Inertsil ods 4

An 8-mL sample of whole blood was collected from each participant before noon and placed into BD Vacutainer® CPT™ cell preparation tubes containing sodium heparin (Becton, Dickinson Co., Plainfield, NJ, USA). Plasma samples were isolated from the whole blood samples according to the tube manufacturer's instructions and subsequently stored at −80 °C. The concentrations of glutamine, glutamate, and glycine were measured using an H-class high-performance liquid chromatography (HPLC) system (Waters Corporation) equipped with a quaternary pump, autosampler with the cooler set at <10 °C, column oven, and fluorescence detector. The analytical HPLC column used was Inertsil ODS-4 (2 μm, 2.1 × 100 mm, G.L. Sciences Inc.) with a guard column packed with Inertsil ODS-4 material (2 μm, 2.1 × 10 mm, G.L. Sciences Inc.), maintained at 40 °C. The plasma levels of l-serine and d-serine were also measured using the same HPLC system, except the analytical HPLC column used was Sumichiral OA2500S (5 μm, 4.6 × 250 mm, Sumika Analytical Center), maintained at 40 °C. Amino acids were labeled with 4-fluoro-7-nitro-benzoxazole, while homocysteine was labeled with 4-fluoro-7-sulfamoylbenzofurazan; they were measured according to the protocol presented in a previous study [18 (link)]. All chemicals for HPLC analyses were purchased from Sigma-Aldrich Japan (Tokyo, Japan), unless otherwise indicated.

The fluorescence intensity of SOSG (Thermo Fisher Scientific, Waltham, MA, USA) oxidized by ¹O₂ was determined from the fluorescence of the HPLC peaks. The photocatalyst (2 µM) and SOSG (10 µM) were added to 50% CH₃CN solution in 10 mM MES buffer (pH 7.4) in a 1.5 mL tube. The solution was irradiated with blue light (RELYON, Twin LED light, 455 nm) for 30 s on ice. After irradiation, the solution was diluted 2.6-fold with 0.1% aqueous formic acid and analyzed using HPLC. Analytical HPLC was carried out on a JASCO PU-4580 HPLC Pump, JASCO LG-4580 Quaternary Gradient Unit (Tokyo, Japan), and JASCO DG-4580 Degassing Unit with a JASCO MD-2018 Plus Photodiode Array Detector, JASCO CO-4060 Column Oven, JASCO As-455 HPLC Autosampler, and JASCO LC-NetII/ADC Interface Box using a C18 reverse phase column (Inertsil ODS-4, 150 × 4.6 mm, 5 μm (GL Science, Inc., Tokyo, Japan)). The HPLC conditions were as follows: mobile phase A, 0.1% formic acid in H₂O, mobile phase B, 0.1% formic acid in CH₃CN. 0−5 min, 5% B; 5−27 min, 5−100% B; 27−32 min, 100% B. The fluorescence of the separated peaks was detected using HPLC (Ex 504 nm/Em 525 nm).