Immediately after blood withdrawal, plasma was separated and stored at -70C. Fatty acid composition
was determined as described previously [11]. Briefly, plasma samples (200 ml) were mixed with 300 ml portions
of 0.67M phosphate buffer and with 5 mg of C17:0 (internal standard). Two milliliter portions of chloroform: methanol, 3:1 (v/v) were added and lipids were extracted by vortexing for 1 min at full speed. After centrifugation (2000g, 10 min), the bottom organic layer was removed and evaporated to dryness under nitrogen. The residue was mixed with 300 ml of 0.5M KOH in 95% ethanol and samples were hydrolysed by 30 min incubation at 851C. After cooling samples to room
temperature free fatty acids were precipitated by addition of 1.5 ml of 0.12M HCl. Free fatty acids were then extracted with 1ml of isooctane and the organic layer was separated by centrifugation (10 min, 2000g).
Fatty acid methyl esters (FAME) were prepared by incubation of dried extracts for 30 min in room
temperature with 0.5 ml of methanol, 0.5 ml of 2,2-dimethoxypropane, 50 ml of water and 20 ml of 12M
HCl. Samples were evaporated to near dryness and vortexed for 1 min with 2.0 ml of water and 1.0 ml
isooctane. After centrifugation (10 min at 2000g), the isooctane layer containing FAME were collected and
750 ml portions were concentrated and used for analysis.