SUIT-019 O2 pfi D045

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SUIT-019 O2 pfi D045

Description

1PalM;2D;2c;3Oct;4P;5G;6U;7S;8Rot;9Ama.png

Abbreviation: FNS(PalOct,PGM)

Reference: A SUIT-019

SUIT number: D045_1PalM;2D;2c;3Oct;4P;5G;6U;7S;8Rot;9Ama

O2k-Application: O2

SUIT-category: FNS(PalOct,PGM)
SUIT protocol pattern: diametral 1PalM;2D;2c;3Oct;4P;5G;6U;7S;8Rot-

SUIT-019 O2 pfi D045 gives information on F-pathway in LEAK and OXPHOS states in permeabilized fibers. Moreover, this SUIT protocol allows the pathway control evaluation of FN in OXPHOS state and FN, FNS and S in ET state. 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 Cardoso LHD, Doerrier C and Gnaiger E (last update 2019-03-06)

Representative traces

D045 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

1PalM;2D;2c;3Oct;4P;5G;6U;7S;8Rot;9Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1PalM PalML(n) F(N) CETF 1PalM
2D PalMP F(N) CETF 1PalM;2D
2c PalMP F(N) CETF 1PalM;2D;2c
3Oct PalOctMP F(N) CETF 1PalM;2D;2c;3Oct
4P PalOctPMP FN CETF&CI 1PalM;2D;2c;3Oct;4P
  • 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.
5G PalOctPGMP FN CETF&CI 1PalM;2D;2c;3Oct;4P;5G
  • 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.
6U PalOctPGME FN CETF&CI&II 1PalM;2D;2c;3Oct;4P;5G;6U
7S PalOctPGMSE FNS CI&CII 1PalM;2D;2c;3Oct;4P;5G;6U;7S
  • 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.
  • Noncoupled electron transfer state, ET state, with ET capacity E.
8Rot SE S CII 1PalM;2D;2c;3Oct;4P;5G;6U;7S;8Rot
9Ama ROX 1PalM;2D;2c;3Oct;4P;5G;6U;7S;8Rot;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-019 provides an estimation of F OXPHOS capacity, physiologically relevant estimate of maximal Complex I capacity as well as of FNS ET capacity.
+ The protocol provides information on FAO capacity in the absence of other, potentially interfering pathways, both in the LEAK state and in OXPHOS.
+ F OXPHOS capacity is estimated in presence of both Oct and Pal therefore it is more accurate than an estimate in the presence of only one of the substrates. In human heart muscle addition of Oct to palmitoylcarnitine (Pal) + malate increased OXPHOS by 26% (Lemuieux et al 2011).
+ FN OXPHOS capacity comprises the most important Complex I-linked pathways in many cell types and thus provides a physiologically relevant estimate of maximum CI capacity.
+ FNS ET capacity is a good estimate of overall ET capacity in many cell types
+ The presence of PMG and S establishes fully operative TCA cycle activity.
+ Reasonable duration of the experiment.
- Oct and Pal can have an uncoupling effect during F OXPHOS and FN OXPHOS states if their concentration is not optimized.
- SRot(E) may be underestimated if S is not saturating.
- CIV activity is not measured, to save experimental time.

Compare SUIT protocols

  • SUIT-002 (RP2): SUIT protocol 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.

Chemicals and syringes

Step Chemical(s) and link(s) Comments
1PalM Palmitoylcarnitine (Pal) and Malate (M)
2D ADP (D)
2c Cytochrome c (c)
3Oct Octanoylcarnitine (Oct)
4P Pyruvate (P)
5G Glutamate (G)
6U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler
7S Succinate (S)
8Rot Rotenone (Rot)
9Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.

References

 YearReferenceOrganismTissue;cell
Lemieux 2011 Int J Biochem Cell Biol2011Lemieux H, Semsroth S, Antretter H, Höfer D, Gnaiger E (2011) Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int J Biochem Cell Biol 43:1729–38.HumanHeart


MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry