SUIT-001 O2 mt D001

high-resolution terminology - matching measurements at high-resolution

SUIT-001 O2 mt D001

Description

Abbreviation: FNSGp(Oct,PGM)

Reference: A Reference protocol RP1; mt: isolated mitochondria and tissue homogenate - SUIT-001

SUIT number: D01_1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd

O2k-Application: O2

SUIT-category: FNSGp(Oct,PGM)

SUIT protocol pattern: orthogonal 1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp;9Ama

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). 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]).
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.
4G	PGME	N	CI	1PM;2D;3U;4G NADH-linked substrates (type N-pathway to Q). Noncoupled electron transfer state, ET state, with ET capacity E.
5S	PGMSE	NS	CI&II	1PM;2D;3U;4G;5S 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.
6Oct	OctPGMSE	FNS	FAO&CI&II	1PM;2D;3U;4G;5S;6Oct Respiratory stimulation by simultaneous action of the F-pathway, N-pathway, and S-pathway, with convergent electron flow in the FNS-pathway for reconstitution of TCA cycle function and additive or inhibitory effect of F. Noncoupled electron transfer state, ET state, with ET capacity E.
7Rot	SE	S	CII	1PM;2D;3U;4G;5S;6Oct;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.
8Gp	SGpE	SGp	CII&GpDH	1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp Respiratory stimulation by action of succinate and glycerophosphate, Gp, with convergent electron flow in the SGp-pathway (CII&GpDH-linked pathway to the Q-junction). Noncoupled electron transfer state, ET state, with ET capacity E.
9Ama	ROX			1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp;9Ama 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.

+ 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

• SUIT-002 O2 mt D005 (RP2): Harmonized SUIT protocol for isolated mitochondria and tissue homogenate.

• SUIT-004: Short version of RP1; without G, F and Gp.
• SUIT-006: Very short initial coupling control module.

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry 

Labels:

SUIT-001