SUIT-004 O2 pfi D010

high-resolution terminology - matching measurements at high-resolution

SUIT-004 O2 pfi D010

Description

Abbreviation: NS(PM)

Reference: A: SUIT-004 short protocol linked to SUIT-001 O2 pfi D002 - SUIT RP1 (human skeletal muscle)

SUIT number: D010_1PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8Azd

O2k-Application: O2

SUIT-category: NS(PM)

SUIT protocol pattern: orthogonal 1PM;2D;2c;3U;4S;5Rot

• Linked to SUIT-001 O2 pfi D002 - SUIT RP1, specifically for human skeletal muscle mitochondria.
• Harmonized with SUIT-005 O2 pfi D011.

Communicated by Iglesias-Gonzalez J and Gnaiger E (last update 2019-01-21)

References

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).
2D	PMP	N	CI	1PM;2D NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	PMcP	N	CI	1PM;2D;2c NADH-linked substrates (type N-pathway to Q). 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]). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3U	PME	N	CI	1PM;2D;3U 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. Noncoupled electron transfer state, ET state, with ET capacity E.
4S	PMSE	NS	CI&II	1PM;2D;3U;4S 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.
5Rot	SE	S	CII	1PM;2D;3U;4S;5Rot 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.
6Ama	ROX			1PM;2D;3U;4S;5Rot;6Ama 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 of >10 mM may be required for saturating SE capacity.

• CIV assessment is skipped if a prolonged protocol is not practical due to time limitation.

1. RP1-6Ama may be skipped since correction of the initial states by ROX measured in RP1-6 Ama may represent an over-correction, and inhibition takes a very long time in several cases (Pesta 2012 Methods Mol Biol).

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

+ Pathway control in ET state (N, NS and S pathways).

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

+ Short experimental time.

- The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-004 in favor of reducing the time consumed during the experiment.

Compare SUIT protocols

• SUIT-001 O2 pfi D002

Specific cases

• imt, ROX: NS(PM)01_imt,1PM,2D(c),3U,4S,5Rot,6Ama
• pfi, high O₂, no ROX: NS(PM)01_pfiO2,1PM,2D(c),3U,4S,5Rot

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry