Glc 110 mixture

The total lipids were quantified following the method of Folch et al. (60 (link)), modified by using dichloromethane:methanol (2:1) instead of trichloromethane:methanol (2:1). The fatty acids were transesterified to fatty acid methyl esters by acid-catalyzed methylation (61 (link)). Non-adecanoic acid (C19:0, Matreya LLC, State College, PA, USA) was added as an internal standard. Fatty acid methyl esters were analyzed by gas chromatography, using a Shimadzu GC-2010 Plus gas chromatograph (Shimadzu Europe GmbH, Duisburg, Germany) equipped with a capillary column (Omegawax 250, 30 m × 0.25 mm × 0.25 μm; Supelco, Bellefonte, PA, USA) and a flame-ionization detector. The carrier gas was helium at 1.30 ml min⁻¹, with a split ratio of 1:100, and the injection volume was 1.0 μl. The initial column temperature of 150°C was held for 7 min, increased at 3°C min⁻¹ to 170°C and held for 25 min, and then increased at 3°C min⁻¹ to 220°C and held for 30 min. The injector and detector temperatures were 250 and 260°C, respectively. Fatty acids were identified by comparing retention times with those of commercially available standards (Supelco 37 Component FAME Mix, BAME Mix, PUFA No.1, PUFA No.2, PUFA No.3, Sigma-Aldrich Co. LLC; GLC-110 Mixture, Matreya LLC) and quantified by using the internal standard (C19:0). Analyses were run in duplicate.

RBCs were separated from plasma by centrifugation at 3,000 rpm for 15 min at 4°C and fatty acids transesterified as previously described by Harris, Pottala (46 (link)). The resulting fatty acid methyl esters were analyzed by gas chromatography using a Shimadzu GC-2010 Plus (Shimadzu Corporation, Kyoto, Japan) equipped with a capillary column (SP-2560, 100 m × 0.25 mm × 0.20 μm; Supelco, Bellefonte, PA, United States of America) and a flame-ionization detector. The oven’s initial temperature of 50°C was held for 1 min, then increased at a rate of 50°C/min to 150°C and held for 20 min, then increased at 1°C/min to 190°C and held for 3 min, and further increased at 1°C/min to 220°C and held for 20 min. Injector and detector temperatures were set at 250 and 280°C, respectively. Hydrogen was the carrier gas at a linear velocity of 17.2 cm/s. Fatty acids were identified by comparing retention times to standards (Supelco 37 Component FAME Mix, BAME Mix, PUFA No.1, PUFA No.2, PUFA No.3, Sigma-Aldrich, St. Louis, MO, United States of America; GLC-110 Mixture, Matreya, Pleasant Gap, PA, United States of America). The omega-3 index was calculated as the sum of EPA and DHA expressed as a proportion of total identified fatty acids.

Microalgal fatty acid methyl esters were prepared by acid-catalyzed transesterification with methanolic HCl [20 (link)] and analyzed by gas chromatography as reported by Maia, et al. [21 (link)], using a Shimadzu GC-2010 Plus (Shimadzu Corporation, Kyoto, Japan) equipped with a capillary column (Omegawax 250, 30 m × 0.25 mm × 0.25 μm; Supelco, Bellefonte, PA, USA), and a flame-ionization detector. Fatty acids were identified by comparing retention times to standards (Supelco 37 Component FAME Mix, BAME Mix, PUFA No.1, PUFA No.2, PUFA No.3, Sigma-Aldrich, St. Louis, MO, USA; GLC-110 Mixture, Matreya, Pleasant Gap, PA, USA), and quantified with the internal standard (C19:0, nonadecanoic acid; Matreya). Analyses were carried out in duplicate.