GC-MS Analysis of Metabolites

These measurements were performed with reference to the previously reported methods [43 (link),44 (link),45 (link)]. GC-MS analysis was performed using a GC-MS QP2010 Ultra (Shimadzu, Kyoto, Japan) with a fused silica capillary column (BPX-5; 30 m × 0.25 mm inner diameter, film thickness: 0.25 μm; Shimadzu), a front inlet temperature of 250 °C, and a helium gas flow rate through the column of 39.0 cm/s. The column temperature was maintained at 60 °C for 2 min, then raised by increments of 15 °C/min to 330 °C, and maintained at that temperature for 3 min. The interface and ion source temperatures were 280 °C and 200 °C, respectively. All data obtained by GC-MS analysis were analyzed using MetaboAnalyst software (v. 5.0; Reifycs, Inc., Tokyo, Japan). The retention times indicated in the Smart Metabolites Database (Shimadzu) were used as references to create a library for data analysis. To perform a semi-quantitative assessment, the peak area of each quantified compound was calculated and normalized using the 2-isopropylmalate peak area.