SUIT-019

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SUIT-019

Description

Abbreviation: FNS(PalOct,PGM)

Reference: A  »Versions

SUIT-category: FNS(PalOct,PGM)

SUIT protocol pattern: diametral 1PalM;2D;3Oct;4P;5G;6U;7S;8Rot-

Communicated by Cardoso LH Doerrier C, Komlodi T and Gnaiger E (last update 2019-02-12)

Specific SUIT protocols

• SUIT-019 O2 pfi D045 for permeabilized fibers

References

	Year	Reference	Organism	Tissue;cell
Lemieux 2011 Int J Biochem Cell Biol	2011	Lemieux H, Semsroth S, Antretter H, Höfer D, Gnaiger E (2011) Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int J Biochem Cell Biol 43:1729–38. https://doi.org/10.1016/j.biocel.2011.08.008	Human	Heart

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1PalM	PalML(n)	F(N)	CETF	1PalM 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. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) the F-pathway capacity is overestimated, if there is an added contribution of NADH-linked respiration, F(N) (see SUIT-002). The FA concentration has to be optimized to saturate the FAO-pathway, without inhibiting or uncoupling respiration. Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	PalMP	F(N)	CETF	1PalM;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. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) the F-pathway capacity is overestimated, if there is an added contribution of NADH-linked respiration, F(N) (see SUIT-002). The FA concentration has to be optimized to saturate the FAO-pathway, without inhibiting or uncoupling respiration. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	PalMP	F(N)	CETF	1PalM;2D;2c 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]). 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. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) the F-pathway capacity is overestimated, if there is an added contribution of NADH-linked respiration, F(N) (see SUIT-002). The FA concentration has to be optimized to saturate the FAO-pathway, without inhibiting or uncoupling respiration. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3Oct	PalOctMP	F(N)	CETF	1PalM;2D;2c;3Oct 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. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) the F-pathway capacity is overestimated, if there is an added contribution of NADH-linked respiration, F(N) (see SUIT-002). The FA concentration has to be optimized to saturate the FAO-pathway, without inhibiting or uncoupling respiration. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4P	PalOctPMP	FN	CETF&CI	1PalM;2D;2c;3Oct;4P 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.
5G	PalOctPGMP	FN	CETF&CI	1PalM;2D;2c;3Oct;4P;5G 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.
6U	PalOctPGME	FN	CETF&CI&II	1PalM;2D;2c;3Oct;4P;5G;6U 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. 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.
7S	PalOctPGMSE	FNS	CI&CII	1PalM;2D;2c;3Oct;4P;5G;6U;7S 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.
8Rot	SE	S	CII	1PalM;2D;2c;3Oct;4P;5G;6U;7S;8Rot 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.
9Ama	ROX			1PalM;2D;2c;3Oct;4P;5G;6U;7S;8Rot;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-019 allows the study of OXPHOS capacity in the fatty acid oxidation (F) and fatty acid oxidation&NADH pathway (FN) control states.

+ The protocol provides information on F-pathway (in presence of palmitoylcarnitine, and palmitoylcarnitine&octanoylcarnitine) in LEAK and OXPHOS states. In human heart muscle, the addition of octanoylcarnitine to palmitoylcarnitine&malate increased OXPHOS by 26% (Lemieux H et al 2011).

+ SUIT-019 provides information of the linear coupling (L-P) in FN-pathway.

+ Maximum ET capacity in FNS-pathway is obtained in SUIT-019.

+ P/E for FN-pathway (5G/6U) can be calculated.

+ Reasonable duration of the experiment.

- Octanoylcarnitine and palmitoylcarnitine can have an uncoupling effect on OXPHOS state if their concentration is not optimized.

- In the presence of anaplerotic pathways F-pathway is overestimated.

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

Compare SUIT protocols

• SUIT-002 (RP2): SUIT protocol specially designed to give information on F-pathway in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways.

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 

MitoPedia methods: Respirometry