4.7. Metabolomics of Fecal Samples and Statistical Analysis of Metabolomics Data
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REGRESSION
Targeted metabolomics analysis was performed on stool samples of 84 infants (control mean age: 102 days, test mean age: 104 days at baseline) collected at V0, V1, V3, and V6 to measure bile acids, other host-gut microbial metabolites, lipids and HMO. The samples were extracted and prepared according to previously published methods [Metabolomic data for the EE and LE cohorts were analyzed separately using a multivariate approach in SIMCA (SIMCA-P16, SARTORIUS STEDIM, Umea, Sweden). Partial least squares regression (PLS) and discriminant analysis (PLS-DA) were employed to model either age, treatment effect, or both at each time timepoint (V1, V3, or V6) or at combined time points (V1,V3,V6 or V3,V6). The model robustness was evaluated based on the value of the cross-validation parameter (Q2Y) and permutation tests. The model predicting age and treatment and combining V3 and V6 time points was the most robust and allowed us to maximize the metabolic differences between the control and test groups. The variable importance in projection (VIP) score was calculated for each feature. The VIP sore corresponds to the weighted sum of squares of the PLS weights, considering the amount of explained Y-variance in each component. The variable with a VIP > 1 is considered significant as its influence on the explanation of the outcomes is above average.Univariate Wilcoxon–Mann–Whitney (WMW) tests on metabolites were also performed at each time point. Due to the low sample size and the exploratory nature of this study, no adjustment for the false discovery rate was applied. The metabolites were considered the most important in the treatment group discrimination if they had VIP score > 1, |PLS coefficient (To test the hypothesis that HMO supplementation exerts an effect on fecal bile acids and SCFA content over time, the differences in the abundance of SCFA and unconjugated/conjugated bile acid ratio between a given time point (V1, V3, or V6) and baseline (V0) were evaluated using WMW tests in the control and the test group individually.