Quantifying Vitamin E Levels Across Tissues

Each VE concentration was measured by high performance liquid chromatography (HPLC) with electrochemical detection (ECD), as described previously with some modifications [18 (link)]. Brain, serum, liver, and each diet were mixed with 6% pyrogallol (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) and 35% KOH solution (FUJIFILM Wako Pure Chemical Corp.), respectively. After mixing, a 2,2,5,7,8-Pentamethyl 6-chromanol (PMC) (Shigma Aldrich Corp., St Louis, MO, USA) solution was added as an internal standard, and then all samples were heated at 100 °C. After heating, all samples were cooled until room temperature (R/T). Then, a 1% NaCl solution and a mixed solution for VE extraction were added, and these mixtures were shaken. After centrifugation (3000 rpm and 4 °C), the upper layer was collected and evaporated. The residue was dissolved by methanol, and VE concentrations in this solution were measured using HPLC (Shiseido Co., Ltd., Tokyo, Japan).