Longitudinal Profiles of Dietary and Microbial Metabolites in Formula- and Breastfed Infants.

The early-life metabolome of the intestinal tract is dynamically influenced by colonization of gut microbiota which in turn is affected by nutrition, i.e. breast milk or formula. A detailed examination of fecal metabolites was performed to investigate the effect of probiotics in formula compared to control formula and breast milk within the first months of life in healthy neonates. A broad metabolomics approach was conceptualized to describe fecal polar and semi-polar metabolites affected by feeding type within the first year of life. Fecal metabolomes were clearly distinct between formula- and breastfed infants, mainly originating from diet and microbial metabolism. Unsaturated fatty acids and human milk oligosaccharides were increased in breastfed, whereas Maillard products were found in feces of formula-fed children. Altered microbial metabolism was represented by bile acids and aromatic amino acid metabolites. Elevated levels of sulfated bile acids were detected in stool samples of breastfed infants, whereas secondary bile acids were increased in formula-fed infants. Microbial co-metabolism was supported by significant correlation between chenodeoxycholic or lithocholic acid and members of Clostridia. Fecal metabolites showed strong inter- and intra-individual behavior with features uniquely present in certain infants and at specific time points. Nevertheless, metabolite profiles converged at the end of the first year, coinciding with solid food introduction.

Disturbed gut microbiota and bile homeostasis in Giardia-infected mice contributes to metabolic dysregulation and growth impairment.

Although infection with the human enteropathogen Giardia lamblia causes self-limited diarrhea in adults, infant populations in endemic areas experience persistent pathogen carriage in the absence of diarrhea. The persistence of this protozoan parasite in infants has been associated with reduced weight gain and linear growth (height-for-age). The mechanisms that support persistent infection and determine the different disease outcomes in the infant host are incompletely understood. Using a neonatal mouse model of persistent G. lamblia infection, we demonstrate that G. lamblia induced bile secretion and used the bile constituent phosphatidylcholine as a substrate for parasite growth. In addition, we show that G. lamblia infection altered the enteric microbiota composition, leading to enhanced bile acid deconjugation and increased expression of fibroblast growth factor 15. This resulted in elevated energy expenditure and dysregulated lipid metabolism with reduced adipose tissue, body weight gain, and growth in the infected mice. Our results indicate that this enteropathogen's modulation of bile acid metabolism and lipid metabolism in the neonatal mouse host led to an altered body composition, suggesting how G. lamblia infection could contribute to growth restriction in infants in endemic areas.

Milk-Derived Amadori Products in Feces of Formula-Fed Infants.

Food processing of infant formula alters chemical structures, including the formation of Maillard reaction products between proteins and sugars. We detected early Maillard reaction products, so-called Amadori products, in stool samples of formula-fed infants. In total, four Amadori products (N-deoxylactulosyllysine, N-deoxyfructosyllysine, N-deoxylactulosylleucylisoleucine, N-deoxyfructosylleucylisoleucine) were identified by a combination of complementary nontargeted and targeted metabolomics approaches. Chemical structures were confirmed by preparation and isolation of reference compounds, LC-MS/MS, and NMR. The leucylisoleucine Amadori compounds, which most likely originate from ß-lactoglobulin, were excreted throughout the first year of life in feces of formula-fed infants but were absent in feces of breastfed infants. Despite high inter- and intraindividual differences of Amadori products in the infants' stool, solid food introduction resulted in a continuous decrease, proving infant formula as the major source of the excreted Amadori products.

Development and application of a HILIC UHPLC-MS method for polar fecal metabolome profiling.

The fecal metabolome is a complex mixture of endogenous, microbial metabolites, and food derived compounds. Hydrophilic interaction liquid chromatography (HILIC) enables the analysis of polar compounds, which is a valuable alternative to reversed-phase liquid chromatography in the field of metabolomics due to its ability to retain a greater portion of the polar metabolome. The objective of the study was to find the optimal chromatographic solution to perform non-targeted metabolomics of feces by means of HILIC ultra-high-pressure liquid chromatography mass spectrometry (UHPLC-Q-TOF-MS). The performance was systematically investigated analyzing a pooled fecal sample, and mixtures of 150 metabolites from different families, including for example amino acids, amines, indole derivatives, fatty acids and carbohydrates. Three different stationary phases (zwitterionic, amide and unbonded silica) were operated at three pH values (4.6, 6.8 and 9.0), and three salt gradient conditions (5-5, 5-10 and 5-25 mM ammonium acetate). Amide and zwitterionic stationary phases performed similarly at low pH, with highest number of detected standards, which increased by increasing the salt gradient. The amide column showed slightly better performance in terms of separation of isomers and peak widths and remarkably good performance at basic pH, with highest number of metabolite features in the non-targeted analysis. The zwitterionic column operated best in terms of number of detected standards, retention time distribution of standards and metabolite feature across whole chromatographic run. Thus, the zwitterionic column was proven to suit for non-targeted analysis of fecal samples, resulting in good coverage of especially amino acids and carbohydrates.

Metformin impacts cecal bile acid profiles in mice.

Bile acids (BAs) are major components of bile synthesized from cholesterol and take part in the digestion of dietary lipids, as well as having signaling functions. They undergo extensive microbial metabolism inside the gastrointestinal tract. Here, we present a method of ultra-high pressure liquid chromatography coupled to ion trap mass spectrometry for quantification of 45 BAs in mouse cecum. The system was validated in regard to sensitivity with limits of detection and quantification (0.6-24.9 nM), interday accuracy (102.4%), interday precision (15.2%), recovery rate (74.7%), matrix effect (98.2%) and carry-over effect (<1.1%). Afterwards, we applied our method to investigate the effect of metformin on BA profiles. Diabetic mice were treated with metformin for 1 day or 14 days. One day of treatment resulted in a significant increase of total BA concentration (2.7-fold increase; db/db metformin 5.32 µmol/g, db/db control mice 1.95 µmol/g), most notable in levels of 7-oxodeoxycholic, 3-dehydrocholic and cholic acid. We observed only minor impact on BA metabolism after 14 days of metformin treatment, compared to the single treatment. Furthermore, healthy wild type mice had elevated concentrations of allocholic and ω-muricholic acid compared to diabetic mice. Our method proved the applicability of profiling BAs in cecum to investigate intestinal BA metabolism in diabetes and pharmacological applications.

The Microbial Metabolite Butyrate Induces Expression of Th1-Associated Factors in CD4+ T Cells.

Short-chain fatty acids (SCFAs), which are generated by the bacterial fermentation of dietary fibers, promote expansion of regulatory T cells (Tregs). Potential therapeutic value of SCFAs has been recently highlighted in the experimental models of T cell-mediated autoimmunity and allergic inflammation. These studies suggest that physiological intestinal concentrations of SCFAs within the millimolar range are crucial for dampening inflammation-mediated processes. Here, we describe opposing effects of SCFAs on T cell-mediated immune responses. In accordance with published data, lower butyrate concentrations facilitated differentiation of Tregs in vitro and in vivo under steady-state conditions. In contrast, higher concentrations of butyrate induced expression of the transcription factor T-bet in all investigated T cell subsets resulting in IFN-γ-producing Tregs or conventional T cells. This effect was mediated by the inhibition of histone deacetylase activity and was independent of SCFA-receptors FFA2 and FFA3 as well as of Na+-coupled SCFA transporter Slc5a8. Importantly, while butyrate was not able to induce the generation of Tregs in the absence of TGF-ß1, the expression of T-bet and IFN-γ was triggered upon stimulation of CD4+ T cells with this SCFA alone. Moreover, the treatment of germ-free mice with butyrate enhanced the expression of T-bet and IFN-γ during acute colitis. Our data reveal that, depending on its concentration and immunological milieu, butyrate may exert either beneficial or detrimental effects on the mucosal immune system.