SUIT-015

SUIT-015

Description

Abbreviation: F+G+P+S_OXPHOS+Rot_ET

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

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

SUIT-015 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 FNS and S in ET state can be evaluated. SUIT-015 can be extended with the CIV assay module.

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

Specific SUIT protocols

SUIT-015 O2 pti D043 for permeabilized tissue

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1OctM	OctM_L	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. Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	OctM_P	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. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3G	OctGM_P	FN	CETF&CI	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.
3c	OctGMc_P	FN	CETF&CI	1OctM;2D;3G;3c 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. 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.
4P	OctPGM_P	FN	CETF&CI	1OctM;2D;3G;3c;4P 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.
5S	OctPGMS_P	FNS	CETF&CI&II	1OctM;2D;3G;3c;4P;5S 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.
6U	OctPGMS_E	FNS	CETF&CI&II	1OctM;2D;3G;3c;4P;5S;6U 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. 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.
7Rot	S_E	S	CII	1OctM;2D;3G;3c;4P;5S;6U;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.
8Ama	ROX			1OctM;2D;3G;3c;4P;5S;6U;7Rot;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 entry into Q-junction	Comment
## AsTm	AsTm_E	CIV	CIV
## Azd	ROX

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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

+ The protocol provides information on FAO capacity in the absence of other, potentially interfering pathways, both in the LEAK state and in OXPHOS.

+ 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.

+ The presence of PMG and S establishes a fully operative TCA cycle activity.

+ Reasonable duration of the experiment.

- Application of the cytochrome c test after 3G in the protocol does not ensure comparability of all states in case of any effect of c. However, it allows to analyse whether the outer mt-membrane integrity is compromised.

- F OXPHOS capacity may be underestimated. In human heart muscle addition of Oct to palmitoylcarnitine (Pal) + malate increased OXPHOS by 26% (Lemieux et al 2011).

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

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

Compare SUIT protocols

References

	Year	Reference	Organism	Tissue;cell
Schoepf 2016 FEBS J	2016	Schöpf B, Schäfer G, Weber A, Talasz H, Eder IE, Klocker H, Gnaiger E (2016) Oxidative phosphorylation and mitochondrial function differ between human prostate tissue and cultured cells. https://doi.org/10.1111/febs.13733	Human	Endothelial;epithelial;mesothelial cell Genital Other cell lines Fibroblast

MitoPedia concepts: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Respirometry