Db 23 123 2332

RBC and PFC total fatty acid composition were determined with an Agilent 9820A equipped with an auto-injector (Agilent Technologies, Santa Clara, CA). The column was a DB-23 (123-2332): 30 m (length), I.D. 0.32 mm wide bore, film thickness of 0.25 μM (J&W Scientific, Folsom CA). Fatty acid identification was determined using retention times of authenticated fatty acid methyl ester standards (Matreya LLC Inc., Pleasant Gap PA). Analysis of fatty acid methyl esters is based on areas calculated with OpenLab CDS ChemStation software. Fatty acid composition is expressed as weight percent of total fatty acids (mg fatty acid/100 mg fatty acids). Primary measures of interest were DHA, AA, and the AA/DHA ratio. Samples were processed by a technician blinded to diet assignment.

Whole venous blood (4 ml) was collected into EDTA-coated BD Vacutainer tubes, and centrifuged at 4°C for 20 min (1,500 xg). Plasma and buffy coat were removed and erythrocytes washed 3 times with sterile 0.9% NaCl and stored at −80°C. Total erythrocyte membrane fatty acid composition was determined with a Shimadzu GC-2010 equipped with an auto-injector (Shimadzu Scientific Instruments Inc., Columbia MD), using the direct saponification method described previously (McNamara et al., 2010 (link), 2014 ). The column was a DB-23 (123–2332): 30m (length), I.D. (mm) 0.32 wide bore, film thickness of 0.25 μM (J&W Scientific, Folsom CA). The carrier gas was helium with a column flow rate of 2.5 ml/min. Fatty acid identification was determined using retention times of authenticated fatty acid methyl ester standards (Matreya LLC Inc., Pleasant Gap PA). Analysis of fatty acid methyl esters was based on areas calculated with Shimadzu Class VP 4.3 software. The lower limit of detection with a threshold area of 500 and a 1 μl injection volume was approximately 200 ng of an individual fatty acid. Data are expressed as weight percent of total fatty acids (mg fatty acid/100 mg fatty acids) which we have previously found in pilot studies to be highly correlated with fatty acid total mass (μmol/g). Samples were processed by a technician blinded to group assignment.