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SUIT-015 O2 pti D043
Description
Abbreviation: FNS(Oct,PGM)
Reference: A pti: permeabilized tissue- SUIT-015
SUIT number: D043_1OctM;2D;2c;3G;4P;5S;6U;7Rot;8Ama
O2k-Application: O2
- 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. 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 Gnaiger E (last update 2019-02-04)
Representative traces
Steps and respiratory states
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 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.
|
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 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;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;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;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
|
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 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
Chemicals and syringes
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
MitoPedia concepts:
SUIT protocol,
SUIT A,
Find
MitoPedia methods:
Respirometry