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SUIT-015
Description
Abbreviation: FNS(Oct,PGM)
Reference: A Schoepf 2016 FEBS J
O2k-Application: O2
MitoPedia concepts:
SUIT A
MitoPedia methods:
Respirometry
- SUIT-category: FNS(Oct,PGM)
- SUIT protocol pattern: diametral 1OctM;2D;3G;4P;5S;6U;7Rot
References
Steps and respiratory states
| Step
|
State
|
Pathway
|
Q-junction
|
Comment - Events (E) and Marks (M)
|
| 1OctM
|
OctML
|
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 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.
|
| 3c
|
OctGMcP
|
FN
|
CETF&CI
|
1OctM;2D;3G;3c
- 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.
- 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]).
- OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
|
| 4P
|
OctPGMP
|
FN
|
CETF&CI
|
1OctM;2D;3G;3c;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 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.
|
| 5S
|
OctPGMSP
|
FNS
|
CETF&CI&II
|
1OctM;2D;3G;3c;4P;5S
- 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). & Succinate, S ( type S-pathway to Q).
- 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.
|
| 6U
|
OctPGMSE
|
FNS
|
CETF&CI&II
|
1OctM;2D;3G;3c;4P;5S;6U
- 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). & Succinate, S ( type S-pathway to Q).
- 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.
- Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency.
- Noncoupled electron transfer state, ET state, with ET capacity E.
|
| 7Rot
|
SE
|
S
|
CII
|
1OctM;2D;3G;3c;4P;5S;6U;7Rot
|
| 8Ama
|
ROX
|
|
|
1OctM;2D;3G;3c;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
|
Comment
|
| ## AsTm
|
AsTmE
|
CIV
|
CIV
|
|
| ## Azd
|
CHB
|
|
|
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- » Coupling control state
- » ET capacity
- » OXPHOS capacity
- » LEAK respiration
- Pathway control
- » Electron transfer pathway
- » Fatty acid oxidation pathway control state, F
- » NADH electron transfer-pathway state, N
- » Succinate pathway control state, S
- » NS-pathway control state, NS
- » Glycerophosphate pathway control state, Gp
- » Complex IV single step, CIV
- » Anaplerotic pathway control state
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Glossary
- » List of SUIT states
- » SUIT concept
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 overal ET capacity in may cell types.
- + The presence of PMG and S establishes fully operative TCA cycle activity.
- + 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.
- - 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
1PalM;2D;3Oct;4P;5G;6U;7S;8Rot
MitoPedia concepts:
SUIT protocol, "SUIT X" is not in the list (MiP concept, Respiratory state, Respiratory control ratio, SUIT concept, SUIT protocol, SUIT A, SUIT B, SUIT C, SUIT state, Recommended, ...) of allowed values for the "MitoPedia concept" property.
SUIT X"SUIT X" is not in the list (Enzyme, Medium, Inhibitor, Substrate and metabolite, Uncoupler, Sample preparation, Permeabilization agent, EAGLE, MitoGlobal Organizations, MitoGlobal Centres, ...) of allowed values for the "MitoPedia topic" property.
1PalM;2D;3Oct;4P;5G;6U;7S;8Rot
