Contents of distal SI was collected and frozen by liquid nitrogen. For extracting ionic metabolites, approximately 20 mg of SI contents were dissolved in H2O containing internal standards [H3304-1002, Human Metabolome Technologies (HMT), Tsuruoka, Yamagata, Japan] with a ratio of 1:9 (w/v). After centrifugation, the supernatant was then centrifugally filtered through a Millipore 5000-Da cutoff filter (UltrafreeMC-PLHCC, HMT) to remove macromolecules (9100g, 4°C, 60 min) for subsequent analysis with capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS).
For extracting nonionic metabolites, approximately 20 mg of distal SI contents were dissolved in 1 ml of methanol containing internal standard (H3304-1002, HMT). After centrifugation, 10 μl of the supernatant was transferred into glass vials for evaporation under nitrogen gas and reconstituted with 200 μl of 50% isopropanol (v/v) for subsequent analysis with liquid chromatography TOFMS (LC-TOFMS).
Metabolome analysis was conducted at HMT with a Dual Scan package using CE-TOFMS and LC-TOFMS for ionic and nonionic metabolites, respectively. Hierarchical cluster analysis and principal components analysis were performed by HMT’s proprietary software, PeakStat and SampleStat, respectively (data file S1).
For extracting nonionic metabolites, approximately 20 mg of distal SI contents were dissolved in 1 ml of methanol containing internal standard (H3304-1002, HMT). After centrifugation, 10 μl of the supernatant was transferred into glass vials for evaporation under nitrogen gas and reconstituted with 200 μl of 50% isopropanol (v/v) for subsequent analysis with liquid chromatography TOFMS (LC-TOFMS).
Metabolome analysis was conducted at HMT with a Dual Scan package using CE-TOFMS and LC-TOFMS for ionic and nonionic metabolites, respectively. Hierarchical cluster analysis and principal components analysis were performed by HMT’s proprietary software, PeakStat and SampleStat, respectively (data file S1).