Fatty Acid Extraction and Methylation

We used the modified method of Moilanen [31 (link)], that is itself a modification of the method described by Folch [32 (link),33 (link)]. Fatty acids extraction and preparation of fatty acid methyl esters(FAME) from red blood cells (RBC) and tissue samples were carried out as previously described [16 (link),34 (link)]. Briefly, total lipids from phospholipids of RBC membranes were extracted by adding 0.9 mL of an acidified salt solution (H₂SO₄ 2 × 10⁻⁴ M, NaCl 0.1%). For fatty acids extraction from tissues, about 20 mg wet tissues were homogenized with 0.8 mL of ice cold 0.9% NaCl. All samples received 5.0 mL of chloroform:methanol (2:1, v/v) (Sigma-Aldrich, Milan, Italy) and the samples were mixed thoroughly and centrifuged at 1000× g for 10 min. The lower layer, containing fatty acids, was removed with care, replaced in a new tube and dried by a centrifugal evaporator (Bio-Rad, Milan, Italy). The FAME were obtained by adding toluene and BF₃·MeOH 14% (Sigma-Aldrich, Milan, Italy) and incubating for 2 h at 80 °C. After the addition of toluene and 5% aqueous sodium chloride solution, the samples were centrifuged at 470× g for 10 min. Fatty acid methyl esters contained in the upper layer of the tubes, were collected and transferred into a vial and analyzed.