Total circulating (cohort 1: serum, cohort 2: plasma) fatty acids were assessed as fatty acid methyl esters (FAME) analyzed by gas chromatography with flame ionization detection (GC-FID) using a Hewlett Packard 5890 instrument system with an Agilent J&W DB-23 column (30 m, 0.25 mm ID, 0.25 μm film) fitted with an inert pre-column (1 m, 0.53 mm ID) for cool on-column injection as previously described [27 (link)]. Independent experiments from our lab and others have shown that the fatty acid profiles of serum and plasma from anti-coagulated blood (heparin or EDTA) are comparable [28 (link), 29 (link)]. Fatty acids were cleaved from complex lipids and converted to methyl esters in duplicate samples (100 μl) utilizing a modification of the protocol developed by Metcalfe et al. [30 (link)] and Sergeant et al. [27 (link)]. Fatty acids in samples were identified based on retention times of commercially available standards. Triheptadecanoin (100 μg; NuChek Prep, Elysian, MN) was used as an internal standard. Fatty acid peaks (23–29 peaks) were identified and accounted for > 99% of the total fatty acids in the sample. Fatty acid data are presented as the mass percent of total fatty acids from serum (cohort 1) or plasma (cohort 2).
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