Cookies help us deliver our services. By using our services, you agree to our use of cookies. More information

Difference between revisions of "SUIT-002 O2 ce-pce D007a"

From Bioblast
Line 9: Line 9:
::: '''[[Categories of SUIT protocols|SUIT-category]]:''' FNSGp(Oct,PGM)
::: '''[[Categories of SUIT protocols|SUIT-category]]:''' FNSGp(Oct,PGM)
::: '''[[SUIT protocol pattern]]:''' diametral 1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot-
::: '''[[SUIT protocol pattern]]:''' diametral 1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot-
The SUIT-002 O2 pce D007a protocol in combination with [[SUIT-001_O2_pce_D004]] provides a common reference for comparison of respiratory control of [[PBMC| PBMCs]] and [[Platelet| Platelets]] in a wide variety of species. SUIT-002 O2 pce D007a is specially designed to give information on [[Fatty_acid_oxidation_pathway_control_state|F-pathway]] in [[Oxidative phosphorylation|OXPHOS state]] avoiding FAO overestimation in the presence of [[Anaplerosis|anaplerotic]] pathways. Moreover, the pathway control in OXPHOS state ([[Fatty_acid_oxidation_pathway_control_state|F]], F(N), [[FN]], [[FNS]], [[FNSGp]] pathways) and in [[ET-capacity| ET state]] ([[FNSGp]] and [[SGp-pathway control state|SGp]]) can be evaluated by using this SUIT protocol. SUIT-002 O2 pce D007a can be extended with the CIV assay module.
__TOC__
__TOC__
{{Template:SUIT-002 pce}}
{{Template:SUIT-002 pce}}

Revision as of 09:56, 6 March 2019


high-resolution terminology - matching measurements at high-resolution


SUIT-002 O2 ce-pce D007a

Description

Ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png

Abbreviation: FNSGp(Oct,PGM)

Reference: A Reference protocol RP2; specific for PBMC and platelet cells - SUIT-002

SUIT number: D007a_ce1;1Dig;1D;2M;3Oct;3c;4M;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd

O2k-Application: O2

MitoPedia: SUIT - SUIT reference protocol RP2 for PBMCs and platelets
SUIT-category: FNSGp(Oct,PGM)
SUIT protocol pattern: diametral 1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot-

The SUIT-002 O2 pce D007a protocol in combination with SUIT-001_O2_pce_D004 provides a common reference for comparison of respiratory control of PBMCs and Platelets in a wide variety of species. SUIT-002 O2 pce D007a is 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. SUIT-002 O2 pce D007a can be extended with the CIV assay module.


MitoPedia: SUIT

Steps and respiratory states

Ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
ce1 ROUTINE ce1
  • ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
1Dig REN ce1;1Dig
  • Optimum effective digitonin concentration for complete plasma membrane permeabilization.
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1D REN 1D
  • ADP is added to stimulate the consumption of endogenous fuel-substrates.
2M.1
3Oct OctMP F FAO 1D;2M.1;3Oct
  • 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.
3c OctMcP F FAO 1D;2M.1;3Oct;3c
  • 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.
  • Addition of cytochrome c yields a test for integrity of the mtOM (cytochrome c control efficiency). 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 (OXPHOS capacity P with saturating [ADP]).
4M2 OctMP F(N) FAO 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot
  • 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.
  • High concentration of malate, typically 2 mM, saturates the N-pathway.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5P OctPMP FN F&CI 1D;2M.1;3Oct;4M2;5P
6G OctPGMP FN F&CI 1D;2M.1;3Oct;4M2;5P;6G
7S OctPGMSP FNS F&CI&II 1D;2M.1;3Oct;4M2;5P;6G;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.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
8Gp OctPGMSGpP FNSGp F&CI&II&GpDH 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp
9U OctPGMSGpE FNSGp F&CI&II&GpDH 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U
10Rot SGpE SGp CII&GpDH 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot
  • 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.
11Ama ROX 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama
  • 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 Comment
## AsTm AsTmE CIV CIV
## Azd CHB


Questions.jpg


Click to expand or collaps

Strengths and limitations

  • SUIT-002 in combination with SUIT-001 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.
+ SUIT-002 allows the depletion of endogenous substrates with ADP (1D).
+ The protocol provides information on F-pathway in OXPHOS state. The low concentration of malate used in this protocol, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
+ F-pathway (3Oct-2M.1) can be compared to FN-pathway (5P) in OXPHOS state.
+ Pathway control in OXPHOS (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be observed.
+ Harmonization with SUIT-001 allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements.
+ Harmonization with many SUIT protocols (up to step 7S).
+ In SUIT-002, the full set of pathways converging into Q (FNSGp) is obtained in OXPHOS and ET states. Therefore, P/E (8Gp/9U) at high ET capacity can be calculated.
+ This protocol can be extended with the Complex IV module.
- S-pathway in ET state is not obtained (it is obtained in SUIT-001).
- Lengthy duration of the experiment.

Compare SUIT protocols

  • SUIT-002 O2 pce D007a (SUIT-002 for permeabilized PBMCs and PLTs) differs from SUIT-002 O2 pce D007 (general SUIT-002 for permeabilized cells) in the concentration and volume of the titrations.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry 


Labels:






SUIT-002