Fecal Long-Chain Fatty Acid Profile Analysis

To determine the LCFA profile in feces, we used 40 mg of previously lyophilized feces during 48 h (Labconco, freeze dryer 4.5). Forty microliter of C19 were added as extraction standard for homogenization with methanol:chloroform (1:2 V/V) by brief sonication in ice (Labsonic U, B. Braun). Homogenates were then filtered with Whatman filters n°1 (10 μm of porosity). Filters were rinsed with 2 ml of chloroform and 1 ml of methanol. Homogenates were purified with KCl 0.88% and KCl 0.88%: methanol (1:1 V/V). After centrifugation (1500 g, 5 min), the chloroform phase was collected in new tubes and evaporated under nitrogen flux until samples were completely dry. The esterified fatty acids were then subjected to alkaline hydrolysis (saponification) and free fatty acids were methylated and quantified by gas chromatography with flame ionization detector as previously described^{50 (link)}.