FAO capacity in type I–dominant red muscle was measured as previously reported (52 (link)) with slight modifications. In brief, isolated soleus muscle was homogenized in distilled deionized water containing 250 mM sucrose, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.4. Then, homogenates were incubated with [14C]-palmitic acid (0.2 μCi/mL; PerkinElmer) in 1 mL of incubation buffer (100 mM sucrose, 10 mM Tris-HCl, pH 7.4, 5 mM KH2PO4, 80 mM KCl, 1 mM MgCl2, 2 mM l -carnitine, 0.1 mM malate, 2 mM ATP, 0.05 mM coenzyme A, 1 mM dithiothreitol, 0.2 mM EDTA, and 0.3% fatty acid–free BSA dissolved in distilled deionized water) at 37°C for 2 hours. After stopping the oxidation by addition of 0.1 mL of 2 M HCl, filter paper soaked in 2 M NaOH was briefly placed into the upper section of the homogenates in the same tube, and the 14CO2 in the filter paper, a final product of oxidated [14C]-palmitic acid in muscle homogenates, was counted using a liquid scintillation counter.
14c palmitic acid
Manufactured by PerkinElmer
Sourced in United States
14C-palmitic acid is a radioactive labeling compound used in research applications. It consists of the fatty acid palmitic acid, with a radioactive carbon-14 atom incorporated into its structure. This compound is typically used as a tracer to study lipid metabolism and other biochemical processes involving fatty acids.
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5 protocols using 14c palmitic acid
1
Fatty Acid Oxidation Capacity Assay
2
Quantifying BAT β-Oxidation Activity
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The β-oxidation ability of BAT homogenates was examined as previously described [35] (link). Twenty milligrams of BAT were manually homogenized using a Potter-Elvehjem homogenizer in 400 µl of ice-cold buffer A (100 mM KCl, 40 mM Tris-HCl, 10 mM Tris base, 5 mM MgCl2-6H2O, 1 mM EDTA, and 1 mM ATP [pH 7.4]). The lysate was centrifuged at 420×g and 4°C for 5 min, and 40 µl of the supernatant was added to 160 µl of the reaction mixture (100 mM sucrose, 10 mM Tris-HCl, 5 mM KH2PO4, 1 mM MgCl2, 2 mM L-carnitine, 0.1 mM malate, 2 mM ATP, 0.05 mM coenzyme A, 1 mM dithiothreitol, 0.2 mM EDTA, and 0.2 µCi 14C-palmitic acid [Perkin Elmer, CT, USA] complexed to fatty-acid-free albumin at a 3∶1 molar ratio [pH 7.4]). The reactions were performed in an Eppendorf tube and were placed in an incubator at 37°C for 60 min. The mixture was transferred to a new Eppendorf tube containing 200 µl of 6 N HCl. Filter paper containing 20 µl of ethanolamine was previously placed in the tube cap. The sample was further incubated at room temperature for 1 h to collect the 14CO2 trapped in the filter paper. The radioactivity in 14CO2 was quantified using liquid scintillation counting. The value was normalized with the lysate protein concentration.
3
Lipid Metabolism Assays in Cells
4
Lipid Extraction and Analysis Protocol
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Butanol, heptane, ethyl acetate, MTBE, methanol, chloroform and acetonitrile were all of HPLC grade and attained from Rathburn Chemicals Ltd (Walkburn, UK). Isopropanol was from Acros Organics (Pittsburgh, PA, USA) and acetic acid was from Merck (Darmstadt, Germany). Non-radiolabelled lipid standards were from Avanti lipids (Alabaster, AL, USA) with the exception of d6-triglyceride (TG, tripalmitate) and d5-cholesteryl ester (CE, oleate), which were from CDN isotopes (Quebec, Canada). Radiolabeled 14C phosphatidylcholine (PC, 1-palmitate, 2-linoleate), 14C lysophosphatidylcholine (LPC, 1-palmitate) and 14C CE (oleate) were from Amersham Biosciences (Little Chalfont Bucks, UK). Radiolabeled 14C TG (tripalmitate) and 14C diglyceride (DG, dioleate) were from American Radiolabeled Chemicals Inc (St Louis, MO, USA) while the 14C free (unesterified) cholesterol (FC), 3H sphingomyelin (SM, palmitate), and 14C palmitic acid were from Perkin-Elmer life science (Boston, MA, USA). Standard serum Seronorm Lipid (freeze-dried bovine serum) was from SERO AS (Billingstad, Norway).
5
Fatty Acid Oxidation Assay in Brown Adipocytes
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Fatty acid oxidation was determined by the sum of 14CO2 liberated to the media and 14C-acid soluble metabolites (ASM) as previously described [34 (link)]. For that purpose, fully differentiated mature brown adipocytes were treated with vehicle or MaR1 (0.1–1 nM; 24 h). Briefly, differentiated brown adipocytes were incubated for 4 h in Krebs–Ringer buffer without glucose, containing 125 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.25 mM KH2PO4, 1.25 mM MgSO4 ⋅7H2O, 25 mM NaHCO3 and 3% fatty acid-free bovine serum albumin (BSA) pH 7.8, 2 mM l -carnitine, 80 μM palmitic acid (Sigma), and 20 μM 14C-palmitic acid (58 μCi/μmol, Perkin Elmer; Waltham, MA). The medium was transferred to a glass vial with a central well containing benzethonium hydroxide (Sigma).
14CO2 was liberated by acidification with 1 M H2SO4 and collected during 2 h in the central well. 14CO2 was measured by scintillation counting in a scintillation counter HIDEX 300 SL (Hidex Oy, Turku, Finland). Cells were washed and then scraped in cold buffer (0.25 M sucrose; 10 mM Tris–HCl; 1 mM EDTA; 1 mM dithiothreitol, pH 7.4). Neutral lipids and ASM were separated by adding 5 vol chloroform/methanol (2:1) and 0.4 vol 1 M KCl/HCl. Specific activity was measured and used to calculate total oxidation as equivalent of oxidized palmitic acid. Results were normalized to total protein content of cell extracts.
14CO2 was liberated by acidification with 1 M H2SO4 and collected during 2 h in the central well. 14CO2 was measured by scintillation counting in a scintillation counter HIDEX 300 SL (Hidex Oy, Turku, Finland). Cells were washed and then scraped in cold buffer (0.25 M sucrose; 10 mM Tris–HCl; 1 mM EDTA; 1 mM dithiothreitol, pH 7.4). Neutral lipids and ASM were separated by adding 5 vol chloroform/methanol (2:1) and 0.4 vol 1 M KCl/HCl. Specific activity was measured and used to calculate total oxidation as equivalent of oxidized palmitic acid. Results were normalized to total protein content of cell extracts.
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