SUIT-002

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SUIT-002

Description

1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot-.png

Abbreviation: RP2

Reference: A: Doerrier 2018 Methods Mol Biol

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 in a wide variety of species, tissues and cell types. SUIT-002 is specially designed to give information on F-pathway in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be evaluated by using this SUIT protocol. SUIT-002 can be extended with the CIV assay module.

Communicated by Doerrier C and Gnaiger E (last update 2019-06-05)

Specific SUIT protocols

SUIT-002 O2 mt D005 and SUIT-002 O2 pfi D006

1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png D005 O2 traces.png

SUIT-002 O2 ce-pce D007

Ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png D07 O2 traces.png

SUIT-002 O2 ce-pce D007a

Ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png D07a O2 traces.png

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


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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:
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 MitoFit Proficiency test for inter-individual and inter-laboratory reproducibility quality control.
  • A succinate concentration of >10 mM may be required for saturating SE capacity.
+ SUIT-002 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.
+ F-pathway (3Oct-2M.1) can be compared to FN-pathway (5P) in OXPHOS state.
+ Pathway control in OXPHOS (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be observed.
+ Harmonization with SUIT-001 allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements.
+ Harmonization with many SUIT protocols (up to step 7S).
+ 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).
- Lengthy duration of the experiment.


Compare SUIT protocols


  • SUIT-001 (RP1): Harmonized SUIT protocol.
  • SUIT-005
  • SUIT-025 SUIT-025 allows the evaluation of different ET-pathway states (F, F(N), FN, FNS and S) in OXPHOS state.

References

 YearReferenceOrganismTissue;cell
MiPNet21.06 SUIT RP2018-06-25
O2k-Protocols
SUIT reference protocol for OXPHOS analysis by high-resolution respirometry.
Doerrier 2018 Methods Mol Biol2018Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70.Human
Mouse
Rat
Saccharomyces cerevisiae
Heart
Skeletal muscle
Endothelial;epithelial;mesothelial cell
Blood cells
HEK
Platelet


MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry