Difference between revisions of "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 Doerrier C, Komlodi T and Gnaiger E (last update 2019-01-30)

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	PalM_L(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	PalM_P	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	PalM_P	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	PalOctM_P	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	PalOctPM_P	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	PalOctPGM_P	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	PalOctPGM_E	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	PalOctPGMS_E	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	S_E	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	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

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

@@ Line 29: / Line 29: @@
 == Strengths and limitations ==
-:::*SUIT-019 provides an estimation of F OXPHOS capacity, physiologically relevant estimate of maximal Complex I capacity as well as of FNS ET capacity.
-:::+ The protocol provides information on FAO capacity in the absence of other, potentially interfering pathways, both in the LEAK state and in OXPHOS.
+:::* SUIT-019 allows the study of OXPHOS capacity in the fatty acid oxidation (F) and fatty acid oxidation&NADH pathway (FN) control states.
-:::+ F OXPHOS capacity is estimated in presence of both Oct and Pal therefore it is more accurate than an estimate in the presence of only one of the substrates. In human heart muscle addition of Oct to palmitoylcarnitine (Pal) + malate increased OXPHOS by 26% (Lemuieux et al 2011).
+:::+ 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 2011 Int J Biochem Cell Biol |Lemieux H et al 2011]]).
-:::+ FN OXPHOS capacity comprises the most important Complex I-linked pathways in many cell types and thus provides a physiologically relevant estimate of maximum CI capacity.
-:::+ FNS ET capacity is a good estimate of overall ET capacity in many cell types
+:::+ SUIT-019 provides information of the linear coupling (''L''-''P'') in FN-pathway.
-:::+ The presence of PMG and S establishes fully operative TCA cycle activity.
+:::+ 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.
-:::- Oct and Pal can have an uncoupling effect during F OXPHOS and FN OXPHOS states if their concentration is not optimized.
+:::- Octanoylcarnitine and palmitoylcarnitine can have an uncoupling effect on OXPHOS state if their concentration is not optimized.
-:::- SRot(E) may be underestimated if S is not saturating.
+:::- In the presence of anaplerotic pathways F-pathway is overestimated.
 :::- CIV activity is not measured, to save experimental time.
 == Compare SUIT protocols ==
-::::*[[SUIT-002]]
+::::* [[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