Colonic microbial metabolite and flux analysis

Abstract

The colon is one of the body’s metabolically most active organs. While insight in colonic microbial diversity increases, little is known of the metabolic function of the microbes, the way diet affects metabolic fluxes, and how produced metabolites affect human health. Therefore, as a first step in detailed metabolomic characterization of colonic carbohydrate metabolism, we studied the conversion by human gut bacteria of the model substrate glucose. An in vitro model of the colon was inoculated with a standardised human fecal microbiota. D-[U-13C]glucose was added at t=0, and lumen as well as dialysate samples were taken at various time points. GC-MS, enzymatic methods and 1- and 2-dimensional NMR techniques at 500 MHz were used to identify microbial metabolites and to analyze their 13C contents. SCFA (short-chain fatty acids), formate and lactate were determined as the principal glucose degradation products. Carbon balancing revealed a 95% recovery of the 13C glucose carbon. Several minor components, including succinate, aspartate and alanine, were identified using 2D NMR experiments. Quantitative insight in bacterial metabolic routes was obtained from analysis of 13C-13C coupling patterns in 2D HSQC spectra that resulted from incorporation of intact 13C glucose backbone fragments in SCFA. In conclusion, this stable isotope study underlines the importance of colonic bacterial SCFA metabolism.