MiPNet21.06 SUIT RP

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Publications in the MiPMap
O2k-Protocols
SUIT reference protocol for OXPHOS analysis by high-resolution respirometry.

» [In progress]

Oroboros (2018-06-25) Mitochondr Physiol Network

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

» MitoPedia: SUIT reference protocol
» Instrument: Oroboros O2k, O2k-Catalogue


O2k-Network Lab: AT_Innsbruck_Oroboros


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, O2k-Protocol 

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 

SUIT-catg

SUIT reference protocol RP1

SUIT-RP1


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

MitoPedia: SUIT

Steps and respiratory states

SUIT-001

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1PM PML(n) N CI 1PM
2D PMP N CI 1PM;2D
2c PMcP 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. 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 (P, OXPHOS-capacity with saturating [ADP]).
3U PME N CI 1PM;2D;3U
4G PGME N CI 1PM;2D;3U;4G
5S PGMSE 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 OctPGMSE FNS FAO&CI&II 1PM;2D;3U;4G;5S;6Oct
  • Respiratory stimulation by simultaneous action of fatty acid (F), type N substrates (N), and succinate (S), 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 SE S CII 1PM;2D;3U;4G;5S;6Oct;7Rot
8Gp SGpE 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 entry into Q-junction Comment
## Tm TmE CIV CIV
## Azd ROX


Questions.jpg


Click to expand or collaps

SUIT reference protocol RP2

SUIT-RP2

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

MitoPedia: SUIT

Steps and respiratory states

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

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1D ROX 1D
  • ADP is added to stimulate consumption of endogenous fuel-substrates.
  • 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.
2M.1
3Oct OctMP 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 OctMcP 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. 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 (P, OXPHOS-capacity with saturating [ADP]).
4M2 OctMP F(N) FAO 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot
5P OctPMP FN FAO&CI 1D;2M.1;3Oct;4M2;5P
  • Respiratory stimulation by simultaneous action of fatty acid (F) and type N substrates (N) with convergent electron flow in the FN-pathway for reconstitution of TCA cycle function and additive or inhibitory effect of F.
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
6G OctPGMP FN FAO&CI 1D;2M.1;3Oct;4M2;5P;6G
  • Respiratory stimulation by simultaneous action of fatty acid (F) and type N substrates (N) with convergent electron flow in the FN-pathway for reconstitution of TCA cycle function and additive or inhibitory effect of F.
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
7S OctPGMSP FNS FAO&CI&II 1D;2M.1;3Oct;4M2;5P;6G;7S
  • Respiratory stimulation by simultaneous action of fatty acid (F), type N substrates (N), and succinate (S), 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 OctPGMSGpP FNSGp FAO&CI&II&GpDH 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp
9U OctPGMSGpE FNSGp FAO&CI&II&GpDH 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U
10Rot SGpE 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 entry into Q-junction Comment
## Tm TmE CIV CIV
## Azd ROX


Questions.jpg


Click to expand or collaps

Harmonized SUIT protocols

RP1 and RP2 in mt-preparations

SUIT RP1 SUIT-RP1 SUIT RP2 SUIT-RP2

SUIT RP1:1PM;2D:2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd
SUIT RP2:1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd

RP1&RP2.png Harmonization between RP1 and RP2

RP1 and RP2 in cells

RP1 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 intact 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 differentet Electron transfer-pathway states.

SUIT RP1 in cells:ce1;1Dig;1PM;2D:2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10Tm;11Azd
SUIT RP2 in cells:ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azdd

RP1&RP2 pce(Dig).png Harmonization between RP1 and RP2 in cells

RP1 and RP2 in PBMCs and PLTs

In PBMCs and PLTs the reference protocol RP1 is performed in the absense of Oct: SUIT 1 for PBMCs and PTLs

SUIT RP1 for PBMCs and PLTs:1PM;2D;2c;3U;4G;5S;6Rot;7Gp;8Ama;9AsTm;10Azd
SUIT RP2:1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd


Reference protocol RP1&RP2 pce PBMCs PTLs.png Harmonization between RP1 and RP2 in PBMC and PTL cells

RP1 and RP2 in cells

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 intact 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 differentet Electron transfer-pathway statea.

SUIT RP1 in cells: ce1;1Dig;1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10Tm;11Azd
SUIT RP2 in cells: ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azdd

RP1&RP2 pce(Dig).png Harmonization between RP1 and RP2 in cells


RP1 and RP2 in PBMCs and PLTs

In PBMCs and PLTs the reference protocol RP1 is performed in the absense of Oct: SUIT 1 for PBMCs and PLTs

SUIT RP1 in PBMCs and PTLsSUIT RP1 for PBMCs and PLTs:1PM;2D;2c;3U;4G;5S;6Rot;7Gp;8Ama;9AsTm;10Azd
SUIT RP2 PBMCs and PLTs: 1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd

Reference protocol RP1&RP2 pce PBMCs PTLs.png Harmonization between RP1 and RP2 in PBMC and PLT 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 PBMCs and platelets. - Gnaiger Erich 18:47, 19 January 2016 (CET)
K-Regio MitoFit

Further details

» Introduction: Gnaiger 2014 MitoPathways
» MitoPedia: SUIT
» Definition: Substrate-uncoupler-inhibitor titration
» Context: SUIT protocol library
» Abbreviations: MitoPedia