Metabolite Profiling and Statistical Analysis

Preprocessed data obtained from the Leco Chroma TOF software (ver. 4.50; Leco) were processed using BinBase, an in-house database [16 (link)–18 (link)]. The peak intensity of the identified metabolites was normalized by the median of the sum of the peak intensities of all identified metabolites in each sample. STATISTICA (ver. 7.1; StatSoft, Tulsa, OK) was used for univariate analyses such as principal component analysis (PCA), nonparametric Wilcoxon-Mann-Whitney test, and breakdown and the one-way analysis of variance (ANOVA) [18 (link),19 (link)]. Orthogonal partial least square-discriminative analysis (OPLS-DA) and seven-fold internal cross validation was performed using SIMCA-P+ (version 12.0; Umetric AB, Umea, Sweden). MedCalc software (Broekstraat, Mariakerke, Belgium) was used to obtain a receiver operating characteristic (ROC) curve for further evaluation of the diagnostic properties of identified metabolites.