Plasma choline level
Plasma free and fat-soluble choline levels were analyzed, respectively. 100 μL of plasma was mixed with 900 μL of methanol. Following sonication for 10 minutes, the samples were centrifuged at 15,000 rpm for 10 min. at 4°C, and the supernatant was aliquoted. Filter filtration was performed immediately before the analysis, and plasma free choline levels was measured by LC/MS/MS quantitative analysis (Waters, QTRAP4500, 40 °C, A: acetonitrile /B:5 mM ammonium acetate [pH 4.0]), and fat-soluble choline levels were also measured by LC/MS/MS quantitative analysis (BRUKER, micrOTOF-QII, 30°C, A: acetonitrile /B:5 mM ammonium acetate [pH4.0]). The analyses were conducted by the specialist in charge of the analyses of Kewpie Corporation (Tokyo, Japan).