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

Abstract

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.

Figure 1

Schematic of the rapid-throughput SCFA quantification platform.

Figure 2

(A) Instrumental analysis workflow. (B) Chromatogram of standards and (C) bioreactor fermentation samples. To facilitate comparison across chromatograms, we scaled each chromatogram by the highest signal in the corresponding MRM transitions.

Figure 3

Absolute quantification results of cecal SCFA concentrations in high-fat-diet mice (n = 17) and low-fat-diet mice (n = 10). Significant differences were determined by Student’s t-test. (*, p < 0.05; **, p < 0.01).

Figure 4

SCFA production in bioreactors fermenting the feces of five different subjects after 3 days (n = 3). The results showed variations in the levels of SCFAs produced among the subjects, indicating individual differences in the gut microbiota and their active metabolic pathways. The statistical significance between subjects was calculated by a one-way ANOVA followed by Tukey’s test. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001).

Figure 5

(A) Experimental design of isotope-labeled fermentations. Unlabeled and labeled glucose/starch were fermented by either a single strain of Bifidobacterium or a complex microbiome in human feces. (B) Glucose/¹³C₆ glucose quantification results before and after 36 h of fermentation after performing acid hydrolysis on each supernatant. (C) Quantification results of isotopically labeled SCFAs.