SUIT-016

high-resolution terminology - matching measurements at high-resolution

SUIT-016

Description

Abbreviation: F+G+S+Rot_OXPHOS+Omy

Reference: A: F-pathway in LEAK state and OXPHOS state

SUIT protocol pattern: 1OctM;2D;3G;4S;5Rot;6Omy;7U-

SUIT-016 gives information on F-pathway in LEAK state and OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. In addition, the pathway control of FN and FNS in OXPHOS state and of S in LEAK state and ET state is evaluated.

Communicated by Doerrier C, Huete-Ortega M, Cardoso LHD and Gnaiger E (last update 2019-06-05)

Specific SUIT protocols

• SUIT-016 O2 pfi D044 for permeabilized fibers

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1OctM	OctML(n)	F(N)	CETF	1OctM Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway. Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	OctMP	F(N)	CETF	1OctM;2D Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3G	OctGMP	FN	CETF&I	1OctM;2D;3G Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. & NADH-linked substrates (type N-pathway to Q). Respiratory stimulation by simultaneous action of the F-pathway and N-pathway with convergent electron flow in the FN-pathway for evaluation of an additive or inhibitory effect of F. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4S	OctGMSP	FNS	CETF&CI&II	1OctM;2D;3G;4S Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. & NADH-linked substrates (type N-pathway to Q). & Succinate, S ( type S-pathway to Q). 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. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5Rot	SP	S	CII	1OctM;2D;3G;4S;5Rot Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway. Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
6Omy	SL(Omy)	S	CII	1OctM;2D;3G;4S;5Rot;6Omy Succinate, S ( type S-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
7U	SE	S	CII	1OctM;2D;3G;4S;5Rot;6Omy;7U Succinate, S ( type S-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.
8Ama	ROX			1OctM;2D;3G;4S;5Rot;6Omy;7U;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).

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

+ This protocol provides information on FAO capacity in the absence of other potentially interfering pathways both in LEAK and OXPHOS coupling-control states.

+ FNS OXPHOS capacity comprises the most important pathways in many cell types and thus provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.

+ FNS ET capacity is a good estimate of overall ET capacity in many cell types.

+ This is a good protocol to analyse coupling control at S-pathway, which is an advantage compared to SUIT-015 and SUIT-017.

+ Glutamate is easier to prepare compared to pyruvate.

- In some tissues GM is not sufficient to fully support N-pathway capacity

- SRot(E) may be underestimated if S is not saturating.

- SRot(E) may be underestimated in the presence of Omy in some tissues and cell types

- CIV activity is not measured, to save experimental time.

• It is possible to add cytochrome c in different steps:

• (2c), when cytochrome c is added after 2D, it allows to compare all steps in OXPHOS even if there is damage of the mitochondrial outer membrane. This approach can be chosen when the cytochrome c effect is not an exclusion criteria.
• (3c) when cytochrome c is added after 3G, due to the higher flux, it is easier to evaluate whether there is an increase in respiration indicating damages in the mitochondrial outer membrane. However, if there is a strong cytochrome c effect, it is not possible to compare OctM_P with the next steps in OXPHOS.
• (7c) when cytochrome c is added after 7U: if there is no cytochrome c effect, it indicates that even after the whole lenght of the protocol, there were no damages to the mitochondrial outer membrane. This approach can be used as an exclusion criteria for samples that normally do not present mitochondrial outer membrane damage after the sample preparation, e.g. human permeabilized muscle fibers.

Compare SUIT protocols

• SUIT-017
• SUIT-015

References

	Year	Reference	Organism	Tissue;cell
Gnaiger 2015 Scand J Med Sci Sports	2015	Gnaiger E, Boushel R, Søndergaard H, Munch-Andersen T, Damsgaard R, Hagen C, Díez-Sánchez C, Ara I, Wright-Paradis C, Schrauwen P, Hesselink M, Calbet JAL, Christiansen M, Helge JW, Saltin B (2015) Mitochondrial coupling and capacity of oxidative phosphorylation in skeletal muscle of Inuit and caucasians in the arctic winter. https://doi.org/10.1111/sms.12612	Human	Skeletal muscle

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 

MitoPedia methods: Respirometry