Cp7420 100 m column

The FA profile in erythrocyte membrane was measured at the Department of Laboratory Medicine and Pathology, University of Minnesota, using the method by Cao et al [29 (link)]. Previous studies have shown that long-term storage of frozen blood samples did not influence FA profiles.[30 (link), 31 (link)] After thawing and adding 50 uL of 17:0 internal standard, FAs were extracted from erythrocyte membranes with a mixture of chloroform and methanol (2:1, v/v), dissolved in heptane, and injected onto a capillary Varian CP7420 100-m column with a Hewlett Packard 5890 gas chromatograph (GC) equipped with a HP6890A autosampler. The GC was configured for a single capillary column with a flame ionization detector and interfaced with HP chemstation software. Adequate separation of FA methylesters was obtained over a 50-min period with an initial temperature of 190°C followed by subsequent temperature gradually increased to 240°C. FAs from 12:0 through 24:1ω9 were separated, identified and expressed as percent of total FA. FA subtypes, including ω3 FA, ω6 FA, and trans FA, were calculated as sum of the respective individual FA. The ratio of ω6 to ω3 FA was calculated. The coefficients of variation on 51 blind triplicates from 17 individual samples were 5.1% for ω3 FA, 3.0% for ω6 FA, and 3.6% for trans FA.

Plasma phospholipid fatty acids were measured using a method described previously [14 (link),16 (link)]. In summary, lipids were extracted from plasma with a mixture of chloroform:methanol (2:1, v/v). Phospholipid subclasses were then separated from cholesterol and triglycerides on a silica thin-layer chromatography plate. Fatty acid methyl esters products were dissolved in heptane and injected onto a capillary Varian CP7420 100-m column with a Hewlett Packard 5890 gas chromatograph (GC) equipped with a HP6890A autosampler. Cases and matching controls were sent to the laboratory in the same batch and assayed concurrently. Lastly, laboratory personnel were blinded on the case-control status of each subject to enhance validity.

Blood samples from each participant were collected, stored frozen at −70°C, and analyzed at the same time to eliminate interassay variability. IL-6, IL-2-soluble receptor α, tumor necrosis factor (TNF) α, and monocyte chemoattractant protein 1 (MCP-1) were measured using quantitative sandwich enzyme immunoassay techniques (ELISA kit assays, R&D System Inc., Minneapolis, MN, USA) as described previously [18 (link)]. High-sensitivity C-reactive protein was measured using a latex particle enhanced immunoturbidimetric assay (Kamiya Biomedical Company, Seattle, WA, USA) as described previously [19 (link)]. Plasma adiponectin was measured using competitive RIA (Linco Research, St Charles, MO, USA) as described previously [20 (link)]. Fatty acids in the erythrocyte membranes were measured by a capillary Varian CP7420 100 m column with a Hewlett Packard 5890 gas chromatograph equipped with a HP6890A autosampler [21 (link)]. The measurements of fatty acids were reliable and have been validated against a diet history questionnaire [22 (link),23 (link)].