Fecal N500 targeted metabolomics

Accurately weigh each metabolite standard, prepare mixed standard linear mother liquor, and dilute the linear mother liquor with methanol to obtain a series of concentration working solutions. Prepare a certain concentration of isotope internal standard solution and mix well to obtain the internal standard solution. The mother liquor and working solution of linear, internal standard, and quality control are stored in a -20 ℃ refrigerator. Grind the samples with liquid nitrogen, weigh a certain amount of fecal samples from the ND group and CPD group, add 300 μ L of 80% methanol aqueous solution, vortex and mix well, let it stand for 10 minutes, centrifuge at 4 ℃ and 15000 r/min for 15 minutes, take 50 μ L of supernatant, add 150 μ L of derivatization reagent, and derivatize at 40 ℃ for 40 minutes; Dilute the derived sample to the corresponding multiple, take 90 μ L of supernatant, add 10 μ L of mixed internal standard solution, mix well, and use ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) system to quantify metabolites (targeted metabolomics detection was completed by Beijing Novogene Technology Co., Ltd.). The N500 metabolome is a targeted metabolomics technology developed for high-throughput absolute quantification of small molecule metabolites in vitro. One detection can achieve absolute quantification of nearly 500 substances in biological samples, while also solving the problems of difficult validation of non targeted metabolomics and limited detection of substances in targeted metabolomics. The key pathways involved include bile acid biosynthesis, fatty acid biosynthesis, tricarboxylic acid cycle, amino acid metabolism, short chain fatty acid metabolism, etc., including numerous small molecule metabolites related to gut microbiota. The screening of differential metabolites mainly refers to three parameters: Variable Importance in the Projection (VIP), Fold Change (FC), and P-value (P). VIP refers to the variable projection importance of the first principal component of the Partial Least Squares Discrimination Analysis (PLS-DA) model, which reflects the contribution of each sample's quantitative value to the difference; FC is the ratio of the mean values of all biological replicates for each metabolite in the comparison group; P-value is calculated through t-test and represents the level of significant difference. Set the threshold to VIP>1.0, FC>1.2, or FC<0.833 with P-value<0.05