Table1 metabolites and metabolic pathways

An ultra-high-pressure liquid chromatography system (Agilent 1290, USA) was used for the chromatography. In this experiment, the chromatographic column was ACQUITY UHPLC HSS T3 (1.8μm; 2.1×100mm) (Waters). Formic acid was included in mobile phases A and B at a concentration of 0.1% in water and 0.1% in acetonitrile, respectively [24]. Elution was performed in the following manner: 5% B for 1 min, followed by 10% B within 1 min, followed by 95% B within 12 min, then held for 2 min. Within 1 minute, it was changed linearly to 5% B and held for 3 minutes. A temperature of 35°C was used for the analytical column and a temperature of 4°C was used for the autosampler. Each run consisted of 5 μL of sample, and the column eluent was analysed directly on the MS [24].The cecal contents (0.2g) was mixed with 4 volumes of methanol/acetonitrile (1:1, v/v) for analysis. After shaking for 30 seconds, all samples were ultrasonically treated for 10 minutes in an ice-cold water bath. A 2-hour incubation at -20°C was followed by 15 minutes at 4°C centrifugation with 13,000 x g to facilitate protein precipitation. A supernatant of cecal contents was collected, dried under vacuum, and centrifuged at 4°C. Following resuspension in 200 μL of methanol/acetonitrile (1:1), shaking for 30 s, and centrifugation at 13,000 x g for 15 minutes at 4°C, the aliquots were dried. The supernatant was analysed on a UHPLC system with a Q-TOF system (Agilent 6545, USA) using approximately 150 microliters. A quality control (QC) test was performed after each analysis of five samples.Data were obtained using the auto MS/MS mode from 50 to 1100 m/z. Collision energies for collision-induced dissociation were 20 V and 40 V. MS parameters were set as follows: ion source dry gas temperature, 320°C; N2 gas flow, 8L/min; sheath gas temperature, 350°C; sheath gas flow, 12 L/min; ion spray voltage, 4000 V (positive ion) and 3500 V (negative ion).The Analysis Base File Converter was used to convert data files from the Q-TOF-MS system to .abf format. The following parameters were used for peak detection, chromatogram deconvolution, and other data analyses: alignment-MS1 tolerance, 0.01 Da; retention time tolerance, 0.2 min; identification accurate mass tolerance (MS1), 0.005 Da; MS2, 0.05Da; identification score cut off, 60%. Metabolites and metabolic pathways were identified using HMDB (http://www.hmdb.ca/), METLIN (http://metlin.scripps.edu/), Massbank (http://www.massbank.jp) and KEGG (http://www.kegg.com/) databases. In order to enhance precision, the identified metabolites were cross-validated utilizing a laboratory standard library.SIMCA-P software (version 14.1, Umetrics, Umea, Sweden) was used for the multivariate statistical analysis. A principal component analysis (PCA) was performed on the matrix imported into R to observe the overall distribution of samples and assess the stability of the whole process. Metabolites were distinguished between groups using partial least squares discriminant analysis (PLS-DA). Seven-fold cross-validation and 200 permutation tests (response permutation testing [RPT]) were performed to evaluate the model's quality. A permutation test was used to assess the validity of the model and its degree of overfitting. A VIP >1.0 and a p-value of 0.05 indicate that there is a significant difference. A p-value of .05 indicates that there is a significant difference between two groups compared using the Mann–Whitney U test. For multiple groups, the false discovery rate (FDR) was applied (p < 0.10). 24.        Zhao, X., et al., Serum metabolomics study of polycystic ovary syndrome based on liquid chromatography-mass spectrometry. J Proteome Res, 2014. 13(2): p. 1101-11.