SUIT-017

high-resolution terminology - matching measurements at high-resolution

SUIT-017

Description

Abbreviation: F+G+S_OXPHOS+Rot_ET

Reference: A:

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

SUIT-017 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 is evaluated. This protocol should not be used to analyze FAO in samples that express mitochondrial malic enzyme and, thus, present a significant OXPHOS capacity in the malate-anaplerotic pathway control state.

Communicated by Doerrier C, Huete-Ortega M, Gnaiger E (last update 2024-09-19)

Specific SUIT protocols

SUIT-017 O2 mt D046

SUIT-017 O2 mt D046 evaluation of fatty acids and Q-junction additivity in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)

SUIT-017 O2 pfi D049

SUIT-017 O2 pfi D049 evaluation of fatty acids and Q-junction additivity in permeabilized muscle fibers

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1OctM	OctM_L(n)	F(N)	FAO	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	OctM_P	F(N)	FAO	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.
2c	OctM_P	F(N)	FAO	1OctM;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. 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	OctGM_P	FN	F&CI	1OctM;2D;2c;3G NADH-linked substrates (type N-pathway to Q). 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.
4S	OctGMS_P	FNS	F&CI&II	1OctM;2D;2c;3G;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. 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.
5U	OctGMS_E	FNS	F&CI&II	1OctM;2D;2c;3G;4S;5U Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. 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. Noncoupled electron transfer state, ET state, with ET capacity E.
6Rot	S_E	S	CII	1OctM;2D;2c;3G;4S;5U;6Rot 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.
7Ama	ROX			1OctM;2D;2c;3G;4S;5U;6Rot;7Ama Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.

Step	Respiratory state	Pathway control	ET-Complex	Comment
## AsTm	AsTm_E	CIV	CIV
## Azd	CHB

Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

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

Comparison of GM- with PM-capacity yields important information on N-pathway respiratory control upstream of CI (Lemieux et al. 2017; Votion et al. 2012).

+ Glutamate is easier to prepare compared to pyruvate.

+ Reasonable duration of the experiment.

+ This protocol can be extended with the Complex IV module, which can prolong the experimental time by ~30 min.

Compare SUIT protocols

SUIT-002: A more comprehensive protocol including additional substrate states.
SUIT-005: A comparable protocol evaluating P instead of G.

References

	Year	Reference	Organism	Tissue;cell
Pesta 2012 Methods Mol Biol	2012	Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol 810:25-58. https://doi.org/10.1007/978-1-61779-382-0_3	Human	Skeletal muscle Other cell lines HEK Fibroblast HUVEC

MitoPedia concepts: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Respirometry