Measurement of Fatty Acids

Erythrocyte fatty acid concentrations were identified using gas-liquid chromatography. Samples were analyzed at the University of Pittsburgh's Heinz Laboratory Total lipids (500pl of packed red blood cells) were extracted according to the general technique of Bligh and Dyer. Briefly, the samples were homogenized in 4 ml of methanol, 2 ml of chloroform and 1.1 ml of water. Two ml of chloroform and 2 ml of water were added to the samples after 15 min. The tubes were then centrifuged at 1200 g for 30 min at 16°C and the upper phase discarded. The lower phase was dried under nitrogen and resuspended in 1.5 ml 14% boron trifluoride methanol. The samples were heated at 90°C for 40 min and after cooling extracted with 4.0 ml pentane and 1.5 ml water. The mixtures were vonexed and the organic phase recovered. The extracts were dried under nitrogen, resuspended in 50 pi heptane and 2 ml injected into a capillary column (SP-2380, 105 m x 53 mm ID. 0.20 um film thickness). Gas chromatographic analyses were carried out on a Perkin Elmer Clarus 500 equipped with a (lame ionization detector Operating conditions were as follows the oven temperatures were 140C for 35 min; 8°C/min to 220°C, held for 12 min; injector and detector temperatures were both at 260°C; and helium, the carrier gas, was at 15 psi. Identification of fatty acids was by comparison of retention times with those of authentic standards (Sigma). A random subset of 27 samples was analyzed for reproducibility; laboratory personnel were blinded to duplicate samples and subject identification. The inter-assay coefficients of variation (CV) for the fatty acid measures reported ranged between 1.7-15.2%. CVs's were 4.6% for LA, 3.4% for AA, and 1.7% for total n-6 fatty acids. CV's were higher for the n-3 fatty acids, with CV's of 15.2% for ALA. 5.3% for EPA, 7.5% for DHA and 5.3% for total n-3 fatty acids. The CVs for the total n-6:n-3, LAALA. AA:EPA, and AAEPA+DIIA ratios were 5.2%, 111%, -4.5% and 5.7% respectively. The individual and total n-6 and n-3 fatty acids are expressed as a percentage by weight of the total erythrocyte fatty acid content.