SUIT-005 O2 pfi D011

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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

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

O2k-Application: O2

MitoPedia: SUIT short protocol linked to SUIT-002 O2 pfi D006 - SUIT RP2 (human skeletal muscle)
SUIT protocol pattern: 1OctM;2D;3P;4S;5U;6Rot

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.

Communicated by Doerrier C, Iglesias-Gonzalez J and Gnaiger E (last update 2019-06-05)
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
  • 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 FAO-pathway. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) FAO-pathway could be overestimated due to a contribution of NADH-linked respiration, F(N) (see SUIT-002). The FA concentration has to be optimized to saturate the FAO-pathway, without inhibiting or uncoupling respiration.
  • Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
  • 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]).
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
3P OctPMP FN F&CI 1OctM;2D;2c;3P
  • 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.
4S OctPMSP FNS F&CI&II 1OctM;2D;2c;3P;4S
  • 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.
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


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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

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry