Serum Metabolomics Analysis by GC/MS

A serum metabolomics analysis was performed using GC/MS as described previously [34 (link)] with some modifications. In brief, a sample of 50 μL of serum was mixed with 5 μL of 1 mg/mL 2-isopropylmalic acid (Sigma-Aldrich, St. Louis, MO, USA) in distilled water as an internal standard, and 250 μL of methanol–chloroform–water (2.5:1:1) mixture. Then samples were lyophilized, and added with 40 μL of 20 mg/mL methoxyamine hydrochloride (Sigma-Aldrich), dissolved in pyridine for oximation. After mixing, the samples were shaken for 90 min at 30 °C. Next, 20 μL of N-methyl N-trimethylsilyl-trifluoroacetamide (GL Science, Tokyo, Japan) was added for trimethylsilylation, and the mixture was incubated at 37 °C for 45 min. The sample was subjected to GC/MS (GCMS QP2010-Ultra; Shimadzu, Kyoto, Japan). The Shimadzu Smart Metabolites Database (Shimadzu) was used to identify metabolites. Samples were normalized by a pooled all sample. All data are presented in Supplementary Tables S1 and S2 [35 (link)]. A metabolic pathway analysis was performed using MetaboAnalyst [36 (link)]. Metabolites that significantly differed between two groups were subjected to an enrichment analysis (http://www.metaboanalyst.ca/faces/upload/EnrichUploadView.xhtml, accessed on 1 June 2021).