Measurement of fatty acid oxidation

Gastrocnemius muscles from 12-month-old mice were finely chopped in 2 ml of Krebs-Henseleit bicarbonate medium (pH 7.4) and then incubated in Erlenmeyer flasks containing a final volume of 25 ml of the same medium. Flasks were maintained at 37°C in an oxygenated atmosphere (O₂/CO₂, 95/5). For fatty acid oxidation determination, 1 pCi of ¹⁴C-linoleate was added to the medium and the flask was hermetically sealed with rubber caps with centrally inserted plastic wells. After 20 to 30 min of incubation, 0.4 ml of 40% (w/v) HClO₄ was added through the rubber cap, and 0.4 ml of 1 M solution of hyamine hydroxide was deposed in the center of the well. The flasks were then shaked at room temperature for 1 hour. The radioactivity present in the center wells (corresponding to the formed CO₂) was determined and the flask content was harvested and centrifuged at 700g for 15 min. The resulting supernatant was neutralized with KOH (5 M), and total ¹⁴C-labeled acid-soluble products were counted. Total fatty acid oxidation corresponded to the sum of the catabolism of labeled linoleate into CO₂ and acid-soluble products.