Biodistribution and racemization of gut-absorbed L/D-alanine in germ-free mice

Discovery and characterization of microbiome-derived metabolites are important for characterization of the different functional systems including the microbiome-gut-brain axis and for discovery of new disease treatments. Essential to microorganisms, D-Alanine (D-Ala) is found in many animals and acts as a potent co-agonist of the N-methyl-D-aspartate receptors (NMDAR). These receptors are widely expressed and have a variety of functions in the nervous and endocrine systems. The gut microbiome, diet and putative endogenous synthesis are the potential sources of D-Ala in animals. Understanding the sources of D-Ala and distribution of gut-absorbed D-Ala in mammals is critical for the evaluation of interaction between an animal and its microbiota. In this work, we used germ-free mice fed with stable isotope-labeled L-/D-Ala to study the biodistribution of gut-absorbed D-Ala without interference from microbiota. This also allows the determination of endogenous D-Ala synthesis through enantiomeric conversion. D-Ala is absorbed by both the small intestines and colon; the distribution of gut-absorbed D-Ala is related to the tissue type and treatment time (1-hour and 2-week). The highest levels of D-Ala accumulate in the pancreatic islets and acinar tissues followed by the brain and pituitary gland. Changes in tissue peptide profiles measured after 2-weeks of D-Ala treatment suggest that D-Ala impacts the activity of the hypothalamus-pituitary-adrenal axis. No endogenous synthesis of D-Ala via racemization was reliably observed in germ-free mice. Alanine racemization was readily observed in bacterial culture and regular mice possessing normal microbiota supporting the idea that mice lack a functional alanine racemase enzyme.