*2.5. Measurement of Elaidic Acid Incorporated into HDL Phospholipids*

One hundred microliters of 50 μM 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (19:0 PC; Merck KGaA, Darmstadt, Germany) were added to 200 μL of apoB-depleted serum as an internal standard, and total lipids were extracted using the Bligh and Dyer method as described previously [22] and applied to InertSep SI columns (GL Sciences Inc., Tokyo, Japan). The columns were then washed with 3 mL of chloroform and 3 mL of acetone. PL were eluted from the columns using 6 mL of methanol, dried under N2, and methylated with a commercially available kit (Nacalai Tesque, Kyoto, Japan) according to the manufacturer's protocol. The concentrations of methylated elaidic acid were measured using gas chromatography-mass spectrometry (GC-MS). The GC-MS conditions used for the measurements in this study were described in a previous study [13], except that the split-less injection mode was adopted to increase the sensitivity, and each value was standardized using pooled serum.
