, and S in E can be evaluated by using this protocol.

Communicated by Antunes D, Komlodi T, Gnaiger E (last update 2020-03-06)

Specific SUIT protocols

SUIT-029 O2 mt D066

400px

• 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).

Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

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.

+ Linear coupling control (L-P-E) with N substrates (PM). N 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).