Quantifying Gut Microbial Short-Chain Fatty Acids and Their Isotopomers in Mechanistic Studies Using a Rapid, Readily Expandable LC-MS Platform.

Short-chain fatty acids (SCFAs) comprise the largest group of gut microbial fermentation products. While absorption of most nutrients occurs in the small intestine, indigestible dietary components, such as fiber, reach the colon and are processed by the gut microbiome to produce a wide array of metabolites that influence host physiology. Numerous studies have implicated SCFAs as key modulators of host health, such as in regulating irritable bowel syndrome (IBS). However, robust methods are still required for their detection and quantitation to meet the demands of biological studies probing the complex interplay of the gut-host-health paradigm. In this study, a sensitive, rapid-throughput, and readily expandible UHPLC-QqQ-MS platform using 2-PA derivatization was developed for the quantitation of gut-microbially derived SCFAs, related metabolites, and isotopically labeled homologues. The utility of this platform was then demonstrated by investigating the production of SCFAs in cecal contents from mice feeding studies, human fecal bioreactors, and fecal/bacterial fermentations of isotopically labeled dietary carbohydrates. Overall, the workflow proposed in this study serves as an invaluable tool for the rapidly expanding gut-microbiome and precision nutrition research field.

A multi-glycomic platform for the analysis of food carbohydrates.

Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan-microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health.