Mitochondrial and Peroxisomal Fatty Acid Oxidation

Fresh kidney homogenates (~5 mg) were incubated in 3 mL of reverse Krebs–Henseleit bicarbonate medium with or without rotenone and antimycin A (10 + 50 μmol/L), blockers of mitochondrial respiratory system. Mitochondrial and peroxisomal fatty acid oxidations were measured in the medium using either [1-¹⁴C]-labeled oleic acid (C18:1) or erucic acid (C22:1) purchased from American Radiolabeled Chemicals (ARC; Saint Louis, MO, USA) as substrate. The biochemical and radio-chemical purities of both C18:1 and C22:1 were greater than 99% based on TLC and HPLC analyses. The fatty acids were bound to fatty acid-free BSA (5:1, molar ratio) and dissolved in the reaction medium. The measurements were performed in 25 mL Erlenmeyer flasks containing 2 mL of the reaction medium. The medium was incubated with 2 µmol [1-¹⁴C]-C18:1 (0.98 kBq/µmol) or [1-¹⁴C]-C22:1 (1.37 kBq/µmol). The incubation was stopped after 30 min by the addition of 0.5 mL of 35% HClO₄. The ¹⁴C accumulation in CO₂ and acid-soluble products (ASP) were collected, processed, and analyzed by liquid scintillation spectrometry (Beckman LS 6000IC, Fullerton, CA, USA) according to the procedures by Lin et al. [24 (link)].