SUIT-015 O2 pti D043

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SUIT-015 O2 pti D043

Description

1OctM;2D;3G;4P;5S;6U;7Rot;8Ama.png

Abbreviation: FNS(Oct,PGM)

Reference: A pti: permeabilized tissue- SUIT-015

SUIT number: D043_1OctM;2D;3G;4P;5S;6U;7Rot;8Ama

O2k-Application: O2

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

SUIT-015 O2 pti D043 gives information on F-pathway in LEAK state and OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. In addition, the pathway control of FN and FNS in OXPHOS state and of FNS and S in ET state can be evaluated. SUIT-015 O2 pti D043 can be extended with the CIV assay module.


Communicated by Iglesias-Gonzalez J and Gnaiger E (last update 2019-02-04)
MitoPedia: SUIT

Steps and respiratory states

1OctM;2D;3G;4P;5S;6U;7Rot;8Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1OctM OctML(n) F(N) CETF 1OctM
  • 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 F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration.
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(n), in the absence of ADP, ATP, AMP (no adenylates).
2D OctMP F(N) CETF 1OctM;2D
  • 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 F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration.
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
3G OctGMP FN CETF&CI 1OctM;2D;3G
  • 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 F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. & NADH-linked substrates (type N-pathway to Q).
  • 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.
4P OctPGMP FN CETF&CI 1OctM;2D;3G;4P
  • 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 F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. & NADH-linked substrates (type N-pathway to Q).
  • 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.
5S OctPGMSP FNS CETF&CI&II 1OctM;2D;3G;4P;5S
6U OctPGMSE FNS CETF&CI&II 1OctM;2D;3G;4P;5S;6U
7Rot SE S CII 1OctM;2D;3G;4P;5S;6U;7Rot
8Ama ROX 1OctM;2D;3G;4P;5S;6U;7Rot;8Ama
  • 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
## Tm TmE 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 PMG and S establishes a fully operative TCA cycle activity.
+ Reasonable duration of the experiment.
- 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).
- SRot(E) may be underestimated if S is not saturating.
- CIV activity is not measured, to save experimental time.

Compare SUIT protocols

References

 YearReferenceOrganismTissue;cell
Schoepf 2016 FEBS J2016Schöpf B, Schäfer G, Weber A, Talasz H, Eder IE, Klocker H, Gnaiger E (2016) Oxidative phosphorylation and mitochondrial function differ between human prostate tissue and cultured cells. FEBS J 283:2181-96.HumanEndothelial;epithelial;mesothelial cell
Genital
Other cell lines
Fibroblast


MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry