SUIT-001 O2 pfi D002

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SUIT-001 O2 pfi D002

Description

SUIT-RP1

Abbreviation: RP1 pfi

Reference: SUIT-001

SUIT number: D002_1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd

O2k-Application: O2

MitoPedia: SUIT - SUIT reference protocol Reference protocol RP1 for permeabilized fibers

The SUIT-001 O2 pfi D002 protocol in combination with SUIT-002_O2_pfi_D006 is specially designed to provide a common reference for comparison of respiratory control of permeabilized fibers in a wide variety of species and tissues. SUIT-001 O2 pfi D002 gives information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS, FNS, S and SGp pathways) can be evaluated by using this SUIT protocol. SUIT-001 O2 pfi D002 can be extended with the CIV assay module.

The SUIT-001 O2 pfi D002 protocol in combination with SUIT-002_O2_pfi_D006 is specially designed to provide a common reference for comparison of respiratory control of permeabilized fibers in a wide variety of species and tissues. SUIT-001 O2 pfi D002 gives information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS, FNS, S and SGp pathways) can be evaluated by using this SUIT protocol. SUIT-001 O2 pfi D002 can be extended with the CIV assay module.

Communicated by Doerrier C and Gnaiger E (last update 2019-06-05)
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
## AsTm AsTmE CIV CIV
## Azd ROX


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Strengths and limitations

  • SUIT-001 in combination with SUIT-002 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.
+ Linear coupling control (L-P-E) with N substrates (PM).
+ Pathway control in ET state (N, NS, FNS, S and SGp pathways).
+ Harmonization with SUIT-002 allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements.
+ In mtprep, SUIT-001 and SUIT-002 harmonize in: SGpE (8Gp in SUIT-001 and 10Rot in SUIT-002) and Complex IV.
+ Harmonization with many SUIT protocols (up to step 5S).
+ Oct is added in ET state to avoid uncoupler effect with fatty acids.
+ If NS- and FNS-pathways are similar (NSE ~ FNSE), the additivity index of N- and S-pathways in ET state can be evaluated.
+ This protocol can be extended with the Complex IV module.
- The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-001 in favour of measuring SE.
- Lengthy duration of the experiment.


Compare SUIT protocols

  • SUIT-004: Short version of RP1; without G, F and Gp.
  • SUIT-006: Very short initial coupling control module.

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: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry