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Difference between revisions of "Talk:Fatty acid oxidation"

From Bioblast
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! Name !! Experience with FAO in PFI !! Fatty acids used !! Other FA !! Protocol !! Other tissue !! Model
! Name !! Experience with FAO in PFI !! Fatty acids used !! Other FA !! Protocol !! Other tissue !! Model
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| [[Wright TJ|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 || - || - || - ||
| [[Wright TJ|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 || - || - || - || - ||
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| [[Gorr TA|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?? || - || - || - || - || - ||
| [[Gorr TA|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?? || - || - || - || - || - ||
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| [[Jelenik T|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 || - || - || - || - ||
| [[Jelenik T|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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| [[Veksler V|Veksler Vladimir]] || We studied fatty acid oxidation in permeabilised cardiac and skeletal muscle fibers for 15 years. We use palmitoylcarnitine, palmitoyl-CoA or octanoate || Octanoylcarnitine || palmitoyl-CoA or octanoate || - || - || permeabilised cardiac and skeletal muscle fibers ||
| [[Veksler V|Veksler Vladimir]] || We studied fatty acid oxidation in permeabilised cardiac and skeletal muscle fibers for 15 years. We use palmitoylcarnitine, palmitoyl-CoA or octanoate || Octanoylcarnitine || palmitoyl-CoA or octanoate || - || - || permeabilised cardiac and skeletal muscle fibers ||
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| [[Wuest RC|Wuest Rob]] || compared with just glucose oxidation, the max Oxphos/ETS capacity was slightly (but significantly) lower with PC & Oct. The protocol i used was: PC, M, ADP, OCT, G, P, SUC, (FCCP, rat), ROT, AA. The concentrations were the same as in the blue book || - || - || PC, M, ADP, OCT, G, P, SUC, (FCCP, rat), ROT, AA || permeabilised heart tissue || - ||
| [[Wuest RC|Wuest Rob]] || compared with just glucose oxidation, the max Oxphos/ETS capacity was slightly (but significantly) lower with PC & Oct. The protocol i used was: PC, M, ADP, OCT, G, P, SUC, (FCCP, rat), ROT, AA. The concentrations were the same as in the blue book || - || - || PC, M, ADP, OCT, G, P, SUC, (FCCP, rat), ROT, AA || permeabilised heart tissue || - ||
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| [[Ramsay RR|Ramsay Rona R]] || I recommend that everyone uses palmitoyl-Cn (with BSA of course) for the most physiological measurement. I actually used decanoyl-CoA when I wanted a more soluble substrate. || - || palmitoyl-Cn (with BSA of course) || - || - || - ||
| [[Ramsay RR|Ramsay Rona R]] || I recommend that everyone uses palmitoyl-Cn (with BSA of course) for the most physiological measurement. I actually used decanoyl-CoA when I wanted a more soluble substrate. || - || palmitoyl-Cn (with BSA of course) || - || - || - ||
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| [[Salin K|Salin Karine]] || worked a bit with palmitoyl carnitine on homogenate of white muscle of fish . do not need to be permeabilized chemically as mechanical permeabilization allow to obtain permeabilized cell (ADP-responsiveness the same). The respiration rate were very lower than the pyruvate / malate / succinate substrate, so I stopped to use the lipid substrates. || Palmitoylcarnitine || - || - || homogenate of white muscle of fish || - ||
| [[Salin K|Salin Karine]] || worked a bit with palmitoyl carnitine on homogenate of white muscle of fish . do not need to be permeabilized chemically as mechanical permeabilization allow to obtain permeabilized cell (ADP-responsiveness the same). The respiration rate were very lower than the pyruvate / malate / succinate substrate, so I stopped to use the lipid substrates. || Palmitoylcarnitine || - || - || homogenate of white muscle of fish || - ||
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| [[Baekkerud F|Baekkerud Fredrik Hjulstad]] || Octanoylcarnitine + Palmitoylcarnitine  || - || - || - || - || diabetic db/db mice ||
| [[Baekkerud F|Baekkerud Fredrik Hjulstad]] || Octanoylcarnitine + Palmitoylcarnitine  || - || - || - || - || diabetic db/db mice ||

Revision as of 16:23, 4 November 2015

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 - - - -
Veksler Vladimir We studied fatty acid oxidation in permeabilised cardiac and skeletal muscle fibers for 15 years. We use palmitoylcarnitine, palmitoyl-CoA or octanoate Octanoylcarnitine palmitoyl-CoA or octanoate - - permeabilised cardiac and skeletal muscle fibers
Wuest Rob compared with just glucose oxidation, the max Oxphos/ETS capacity was slightly (but significantly) lower with PC & Oct. The protocol i used was: PC, M, ADP, OCT, G, P, SUC, (FCCP, rat), ROT, AA. The concentrations were the same as in the blue book - - PC, M, ADP, OCT, G, P, SUC, (FCCP, rat), ROT, AA permeabilised heart tissue -
Chavanelle Vivien fatty oxidation in permeabilised db/db mice muscle fibres, in Mir05 + Cr +37°C. We found that ADP-stimulated respiration in presence of 0,02mM of palmitoyl-carnitine (OXPHOS) is very close to the non-ADP-stimulated respiration (LEAK state) when working with these conditions. (Values around 14 to 20 (pmol/s/mg w.t.) for leak state and 15 to 24 (pmol/s/mg w.t.) for fatty acid triggered-OXPHOS). These data suggest either a very poor ability for this animal model to use palmitoyl-carnitine or a very high leak state (or both!) Palmitoylcarnitine - - - mice
Ramsay Rona R I recommend that everyone uses palmitoyl-Cn (with BSA of course) for the most physiological measurement. I actually used decanoyl-CoA when I wanted a more soluble substrate. - palmitoyl-Cn (with BSA of course) - - -
Salin Karine worked a bit with palmitoyl carnitine on homogenate of white muscle of fish . do not need to be permeabilized chemically as mechanical permeabilization allow to obtain permeabilized cell (ADP-responsiveness the same). The respiration rate were very lower than the pyruvate / malate / succinate substrate, so I stopped to use the lipid substrates. Palmitoylcarnitine - - homogenate of white muscle of fish -
Baekkerud Fredrik Hjulstad Octanoylcarnitine + Palmitoylcarnitine - - - - diabetic db/db mice
Roden Michael Great experience Octanoylcarnitine + Palmitoylcarnitine - - - -
Makrecka Marina We were examined fatty acid oxidation in permeabilized muscle fibres and cardiac fibres and in isolated cardiac mitochondria of rats and mice. Since the area of our research is the regulation of energy metabolism, the determination of fatty acid oxidation is one of the typical measurements. Which substrate we use, is dependent on the aim (include CPT I (rate limiting step) or not). In addition, presence of BSA in media and used carnitine concentration are always under discussions (physiological or not physiological). Palmitoylcarnitine + Malate - - - rats and mice
Bouitbir Jamal skeletal muscles and heart Palmitoylcarnitine - - - -
Moellering Douglas R We use submaximal concentrations 40uM of palmitoylcarnitine with our permeabilized muscle fiber preps. Palmitoylcarnitine - - - -
Gottschalk Kirby - Palmitoylcarnitine - - - -
Chicco AJ We have used palmitoylcarnitine for most of our studies, but have found it to behave inconsistently in the past, and differently than OctCrn when the two have been compared. So, we have used both, but would be best represented by Palmitoylcarnitine. - Palmitoylcarnitine - - -
Miller Benjamin - Octanoylcarnitine - - - -
Coen Paul We use Palmitylcarnitine as a substrate in assays of permeabilized fiber bundle respiration. The substrate works well for us and in combination with Pal, Mal, Glut, and Succ we see higher levels of state 3 respiration than without it. Palmitoylcarnitine - - - -
Essop Faadiel - Palmitylcarnitine - - - -
McCurdy Carrie E We have had good success using the Octanoylcarnitine at the recommended dose of 0.2mM in SUIT protocols with PMG and with just GM. Respiration increases with addition of ADP and subsequent substrate additions. We have also used palmitoylcarnitine at a conc of 0.025 -0.05mM with mixed success. Often there is no or little stable increase in ADP stimulated respiration and requires addition of pyr and/or Glut. We primarily test nonhuman primate (japanses macaque) permeabilized muscle fibers. Octanoylcarnitine + Palmitoylcarnitine - - - nonhuman primates
Madlala Hlengiwe We have assessed beta oxidation in permeabilised skeletal muscle fibres. We initially used palmitoylcarnitine in our analysis but we realised that using this substrate for assessing beta oxidation in skeletal muscle of rat does not work (no response in respiration). this is also documented in literature; absence of LCAD in rat skeletal muscle so we decided to use octanoylcarnitine instead and it works well. afterwards the muscle is responsive to ADP and uncoupler titrations. However, we use this in combination with malate. Octanoylcarnitine + Palmitoylcarnitine - - - -
Sparks Lauren We routinely (up to 4 human and mouse studies) measure respiration supported by glycolytic and fatty acid substrates using SUIT protocols in permeabilized muscle fibers. We always use palmitoylcarnitine. In the past, in Dr. Schrauwen’s lab, we used octanoate to titrate for fatty acid induced uncoupling. Palmitoylcarnitine - - - -
Lemieux Helene heart and skeletal muscle Octanoylcarnitine + Malate, Palmitoylcarnitine + Malate Acetylcarnitine + Malate, Palmitoyl-CoA+Malate+carnitine - permeabilized animals (Dugesia tigrina worm) -
Jjubkovic Marko Following your email from yesterday, I am sending you a graph with some mitochondrial respiration measurements obtained from the left ventricular human tissue of patients undergoing CABG surgery. As can be seen from the graph, we used FFA substrate to evoke the State 2 and State 3 respiration, and then followed with other substrates and an uncoupler. FFA substrate was palmitoyl-carnitine (40 ?M concentration). We performed quite a few of experiments using palmitoyl-carnitine, also in the range of concentrations from 1 – 40 ?M. We usually see a gradual increase in State 3 respiration with concentration increments (1, 2.5, 5, 10, 15, 40 ?M). However, the respiration is always lower then when pyruvate is used (although in much higher concentration, from 2 mM up). All of the observations come from the human left ventricular tissue, that we almost exclusively use in past couple of years for our experiments on mitochondria (we also measured activity of some enzymes and protein expression). Palmitoylcarnitine - - - -
Wohlwend Martin Fatty acid oxidation in permeabilized muscle fibers has worked well for us in both human and rat studies. Typically we use Octanoylcarnitine and Malate for skeletal muscle and a combination of Octanoylcarnitine plus Palmitylcarnitine and Malate for human heart tissue. These protocols have given us a good insight into Beta oxidation. Octanoylcarnitine + Malate, Octanoylcarnitine + Palmitoylcarnitine + Malate (for heart) - - - human and rat
Darveau Charles I have done conducted one study examining bumblebee flight muscle permeabilized fibers and used. Palmitylcarnitine - - - -
Ojuka Edward We have used Octanoylcarnitine + malate as fatty acid substrates (at the concentration stipulated in BIOBLAST) with much success. However, Palmitylcarnitine + malate was not responsive to ADP or CCCP. Octanoylcarnitine + Malate - - - -
Senyilmaz Deniz - - palmitoyl-CoA and I include carnitine in the buffer - inverted Drosophila larvae which are permeabilized by digitonin. Their body walls are more or less made of muscles -
Smith Cody D I have extensively tested FAO in PmFBs using the Palmitoyl-Carnitine, Palmitoyl-CoA, and L-Carnitine. For maximal respiration we make sequential additions of 18uM PC, 20uM PCoA, and 5mM Carnitine. Addition of ADP induces a peak rate of O2 consumption that is not increased with FCCP addition. Palmitylcarnitine Palmitoyl-CoA, and L-Carnitine - - -
Schauer Irene We are successfully measuring FA oxidation in permeabilized fibers. We have switched to Octanoyl carnitine (A) because we were seeing evidence of toxicity with palmitoyl carnitine. This evidence was that we were seeing final uncoupled respiration to be lower than state 3 respiration in samples with PC. This issue resolved when we switched to OC. Octanoylcarnitine - - - -
Van Schaardenburgh Michel in my studies on patients with intermittent claudication we used Octanoylcarnitine in the SUIT protocol described by Pesta and you. Octanoylcarnitine - - - -
Lesnefsky Edward To assess role of CPT-1; one can use palmitoyl-CoA + carnitine. We use the method pioneered by Dr. Chuck Hoppel in Cleveland. We use palmitoylcarnitine. To assess role of CPT-1; one can use palmitoyl-CoA + carnitine. Palmitoylcarnitine - - - -
Goodpaster Bret Excellent. Lower overall O2 consumption compared to carbohydrate-supported respiration Palmitoylcarnitine - - - -
Egelandsdal Bjorg We have examined for how long time post mortem the fatty acid oxidation prevails in beef muscles Octanoylcarnitine - - - beef muscles
Votion Dominique Very limited (only preliminary tests on mice) but satisfying. I have planned new experiments on equine muscle tissue in 2016 Octanoylcarnitine, Palmitoylcarnitine Octanoate, Acetylcarnitine , Palmitoyl CoA (+ carnitine) - - -
Kane Daniel Palmitoylcarnitine - - - - -
Ciapaite Jolita Palmitoylcarnitine + malate (25 µM + 2.5 mM) palmitoyl-CoA + L-carnitine + malate (25 µM + 2 mM + 2.5 mM) - - - -
Chou Cheng-Hsien Palmitoylcarnitine - - - - permeabilized platelet & lymphocyte


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

  1. »Fatty acid oxidation#FAO and HRR
  2. 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)