Trust your gut microbiome: intramolecular isotopic fingerprints of amino acids in mouse tissues

Abstract:

The degree to which the gut microbiome contributes to the protein metabolism of its host has not yet been fully explored, yet this information would allow us to assess the functional significance of gut microbes to their host’s health and enhance our ability to quantify the diet composition and trophic position of animals with stable isotope analysis. We investigate this through ongoing controlled feeding experiments in which white-footed deer mice (Peromyscus leucopus) are fed either isotopically (¹³C, ¹⁵N) enriched or natural abundance level diet ingredients (amino acids, sucrose, cellulose). We are generating both inter- and intramolecular isotope data to identify the biochemical origin of amino acids in mice tissues. While numerous feeding studies have previously relied on conventional amino acid-specific isotope analysis to study diet and metabolism, these data cannot distinguish between host and microbiome synthesis of nonessential amino acids. We predict that the intramolecular isotopic fingerprint of nonessential amino acids in mice tissues will reflect the unique biochemical pathways used by mice or microbes to synthesize them. Thus, we can estimate the relative importance of amino acid incorporation via synthesis by the host or microbiome or direct dietary routing.

We have made some of the first intramolecular δ¹³C measurements of amino acids using a newly developed gas chromatograph triple stage quadrupole combustion isotope ratio mass spectrometer. With this instrument, individual amino acids are identified, fragmented, then molecular fragments (e.g. decarboxylated amino acids) are measured quantitatively. We analyzed mixtures of ¹³C-labeled and natural abundance amino acid standards to calibrate the accuracy and precision of this novel technique. We have found that our instrumentation is capable of differentiating between natural and labeled amino acids with an extremely low detection threshold (< 5% label). We will combine this intramolecular analysis with both amino acid-specific δ¹³C and δ¹⁵N for a comprehensive assessment of the role of the gut microbiome in rodent protein homeostasis.