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. Permeabilized muscle fibers are sensitive to oxygen supply due to limited diffusion of oxygen to the fiber bundle core. To counteract this limitation, hyperoxic conditions (400-250 µM O2) must be employed. To set the optimal oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.

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