SUIT-005 O2 pfi D011

From Bioblast
Jump to: navigation, search
Bioblasts - Richard Altmann and MiPArt by Odra Noel
MitoPedia     Terms and abbreviations     Preprints and history     MiP and biochemistry     Concepts and methods     MitoPedia: SUIT     MitoPedia: O2k



MitoPedia

SUIT-005 O2 pfi D011

Description

1OctM;2D;2c;3P;4S;5U;6Rot;7Ama;8AsTm;9Azd.png

Abbreviation: RP2-short pfi

Reference: A: - SUIT-005 short protocol linked to SUIT-002 O2 pfi D006 - SUIT RP2 (human skeletal muscle)

SUIT number: D011_1OctM;2D;3P;4S;5U;6Rot;7Ama;8AsTm;9Azd

O2k-Application: O2

SUIT-005 O2 pfi D011 protocol provides information on the F-pathway, the combined FN pathway, and the convergence FNS pathways in the OXPHOS state. FNS comprises the most important pathways in many cell types and thus provides a physiologically relevant estimate of maximum OXPHOS- and ET capacity. SUIT-005 can be extended with the CIV assay module. This protocol is linked to SUIT-002 O2 pfi D006 - SUIT RP2, specifically for human skeletal muscle mitochondria. SUIT-005 O2 pfi D011 is harmonized with SUIT-004 O2 pfi D010. The F-pathway has to be tested previously. 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, Gnaiger E (last update 2019-06-05)

Representative traces

SUIT-005 O2 pfi D11.png

MitoPedia: SUIT

Steps and respiratory states

1OctM;2D;2c;3P;4S;5U;6Rot;7Ama;8AsTm;9Azd.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1OctM OctML(n) F(N) FAO 1OctM
2D OctMP F(N) FAO 1OCtM;2D
2c OctMcP F(N) FAO 1OCtM;2D;2c
3P OctPMP FN F&CI 1OctM;2D;2c;3P
  • 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.
4S OctPMSP FNS F&CI&II 1OctM;2D;2c;3P;4S
  • 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.
5U OctPMSE FNS F&CI&II 1OctM;2D;2c;3P;4S;5U
6Rot SE S CII 1OctM;2D;3P;4S;5U;6Rot
7Ama ROX 1OctM;2D;3P;4S;5U;6Rot;7Ama
  • 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.
Step Respiratory state Pathway control ET-Complex entry into Q-junction Comment
## AsTm AsTmE CIV CIV
## Azd ROX


Questions.jpg


Click to expand or collaps

Strengths and limitations

+ The protocol provides information on FAO capacity in the absence of other, potentially interfering pathways, both in the LEAK state and in OXPHOS.
+ FNS OXPHOS capacity comprises the most important pathways in many cell types and thus provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
+ FNS ET capacity is a good estimate of overall ET capacity in many cell types.
+ The presence of PM and S establishes a fully operative TCA cycle activity (PMS) if the 2-oxoglutarate carrier does not outcompete the sources of 2-oxoglutarate
+ Application of the cytochrome c test early in the protocol ensures comparability of all states in case of any effect of c.
+ Reasonable duration of the experiment (1.5 to 2 h); 2-3 h when the CIV module is added.
- F- OXPHOS capacity may be underestimated. In human heart muscle addition of Oct to palmitoylcarnitine (Pal) + malate increased OXPHOS by 26% (Lemuieux et al 2011).
- SRotE may be underestimated if S is not saturating.


Compare SUIT protocols

  • SUIT-002: A comparable but more comprehensive protocol comprising additional substrate states.
  • SUIT-002 O2 pfi D006: A comparable but more comprehensive pfi-specific protocol comprising additional substrate states.
  • imt, ROX: 1OctM;2D;2c;3P;4S;5U;6Rot;7Ama
  • pfi, high O2, no ROX: FNS(PM)01_pfiO2,1OctM,2D(c),3P,4S,5U,6Rot

Chemicals and syringes

Step Chemical(s) and link(s) Comments
1OctM Octanoylcarnitine (Oct) and Malate (M)
2D ADP (D)
2c Cytochrome c (c)
3P Pyruvate (P)
4S Succinate (S)
5U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler
6Rot Rotenone (Rot)
7Ama Antimycin A (Ama)
Step Chemical(s) and link(s) Comments
## AsTm Ascorbate (As) and TMPD (Tm)
## Azd Azide (Azd)
Suggested stock concentrations are shown in the specific DL-Protocol.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry