Chemstation a 08

Blood specimens were collected after an overnight fast at baseline and three-month follow-up. Complete blood count and high sensitivity CRP concentration were both assessed at Laboratory Corporation of America (Raritan, NJ), using a Sysmex X-series analyzer (Sysmex Corporation, Kobe, Japan) and a particle enhanced turbidimetric assay on a COBAS INTEGRA system (Roche Diagnostics, Mannheim, Germany), respectively. RBC FAs were quantified in the Department of Nutrition at Harvard School of Public Health (Boston, MA), using gas–liquid chromatography on a fused silica capillary cis–trans column SP2560 (Supelco, Bellefonte, PA) with peak retentions analyzed in Agilent Technologies ChemStation A.08.03 software. Individual RBC FA was reported as a proportion of total phospholipid FAs. Serum IL-6 concentration was measured at the Harvard Catalyst Central Laboratory (Boston, MA) using access chemiluminescent immunoassay (Beckman Coulter, Fullerton, CA). The intra-assay coefficients of variation for CRP, IL-6, palmitic, and stearic acids were 1.3%, 1.7–4.6%, 5%, and 19%, respectively.

Fatty acid levels in RBC membranes were analyzed by gas-liquid chromatography using analytical procedures described previously [39 (link),40 (link)]. Briefly, fatty acids were converted to methyl esters and separated by gas chromatography. Individual fatty acids were identified by comparing their peak retention times to known certified standards (NuCheck Prep, Elysium, MN) using CHEMSTATION A. 0.8.03 software (Agilent Technologies, Santa Clara, CA) [39 (link)]. We expressed the level of each fatty acid as the percentage of the total fatty acids. We assayed case and control RBC sample pairs in the same analytic run and measured coefficients of variation (CVs) by analyzing blinded quality control samples that we embedded throughout the study blood samples. Within-batch CVs ranged from 1.5% for cis 20:4n-6 to 9.9% for cis 22:2n-6. Between-batch CVs ranged from 2.1 % cis 20:3n-6 to 10.1% for cis 22:4n-6 (Supplementary Table 1).