(P06-045-23) Elucidating Gut Microbial Metabolism Through 13C Labeled Dietary Fiber

Objectives: Dietary fiber has been shown to reduce the risk of cardiovascular and metabolic diseases. While inaccessible to digestion in the upper GI track, fiber reaches the colon and is fermented by the gut microbiota. Microbial products of fiber fermentation, such as the short chain fatty acids (SCFAs), are thought to exert local and systemic effects on host physiology. In this work, ¹³C labeled dietary fibers were subjected to anaerobic in vitro fecal fermentations and isotopically labeled SCFAs were quantified by a UHPLC-QqQ platform to enable a deeper understanding of the gut-host-health paradigm.

Methods: ¹³C dietary fibers were used as substrates for in vitro fermentations using adult feces. The cell media was subjected to monosaccharide and SCFA analysis. The monosaccharide analysis consisted of hard acid hydrolysis with 4 M trifluoracetic acid and 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization in ammonia solution. SCFAs were analyzed by derivatizing samples with 2-picolyamine. All sample preparations were conducted in 96-well plates, enabling rapid-throughput, quantitative analysis with UHPLC-QqQ MS.

Results: This proof-of-concept study demonstrates that ¹³C polysaccharides can be leveraged to unambiguously assign gut microbial fermentation outputs. In vitro fecal fermentations were conducted for 24 hrs with fully ¹³C labeled cellulose, inulin, arabinoxylan, and starch as well as with a mixture of all 4 ¹³C polysaccharides. Monosaccharide and SCFA analysis were conducted on the cell media to track consumption of labeled carbohydrates and production of labeled SCFAs.

For most fermentations the most abundant SCFA isotopologues were fully labeled acetate, fully labeled lactate, and doubly and fully labeled butyrate reflecting their biosynthesis from pyruvate a 3-carbon intermediate. Additionally, the SCFA profile for ¹³C starch fermentation was compared for 2 adult donors. Donor 1 showed much greater SCFA diversity than donor 2 demonstrating the importance of the microbial community composition on the observed metabolic outputs.

Conclusions: Proof-of-concept study highlighting ¹³C dietary fiber as a tool for tracing gut microbial metabolic pathways.

Funding Sources: National Institute of Health