Glycine

To test for conjugation of isotopically labeled amines to DCA by B. fragilis P207, 1-mL cultures containing 1 mM (final concentration) of either 4-aminobutyric acid (¹³C4, 97%–99%), L-alanine (¹³C3, 99%), glycine (2-¹³C, 99%; ¹⁵N, 98%+), L-phenylalanine (D₈, 98%), or tyramine:HCl (1,1,2,2-D₄, 98%) (Cambridge Isotope Laboratories, Inc.) were added to BHIS containing 0.1% (wt/vol) DCA (Thermo Fisher Scientific). Control samples contained either no isotopically labeled amines, no DCA, or BHIS media without any additional supplement. Culture supernatants were prepared for metabolomic analysis as described above.

Amino acid standards (alanine, arginine, aspartic acid, cysteine,
glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tyrosine, valine, glutamine,
asparagine, GABA, citrulline, ornithine, taurine, tryptophan, 5-HTP,
norvaline, and kynurenine) were purchased from Sigma (St. Louis, MO).
Isotopically labeled amino acid standards (alanine (¹³C₃,¹⁵N), arginine (¹³C₆, ¹⁵N₄), aspartic acid (¹³C₄, ¹⁵N), cystine (¹³C₆, ¹⁵N₂), glutamic acid (¹³C₅, ¹⁵N), glycine (¹³C₂, ¹⁵N), histidine
(¹³C₆, ¹⁵N₃), isoleucine
(¹³C₆, ¹⁵N), leucine (¹³C₆, ¹⁵N), lysine (¹³C₆, ¹⁵N₂), methionine (¹³C₅, ¹⁵N), phenylalanine (¹³C₉, ¹⁵N), proline (¹³C₅, ¹⁵N),
serine (¹³C₃, ¹⁵N), threonine (¹³C₄, ¹⁵N), tyrosine (¹³C₉, ¹⁵N), valine (¹³C₅, ¹⁵N), citrulline (d4), GABA (¹³C₄), glutamine
(¹³C₅), asparagine(¹³C₄), ornithine (d6), tryptophan (d8), kynurenine (d4)) were obtained
from Cambridge Isotope Laboratories (Andover, MA). Stock solutions
of each compound were prepared at a concentration of 500 ng/μL
and stored at −80 °C. A 10-point calibration curve (0.01–100
pmol/μL) was prepared by serial dilutions and spiked with internal
standards at a final concentration of 5 pmol/μL. Mass spectrometry-grade
formic acid was purchased from Sigma-Aldrich (St Louis, MO), and HPLC-grade
acetonitrile was purchased from Fisher Scientific (Fair Lawn, NJ).