Description
Abbreviation: Optimum [acylcarnitine] test
Reference: A - SUIT-041 - F-pathway and determination of optimum acylcarnitine concentration
SUIT number: D096_1D;2M.1;3AC;3c;4M2;5P;6S;7Rot;8Ama
O2k-Application: O2
The SUIT-041 O2 mt D096 protocol provides a common reference for comparison of respiratory control of mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species, tissues and cell types. SUIT-041 O2 mt D096 is specially designed to give information on F-pathway with titration of acylcarnitine as a FAO substrate to determine the optimal concentration. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS pathways) can be evaluated by using this SUIT protocol.
Communicated by Grings M, Cecatto C and Cardoso LHD (last update 2023-04-12)
Representative traces
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1D | ROX | 1D
| ||
2M.1 | 1D;2M.1 | |||
3AC | ACMP | F | FAO | 1D;2M.1;3AC
|
3c | ACMcP | F | FAO | 1D;2M.1;3AC;3c
|
4M2 | ACMP | F(N) | FAO | 1D;2M.1;3AC;3c;4M2
|
5P | ACPMP | FN | FAO&CI | 1D;2M.1;3AC;4M2;5P
|
6S | ACPMSP | FNS | FAO&CI&II | 1D;2M.1;3AC;4M2;5P;6S
|
7Rot | SE | SGp | CII | 1D;2M.1;3AC;4M2;5P;6S;7Rot
|
8Ama | ROX | 1D;2M.1;3AC;4M2;5P;6S;7Rot;8Ama
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- SUIT-041 has been developed to test the optimum concentration of acylcarnitine for high-resolution respirometry experiments. High concentrations of fatty acids or acylcarnitines might inhibit respiration, therefore, it is recommended to test the optimum concentration for new samples or different acylcarnitines.
- + SUIT-041 allows the depletion of endogenous substrates with ADP (1D).
- + The protocol provides information on F-pathway in OXPHOS state with multiple titrations of acylcarnitines (e.g., octanoylcarnitine, palmitoylcarnitine) until the optimum concentration is reached (respiration reaches its maximum and does not increase further with more acylcarnitine).
- + The low concentration of malate used in this protocol to assess F-pathway, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
- + F-pathway (3Pal-2M.1) can be compared to FN-pathway (5P) in OXPHOS state.
- + Pathway control in OXPHOS (F, F(N), FN, FNS pathways) can be observed.
- - Very long duration of the experiment.
- - Glycerophosphate (Gp) pathway is not investigated.
- - LEAK and ET states are not investigated.
Compare SUIT protocols
- SUIT-036_O2_mt_D089, SUIT-037 O2 mt D090, SUIT-038 O2 mt D091: A combination of protocols to assess FAO-linked respiration using palmitoylcarnitine and octanoylcarnitine, and investigating the presence of malic enzyme with malate kinetics.
- SUIT-002 O2 mt D005: Reference protocol SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized). Allows evaluation of FAO with octanoylcarnitine, without overestimation by using low malate concentration. Besides this, provides information on pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and ET-state (FNSGp, SGp pathways).
- SUIT-025_O2_mt_D057: Allows evaluation of FAO with octanoylcarnitine, without overestimation by using low malate concentration. Besides this, provides information on pathway control in OXPHOS state (F, F(N), FN, FNS pathways).
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1D | ADP (D) | |
2M.1 | Malate (M) | |
3AC | Template:Acylcarnitine | |
3c | Cytochrome c (c) | |
4M2 | Malate (M) | |
5P | Pyruvate (P) | |
6S | Succinate (S) | |
7Rot | Rotenone (Rot) | |
8Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
Labels:
FAT4BRAINSUIT