SUIT-004 O2 pfi D010

high-resolution terminology - matching measurements at high-resolution

SUIT-004 O2 pfi D010

Description

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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;3U;4S;5Rot

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.

References

	Year	Reference	Organism	Tissue;cell
MiPNet21.06 SUIT RP	2018-06-25	SUIT reference protocol for OXPHOS analysis by high-resolution respirometry.
Doerrier 2018 Methods Mol Biol	2018	Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. https://doi.org/10.1007/978-1-4939-7831-1_3	Human Mouse Rat Saccharomyces cerevisiae	Heart Skeletal muscle Endothelial;epithelial;mesothelial cell Blood cells HEK Platelet

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

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

Link to RP1

The 1PM;2D;3U;4S;5Rot- protocol is linked to 1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp- for healthy human skeletal muscle mitochondria on the basis of the following rationale.

RP1

1. The first coupling control steps (1-4) are identical.
2. RP1-5G is skipped, since PM and PGM yield nearly identical OXPHOS capacity. However, PM<PGM is a potential diagnostic for a defect in the PM-linked pathway upstream of CI.
3. RP1-7Oct is skipped, since octanoylcarnitine exerts practically no stimulatory effect on OXPHOS capacity in state PMS or PGMS, but in conjunction with prolonged exposure may even yield a slight decline of respiration.
4. RP1-9Gp is skipped, since glycerolphosphate exerts only a minor stimulatory effect on OXPHOS capacity in state SRot.
5. Omission of the three steps described above shortens the SUIT protocol, thus reducing the risk of diminishing respiratory capacity over time, reducing the number of necessary reoxygenations, and providing more time for detailed evaluation of steady-states. The loss of diagnostic information may be minor relative to the benefits of simplification of the protocol, particularly in studies of exercise in healthy humans.
6. RP1-10Ama may be skipped, since correction of the initial states by ROX measured in RP1-10 Ama may represent an over-correction, and inhibition takes a very long time in several cases (Pesta 2012 Methods Mol Biol).
7. RP1-11Tm and RP1-12Azd are skipped, if a prolonged protocol is not practical due to limination of time.

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry