Talk:Fatty acid oxidation
O2k-Network discussion forum: fatty acids used in permeabilized fibre assays (2015-10-20)
| Name | Experience with FAO in PFI | Fatty acids used | Other FA | Protocol | Other tissue | Model | |
|---|---|---|---|---|---|---|---|
| Wright Traver | We do include octanoylcarnitine in the initial titration of substrates for our SUIT protocols typically, but do not measure its individual contribution to OXPHOS. | Octanoylcarnitine | - | - | - | ||
| Gorr Thomas A | What´s wrong with using simple C16 saturated or C18 monounsaturated fatty acids like palmitate or oleate, respectively, for analysing beta-oxidation rates?? | - | - | - | - | - | |
| Neviere Remi | - | Palmitoylcarnitine | - | - | - | mice and human atria | |
| Ost Mario | right now we´re using octanoyl carnitine (OC) when examining mouse skeletal muscle permeabilized fiber respiration. We ususally start with Malate (2mM) and OC (0.2mM), then add ADP (5mM) in order to assess fatty acid oxidative capacity. For Soleus muscle fibers we got an average respiration of 60 pmol/(s*mg fiber ww). | Octanoylcarnitine | - | - | - | mouse skeletal muscle | |
| Cannon Daniel T | Palmitoylcarnitine + Malate | - | - | - | - | - | |
| West Malcolm | just started | Octanoylcarnitine | - | - | - | - | |
| Fontes-Oliveira Cibely | - | Palmitoylcarnitine | - | - | cells | - | |
| Hickey Anthony | We use Palmitoyl Carnitine, I recall we made it up with fatty acid free BSA at 5:1 (PC:BSA). Worked well with fish and rat heart mitochondria, and permeabilised fibres. | Palmitoylcarnitine | - | - | - | fish and rat heart mitochondria and fibres | |
| Pasdois Philippe | couple palmitoyl-carnitine (10µM) + malate (1mM) on isolated mitochondria and permeabilized fibers. In such case the buffer is always supplemented with 2mg/ml of BSA. | Palmitoylcarnitine | - | - | isolated mitochondria and pfi | - | |
| Jelenik Tomas | we standardly measure FA-linked respiration in the muscle
- we used Octanoyl-C as well as Palmitoyl-C, however, the latter one gives poor response. Thus we use octanoyl. || Octanoylcarnitine || - || - || - || - || | ||||||
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Palmitoylcarnitine Octanoylcarnitine
FA and cell toxicity
I am working at the University of Colorado with Drs Paul MacLean and Matthew Jackman who acquired two OROBOROS this year. Following our collaborators advice (Dr Donal O'Gorman at Dublin City Uni), we are following your protocol to measure fat oxidation. Contrary to results in the literature and from Dr O'Gorman's lab, as soon as we add L-palmitoylcarnitine, the cells die. 2 uM only of L-PC are sufficient to reach a respiration of zero. Adding any other substrates does not allow an increase in O2 consumption. Our local collaborator Dr Jane Reush is encountering the same issue and therefore only use octanoyl carnitine. Octanoyl carnitine does not alter mitochondria/cell. Following advices from Dr Neufer (North Carolina), we added some carnitine. This did not allow maintaining the integrity of the cell. (1)
Because we are in altitude (1600m), we need to use a piO2 of 200-220 (instead of 160) in basal and of 350 (instead of 300) in maximal. We are wondering if this higher piO2 could promote the generation of ROS species or oxidative stress that would kill the mitochondria/cell when adding L-PC? (2)
- Audrey (2015-05-14)
- O2k-Network: US CO Aurora Jackman MR
- Audrey Bergouignan, PhD
- Anschutz Health and Wellness Center Division of Endocrinology, Metabolism and Diabetes
- University of Colorado
- E [email protected]
- http://www.anschutzwellness.com/
Response
- »Fatty acid oxidation#FAO and HRR
- When using permeabilized muscle fibers, we have to increase oxygen levels to nonphysiological hyperoxia to avoid oxygen diffusion limitation to the fibers. Although high oxygen levels stimulate ROS production, mitochondria remain functional during times of measurement up to 3 hours. (Gnaiger E, 2015-05-15)
