SUIT-039 O2 mt D092: Difference between revisions

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SUIT-039 O2 mt D092

Description

Abbreviation: FAO and NS-pathways

Reference: A - SUIT-039 - F-pathway and NS-pathway

SUIT number: D092_1D;2M.1;3Pal;3c;4M2;5P;6G;7S;8U;9Rot;10Ama

O2k-Application: O2

The SUIT-039 O2 mt D092 protocol provides a common reference for comparison of respiratory control of mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species, tissues and cell types. SUIT-039 O2 mt D092 is specially designed to give information on F-pathway with palmitoylcarnitine as a FAO substrate in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS pathways) and in ET state (FNS and S) can be evaluated by using this SUIT protocol.

Communicated by Grings M, Cecatto C and Cardoso LHD (last update 2023-04-13)

Representative traces

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1D	ROX			1D ADP is added to stimulate the consumption of endogenous fuel-substrates.
2M.1				1D;2M.1 Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
3Pal	PalM_P	F	FAO	1D;2M.1;3Pal 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.
3c	PalMc_P	F	FAO	1D;2M.1;3Pal;3c 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. 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]).
4M2	PalM_P	F(N)	FAO	1D;2M.1;3Pal;3c;4M2 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. High concentration of malate, typically 2 mM, saturates the N-pathway. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5P	PalPM_P	FN	FAO&CI	1D;2M.1;3Pal;4M2;5P 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.
6G	PalPGM_P	FN	FAO&CI	1D;2M.1;3Pal;4M2;5P;6G 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.
7S	PalPGMS_P	FNS	FAO&CI&II	1D;2M.1;3Pal;4M2;5P;6G;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. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
8U	PalPGMS_E	FNS	FAO&CI&II	1D;2M.1;3Pal;4M2;5P;6G;7S;8U 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.
10Rot	S_E	S	CII	1D;2M.1;3Pal;4M2;5P;6G;7S;8U;9Rot Succinate, S ( type S-pathway to Q). Noncoupled electron transfer state, ET state, with ET capacity E.
11Ama	ROX			1D;2M.1;3Pal;4M2;5P;6G;7S;8U;9Rot;10Ama 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).

Strengths and limitations

SUIT-039 allows assessing FAO-linked respiration avoiding overestimation by endogenous substrates or malate anaplerosis. Other electron transfer pathways are also analyzed in OXPHOS (FN, FNS) and ET-state (FNS, S).

+ SUIT-039 allows the depletion of endogenous substrates with ADP (1D).

+ The protocol provides information on F-pathway in OXPHOS state with palmitoylcarnitine, a long-chain acylcarnitine commonly found in vivo.

+ The low concentration of malate used in this protocol to assess F-pathway, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.

+ F-pathway (3Pal-2M.1) can be compared to FN-pathway (5P) in OXPHOS state.

+ Pathway control in OXPHOS (F, F(N), FN, FNS pathways) and in ET state (FNS and S) can be observed.

+ Multiple pathways converging into Q (FNS) are assessed in OXPHOS and ET states. Therefore, P/E (7S/8U) at high ET capacity can be calculated.

- LEAK state is not investigated.

- Glycerophosphate (Gp) pathway is not investigated.

Compare SUIT protocols

SUIT-036_O2_mt_D089: Similar protocol, including malate kinetics to assess mitochondrial malic enzyme and anaplerosis.

SUIT-002 O2 mt D005: Reference protocol SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized). Allows evaluation of FAO with octanoylcarnitine, also without overestimation by using low malate concentration. Besides this, provides information on pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and ET-state (FNSGp, SGp pathways).

SUIT-025_O2_mt_D057: Allows evaluation of FAO with octanoylcarnitine, also without overestimation by using low malate concentration. Besides this, provides information on pathway control in OXPHOS state (F, F(N), FN, FNS pathways).

SUIT-041 O2 mt D096: A protocol to determine the optimum concentration of acylcarnitine.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1D	ADP (D)
2M.1	Malate (M)
3Pal	Palmitoylcarnitine (Pal)
3c	Cytochrome c (c)
4M2	Malate (M)
5P	Pyruvate (P)
6G	Glutamate (G)
7S	Succinate (S)
8U	Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)	Can be substituted for other uncoupler
9Rot	Rotenone (Rot)
10Ama	Antimycin A (Ama)

Suggested stock concentrations are shown in the specific DL-Protocol.

References

The project FAT4BRAIN has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 857394

Labels:

FAT4BRAINSUIT

@@ Line 1: / Line 1: @@
 {{MitoPedia
-|abbr=
+|abbr= FAO and NS-pathways
-|description=[[File:1D;2M.1;3Pal;3c;4M2;5P;6G;7S;8U;9Rot;10Ama.png|thumb|1D;2M.1;3Pal;3c;4M2;5P;6G;7S;8U;9Rot;10Ama.png]]
+|description=[[File:1D;2M.1;3Pal;3c;4M2;5P;6G;7S;8U;9Rot;10Ama.png|400px]]
-|info='''A:''' [[Doerrier 2018 Methods Mol Biol]]
+|info='''A''' - '''[[SUIT-039]]''' - '''[[Fatty_acid_oxidation_pathway_control_state|F-pathway]] and [[NS-pathway]]'''
+|application=O2
+|SUIT number=D092_1D;2M.1;3Pal;3c;4M2;5P;6G;7S;8U;9Rot;10Ama
 }}
-::: '''[[SUIT protocol pattern]]:''' 1D;2M;3Oct;4M;5P;6G;7S;8Gp;9U;10Rot-
-The SUIT-002 protocol in combination with [[SUIT-001]] provides a common reference for comparison of respiratory control of [[Mitochondrial preparations| mitochondrial preparations]] in a wide variety of species, tissues and cell types. SUIT-002 is specially designed to give information on [[Fatty_acid_oxidation_pathway_control_state|F-pathway]] in [[Oxidative phosphorylation|OXPHOS state]] avoiding FAO overestimation in the presence of [[Anaplerosis|anaplerotic]] pathways. Moreover, the pathway control in OXPHOS state ([[Fatty_acid_oxidation_pathway_control_state|F]], F(N), [[FN]], [[FNS]], [[FNSGp]] pathways) and in [[ET capacity| ET state]] ([[FNSGp]] and [[SGp-pathway control state|SGp]]) can be evaluated by using this SUIT protocol. SUIT-002 can be extended with the CIV assay module.
+{{Template:SUIT text D092}}
 __TOC__
-  Communicated by [[Doerrier C]] and [[Gnaiger E]] (last update 2019-06-05)
+  Communicated by [[Grings M]], [[Cecatto C]] and [[Cardoso LHD]] (last update 2023-04-13)
-== Specific SUIT protocols ==
+== Representative traces ==
-=== SUIT-002 O2 mt D005 and SUIT-002 O2 pfi D006 ===
-[[File:1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png|400px]]
-[[File:D005_O2_traces.png|400px]]
-*  [[SUIT-002 O2 mt D005]] for [[Isolated mitochondria|isolated mitochondria]], [[Tissue homogenate|tissue homogenate]] and [[Permeabilized cells|permeabilized cells]] (already permeabilized when they are added to the chamber)
-*  [[SUIT-002 O2 pfi D006]] for [[Permeabilized_muscle_fibers|permeabilized muscle fibers]]
-=== SUIT-002 O2 ce-pce D007 ===
+{{Template:SUIT-039}}
-[[File:ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png|400px]]
-[[File:D07_O2_traces.png|400px]]
-*  [[SUIT-002 O2 ce-pce D007]] for [[Living cells|living cells]] (ce) and [[Permeabilized cells|permeabilized cells]] (pce; permeabilization of the plasma membrane occurs into the chambers through digitonin addition)
-=== SUIT-002 O2 ce-pce D007a ===
-[[File:ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png|400px]]
-[[File:D07a_O2_traces.png|400px]]
-*  [[SUIT-002 O2 ce-pce D007a]] for [[Living cells|living cells]] (ce) and [[Permeabilized cells|permeabilized PBMC and PLT]] (pce; permeabilization of the plasma membrane occurs into the chambers through digitonin addition)
-{{Template:SUIT-002}}
 == Strengths and limitations ==
-:::* SUIT-002 in combination with [[SUIT-001]] provides a common reference for comparison of respiratory control in a large variety of species, tissues and cell types. Both SUIT protocols provide a mitochondrial mapping which allows:
+:::* SUIT-039 allows assessing FAO-linked respiration avoiding overestimation by endogenous substrates or malate anaplerosis. Other electron transfer pathways are also analyzed in OXPHOS (FN, FNS) and ET-state (FNS, S).
-:::: 1. to obtain reference values.
-:::: 2. to evaluate mitochondrial physiological diversity, generating a mt-database on comparative mitochondrial physiology.
-:::: 3. to screen specific defects.
-:::* [[SUIT-001]] and SUIT-002 are used in the [[Proficiency_test| MitoFit Proficiency test]] for inter-individual and inter-laboratory reproducibility quality control.
-:::* A succinate concentration of >10 mM may be required for saturating S<sub>''E''</sub> capacity.
-:::+ SUIT-002 allows the depletion of endogenous substrates with ADP (1D).
+:::+ SUIT-039 allows the depletion of endogenous substrates with ADP (1D).
-:::+ The protocol provides information on F-pathway in OXPHOS state. The low concentration of malate used in this protocol, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
+:::+ The protocol provides information on F-pathway in OXPHOS state with palmitoylcarnitine, a long-chain acylcarnitine commonly found ''in vivo''.
-:::+ F-pathway (3Oct-2M.1) can be compared to FN-pathway (5P) in OXPHOS state.
+:::+ The low concentration of malate used in this protocol to assess F-pathway, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
-:::+ Pathway control in OXPHOS (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be observed.
+:::+ F-pathway (3Pal-2M.1) can be compared to FN-pathway (5P) in OXPHOS state.
-:::+ Harmonization with [[SUIT-001]] allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements.
+:::+ Pathway control in OXPHOS (F, F(N), FN, FNS pathways) and in ET state (FNS and S) can be observed.
-:::+ Harmonization with many SUIT protocols (up to step 7S).
+:::+ Multiple pathways converging into Q (FNS) are assessed in OXPHOS and ET states. Therefore, ''P''/''E'' (7S/8U) at high ET capacity can be calculated.
-:::+ In SUIT-002, the full set of pathways converging into Q (FNSGp) is obtained in OXPHOS and ET states. Therefore, ''P''/''E'' (8Gp/9U) at high ET capacity can be calculated.
-:::+ This protocol can be extended with the Complex IV module.
-:::- S-pathway in ET state is not obtained (it is obtained in [[SUIT-001]]).
+:::- LEAK state is not investigated.
-:::- Lengthy duration of the experiment.
+:::- Glycerophosphate (Gp) pathway is not investigated.
+== Compare SUIT protocols ==
+::::* [[SUIT-036_O2_mt_D089]]: Similar protocol, including malate kinetics to assess mitochondrial malic enzyme and anaplerosis.
+::::* [[SUIT-002 O2 mt D005]]: Reference protocol SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized). Allows evaluation of FAO with octanoylcarnitine, also without overestimation by using low malate concentration. Besides this, provides information on pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and ET-state (FNSGp, SGp pathways).
-== Compare SUIT protocols ==
+::::* [[SUIT-025_O2_mt_D057]]: Allows evaluation of FAO with octanoylcarnitine, also without overestimation by using low malate concentration. Besides this, provides information on pathway control in OXPHOS state (F, F(N), FN, FNS pathways).
-::::* [[SUIT-002 O2 ce-pce D007]] (general SUIT-002 for permeabilized cells) differs from [[SUIT-002 O2 ce-pce D007]] (SUIT-002 for permeabilized PBMC and PLT) in the concentration and volume of the titrations.
+::::* [[SUIT-041 O2 mt D096]]: A protocol to determine the optimum concentration of acylcarnitine.
-::::* [[SUIT-002 O2 ce-pce D007]] for permeabilized cells (ROUTINE respiration in [[Living cells|living cells]] is obtained before selectively permeabilizing the plasma membrane).
+== Chemicals and syringes ==
-::::* [[SUIT-002 O2 ce-pce D007a]] for permeabilized PBMC and PLT (ROUTINE respiration in [[Living cells|living cells]] is obtained before selectively permeabilizing the plasma membrane).
+{{Template:Chemicals SUIT-039}}
+::: Suggested stock concentrations are shown in the specific DL-Protocol.
+== References ==
-::::* [[SUIT-001]] (RP1): Harmonized SUIT protocol.
+<!--
-::::* [[SUIT-005]]
+== Download DatLab Protocol ==
-::::* [[SUIT-025]] SUIT-025 allows the evaluation of different ET-pathway states (F, F(N), FN, FNS and S) in OXPHOS state.
+::: DatLab Protocol (DLP) for DatLab 7.4:
-::::* [[SUIT-027]] SUIT-027 focuses on the analysis of the malate-anaplerotic pathway control state in mitochondrial preparations.
+::: Excel analysis template (DatLab 7.4):
+-->
-== References ==
+<br /><br />
-{{#ask:[[Category:Publications]] [[Instrument and method::O2k-Protocol]] [[Additional label::SUIT-002]]
+{{Support_FAT4BRAIN}}
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