Serum sample collection and metabolomics.
Serum samples were collected and immediately frozen at −80°C until metabolite extraction. Total metabolite content was extracted from 0.05 g of each of the serum samples, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses, performed using the ultrahigh-performance liquid chromatography (UHPLC) system (1290, Agilent Technologies) with an ultraperformance (UPLC) HSS T3 column (2.1 mm by 100 mm, 1.8 μm) coupled to the Q Exactive system (Orbitrap MS; Thermo). MS raw data were filtered if the following criteria were met: (i) metabolites present in less than 50% of all samples in a group were removed, and (ii) missing values were replaced by half of the minimum values found by default in the data set. Peaks were annotated against an in-house MS/MS database constructed using HMDB, Metlin, and Mona. Finally, the relative content of each peak was determined by the normalization method of peak area to generate percentage value for each metabolite.