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.

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

• SUIT-001 in combination with SUIT-002 provides a common reference for comparison of respiratory control in a large variety of species, tissues and cell types. Both SUIT protocols provide a mitochondrial mapping which allows:

1. to obtain reference values.

2. to evaluate mitochondrial physiological diversity, generating a mt-database on comparative mitochondrial physiology.

3. to screen specific defects.

• SUIT-001 and SUIT-002 are used in the MitoFit Proficiency test for inter-individual and inter-laboratory reproducibility quality control.
• A succinate concentration of >10 mM may be required for saturating S_E capacity.

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

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

+ Harmonization with SUIT-002 allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statically used as repeat measurements.

+ In mtprep, SUIT-001 and SUIT-002 harmonize in: SGp_E (8Gp in SUIT-001 and 10Rot in SUIT-002) and Complex IV.

+ Harmonization with many SUIT protocols (up to step 5S).

+ Oct is added in ET state to avoid uncoupler effect with fatty acids.

+ If NS- and FNS-pathways are similar (NS_E ~ FNS_E), the additivity index of N- and S-pathways in ET state can be evaluated.

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

- The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-001 in favour of measuring S_E.

Compare SUIT protocols

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

• The SUIT-004 protocol is linked to SUIT-001 O2 pfi D002 for healthy human skeletal muscle mitochondria on the basis of the following rationale.

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