Pathway analysis, metabolite set enrichment analysis (MSEA) and receiver operator characteristic (ROC) curves of dysregulated metabolites

Using Metaboanalyst 4.0 (www.metaboanalyst.ca), the peak areas of all the identified metabolites were subjected to pathway analysis and MSEA to identify potential key significantly altered metabolic pathways (ACO vs asthma and ACO vs COPD) [36, 37]. First, to explore the metabolic pathways that are potentially dysregulated in ACO, a global metabolic pathway analysis was carried out. The default ‘global test’ and ‘relative-betweenness centrality’ for pathway enrichment and pathway topological analyses were selected, respectively. The “current 2019” Kyoto Encyclopedia of Genes and Genomes (KEGG) version pathway library was also used.

For MSEA, quantitative enrichment analysis (QEA) was performed on normalized data for comprehensive screening of affected pathways. QEA is based on the global test algorithm to perform enrichment analysis directly from raw concentration data (peak area in this case) and does not require a list of significantly changed compounds. The QEA algorithm uses a generalized linear model to estimate a ‘Q-stat’ for each metabolite set. In addition to the Q-stat values, the QEA module also provide p-values, Holm adjusted p-values, and estimates of false discovery rate (FDR).

The prediction ability of all significant metabolites was assessed using receiver operating characteristic (ROC) curve and the area under the curve (AUC) calculated (MedCalc Statistical Software version, version 19.2.1, MedCalc Software bvba, Ostend, Belgium).