Evaluation of Normalization Approaches for Quantitative Analysis of Bile Acids in Human Feces

Quantitative analysis of bile acids in human feces can potentially help to better understand the influence of the gut microbiome and diet on human health. Feces is a highly heterogeneous sample matrix, mainly consisting of water and indigestible solid material (as plant fibers) that show high inter-individual variability. To compare bile acid concentrations among different individuals, a reliable normalization approach is needed. Here, we compared the impact of three normalization approaches, namely sample wet weight, dry weight, and protein concentration, on the absolute concentrations of fecal bile acids. Bile acid concentrations were determined in 70 feces samples from healthy humans. Our data show that bile acid concentrations normalized by the three different approaches are substantially different for each individual sample. Fecal bile acid concentrations normalized by wet weight show the narrowest distribution. Therefore, our analysis will provide the basis for the selection of a suitable normalization approach for the quantitative analysis of bile acids in feces.