MiPNet21.06 SUIT RP

MiPNet21.06 SUIT RP

SUIT reference protocol for OXPHOS analysis by high-resolution respirometry.

» [In progress]

Oroboros (2018-06-25) Mitochondr Physiol Network

Abstract: Doerrier C, Sumbalova Z, Gnaiger (2016) SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. Mitochondr Physiol Network 21.06(01):1-12.

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SUIT reference protocol RP1

- - - » SUIT-001

SUIT states: 1-3PM(LPcE) 4PGM 5PGMS 6OctPGMS 7S 8SGp 9ROX 10Tm 11ROX

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1PM	PM_L(n)	N	CI	1PM NADH-linked substrates (type N-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	PM_P	N	CI	1PM;2D NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	PMc_P	N	CI	1PM;2D;2c NADH-linked substrates (type N-pathway to Q). 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]).
3U	PM_E	N	CI	1PM;2D;3U NADH-linked substrates (type N-pathway to Q). 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.
4G	PGM_E	N	CI	1PM;2D;3U;4G NADH-linked substrates (type N-pathway to Q). Noncoupled electron transfer state, ET state, with ET capacity E.
5S	PGMS_E	NS	CI&II	1PM;2D;3U;4G;5S Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. Noncoupled electron transfer state, ET state, with ET capacity E.
6Oct	OctPGMS_E	FNS	FAO&CI&II	1PM;2D;3U;4G;5S;6Oct 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.
7Rot	S_E	S	CII	1PM;2D;3U;4G;5S;6Oct;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.
8Gp	SGp_E	SGp	CII&GpDH	1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp Respiratory stimulation by action of succinate and glycerophosphate, Gp, with convergent electron flow in the SGp-pathway (CII&GpDH-linked pathway to the Q-junction). Noncoupled electron transfer state, ET state, with ET capacity E.
9Ama	ROX			1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp;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

SUIT reference protocol RP2

- - - » SUIT-002

SUIT states: 1REN 2OctM(P) 2OctM(c) 3OctPM 4OctPGM 5OctPGMS 6OctPGMSGp 7E 8SGp 9ROX 10CIV 11CHB

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1D	REN			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.
3Oct	OctM_P	F	FAO	1D;2M.1;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. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration.
3c	OctMc_P	F	FAO	1D;2M.1;3Oct;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	OctM_P	F(N)	FAO	1D;2M.1;3Oct;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	OctPM_P	FN	FAO&CI	1D;2M.1;3Oct;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	OctPGM_P	FN	FAO&CI	1D;2M.1;3Oct;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	OctPGMS_P	FNS	FAO&CI&II	1D;2M.1;3Oct;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.
8Gp	OctPGMSGp_P	FNSGp	FAO&CI&II&GpDH	1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp Respiratory stimulation by simultaneous action of fatty acid (F), type N substrates (N), succinate (S), and glycerophosphate with convergent electron flow in the FNSGp-pathway to the Q-junction. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
9U	OctPGMSGp_E	FNSGp	FAO&CI&II&GpDH	1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U Respiratory stimulation by simultaneous action of fatty acid (F), type N substrates (N), succinate (S), and glycerophosphate with convergent electron flow in the FNSGp-pathway to the Q-junction. 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	SGp_E	SGp	CII&GpDH	1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot Respiratory stimulation by action of succinate and glycerophosphate, Gp, with convergent electron flow in the SGp-pathway (CII&GpDH-linked pathway to the Q-junction). Noncoupled electron transfer state, ET state, with ET capacity E.
11Ama	ROX			1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama 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

Harmonized SUIT protocols

RP1 and RP2 in mt-preparations

SUIT RP1 SUIT RP2

SUIT RP1 in mt-preparations: mt;1PM;2D:2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd

SUIT RP2 in mt-preparations: mt;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd

Harmonization between RP1 and RP2

RP1 and RP2 in permeabilized fibers

To avoid oxygen limitation in pfi, high oxygen concentration levels (250-450 µM O2) are used during the assay.

SUIT RP1 in pfi SUIT RP2 in pfi

SUIT RP1 in pfi: pfi;1PM;2D:2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd

SUIT RP2 in pfi: pfi;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd

Harmonization between RP1 and RP2

RP1 and RP2 in cells

RP1 and RP2 can be assessed in cells after the selective plasma membrane permeabilization. Digitonin is a mild detergent that permeabilizes plasma membranes. The optimum effective digitonin concentration for complete plasma membrane permeabilization of cultured cells can be determined directly in a respirometric protocol (see Digitonin). The use of living cells allows the possibility to evaluate respiration in two different modules: intact cells and permeabilized cells. ROUTINE respiration (ce1) is obtained before digitonin is employed, whereas after the use of digitonin we can address different Electron-transfer-pathway states.

SUIT RP1 in ce-pce SUIT RP2 in ce-pce

SUIT RP1 in cells: ce1;1Dig;1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd

SUIT RP2 in cells: ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd

Harmonization between RP1 and RP2 in cells

RP1 and RP2 in PBMC and PLT

In PBMC and PLT, the reference protocol RP1 is performed in the absence of Oct.

SUIT RP1 in PBMC and PLT SUIT RP2 in PBMC and PLT

SUIT RP1 for PBMC and PLT: ce1;1Dig;1PM;2D;2c;3U;4G;5S;6Rot;7Gp;8Ama;9AsTm;10Azd

SUIT RP2 for PBMC and PLT: ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd

Harmonization between RP1 and RP2 in PBMC and PTL cells

Experimental details

Work in progress: pre-publication protocol

Saturating ADP concentrations

Different concentrations of ADP are sufficient to obtain maximum flux for estimating OXPHOS capacity. The typical range for isolated mitochondria is 1 to 2.5 mM, for permeabilized cells 1 to 5 mM, for permeabilized muscle fibres 2.5 to 10 mM.

SUIT-RP development

RP-T01

RP1: Compared to RP1-T01, move Oct titration after S.
RP2: Compared to RP2-T01, move U titration after Gp.

RP-T02

RP2: Compared to RP2-T02, move D titration after the sample. Titration of M.1 before Oct. M2 is added after Oct.

SUIT RP2 development

Cytochrome c test

In protocol RP2, the cytochrome c test might better be moved from a c-titration after pyruvate (P) to a titration after M2. Otherwise, there is a risk of underestimation of FAO in cases of a cytochrome c effect. On the other hand, at the low flux before titration of P, the sensitivity of the cytochrome c test may be low.

~ Gnaiger Erich 18:26, 23 January 2016 (CET)

Mark names

Even the highly abbreviated names of respiratory states have become too long for DatLab, where mark names are restricted to 8 digits. The visibility in DatLab of long mark names is restricted. The simplest solution for short mark names is the use of a numerical sequence, with the preceeding event name added for information.

~ Gnaiger Erich 16:50, 23 January 2016 (CET)

Pre-publication communication

This communication is a pre-publication, inviting critical feedback and comments. All feedback will be carefully documented and evaluated in terms of justification of co-authorship. We intend to finally publish this topic in a peer-reviewed journal as an original article, with reference to the pre-publication history including reviewer's reports.

Experiments in progress

The SUIT reference protocol is presently applied in permeabilized HEK cells, mouse heart isolated mitochondria, liver homogenate, permeabilized skeletal muscle (mouse and human), and human PBMC and PLT. - Gnaiger Erich 18:47, 19 January 2016 (CET)

AT Innsbruck Gnaiger E and AT Innsbruck Oroboros - MitoFit project.
US NC Winston-Salem Molina AJA - UPBEAT project.

Further details

» Introduction: Gnaiger 2014 MitoPathways

» MitoPedia: SUIT

Table of titrations » MiPNet09.12 O2k-Titrations

» Definition: Substrate-uncoupler-inhibitor titration

» Context: SUIT protocol library

» Abbreviations: MitoPedia

Labels: MiParea: Instruments;methods

Preparation: Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria

Regulation: Substrate Coupling state: LEAK, OXPHOS, ET Pathway: F, N, S, Gp, CIV, NS, Other combinations HRR: Oxygraph-2k

MitoPathways, MitoFitPublication, SUIT-001, SUIT-002, SUIT-001 O2 mt D001, SUIT-001 O2 pfi D002, SUIT-001 O2 ce-pce D003, SUIT-001 O2 ce-pce D004, SUIT-002 O2 mt D005, SUIT-002 O2 pfi D006, SUIT-002 O2 ce-pce D007, SUIT-002 O2 ce-pce D007a