Δ-6 desaturase activity assay

The liver was homogenized in a phosphate buffer containing 0.25 M sucrose to recover the post-mitochondrial supernatant, as previously described (36).

Enzymatic activity was determined using a 1-mL assay mixture containing 100 μL of supernatant (100 μg proteins), 150 mmol/L phosphate buffer (pH 7.16), 6 mmol/L NADH, 6 mmol/L MgCl₂, 7.2 mmol/L ATP and 0.54 mmol/L CoA. The incubation was carried out in duplicate at 37°C for 20 min, after addition of 60 nmol of [1-¹⁴C]-ALA (60 μmol/L, 10 mCi/mmol, ARC, Saint Louis, MO, USA). The reactions were stopped by adding 1 mL of 2 M KOH in ethanol. After 30 min at 70°C, the FA were liberated by acidification, extracted with diethyl ether, converted to FA naphthacyl esters and separated on HPLC (Alliance 2695 integrated system, Waters, Saint-Quentin-en-Yvelines, France) as previously described (44). Peaks corresponding to radiolabeled FA substrate and product of each desaturase assay were collected and subjected to liquid scintillation counting (PerkinElmer 2810, Waltham, MA, USA). The enzyme activity was determined and expressed as pmol of substrate converted to product per min per mg of protein. Protein in the supernatant used for the desaturase assays was determined by Lowry method (45).