Analysis of fatty acids composition in erythrocyte

In this study, fatty acids of RBCs were extracted and methylated using a one-step procedure according to Raquel et al. with modification.[9] Briefly, 150 μL of RBCs sample was added to a screw-cap glass vial containing 25 μg of internal standard heptadecanoic acid (C17:0) and 100 μg of BHT and mixed with 1 ml of methanolic NaOH solution (1N). Saponification was performed at 85°C for 10 min. Transmethylation was then performed by adding 2 ml H₂SO₄ (1N) in methanol, flushing with N₂ and heating at 85°C for 40 min. After cooling, 0.5 ml of saturated solution of NaCl and 1 ml of hexane were added and tubes were vigorously shaken for 1 min. The upper hexane layer containing fatty acid methyl esters (FAMEs) was collected, washed with 1 ml of HPLC grade water and dried with 1 g sodium sulfate anhydrous (Na₂SO₄). The volume of the extract was then reduced to about 100 μl under N2 gas and samples were transferred to GC vials for further analysis.

FAMEs were analyzed by GC-MS (HP6890, Agilent technologies) equipped with a SP-2560 column (100 m × 0.25 mm ID × 0.2 μm film thickness) (Supelco, Bellefonte, PA, USA) and Detector (MSD HP 6890) as described elsewhere.[10]

To assess the reproducibility of the erythrocytes fatty acid analysis, the intra-assay coefficient of variation (CVs) was determined by the analysis of 5 aliquots of a pooled blood sample in the same day and was found to be <5%. The interassay reproducibility was assessed by analysis of 10 aliquots of the same sample on different days spread over 2 months. The interassay imprecision (CV) for all the fatty acids measurement was <6%, except for EPA which had a CV of 9.3%.