Msk caa 1

40,000 A549 cells were plated in 6-well plates in RPMI, allowed to attach overnight, washed twice with PBS, then treated with 2.2 or 6 mL of RPMI or ftABS for 4, 12, 24, 48, or 72 hr. At each time point, media was centrifuged at 845 × g for 3 min to remove cells, then flash frozen. 5 μL of media was mixed with 45 μL of HPLC-grade methanol:water (80:20) including 500 nM ¹³C/¹⁵N labeled amino acids (Cambridge Isotope Laboratories, MSK-CAA-1). Samples were then vortexed for 15 min at 4°C, centrifuged at 16,000 × g for 10 min at 4°C, and 20 μL of the soluble polar metabolite extract was taken for LC/MS analysis. Metabolite measurements were normalized to a labeled amino acid internal standard that eluted at roughly the same retention time. Metabolite concentrations were determined by normalizing to ion counts measured in fresh RPMI or ftABS media with known concentration values.

5 μL of adult bovine serum or ftABS^B was mixed with 45 μL of HPLC-grade methanol:water (80:20) including 500 nM ¹³C/¹⁵N labeled amino acids (Cambridge Isotope Laboratories, MSK-CAA-1). Samples were vortexed for 15 min at 4°C and insoluble material was sedimented by centrifugation at 16,000 × g for 10 min at 4°C. 20 μL of the soluble polar metabolite extract was taken for LC/MS analysis. Untargeted metabolomics data were acquired in LC-MS mode with additional data-dependent MS/MS collected on pooled samples for identification of unknown metabolites. Data analysis was performed using Compound Discoverer 3.2 (Thermo Fisher Scientific) with an in-house mass-list as well as online databases. Background compounds (i.e., compounds present in samples containing only extraction buffer or water) were excluded, as well as compounds with insufficient identification confidence. The median chromatographic peak area per group were used to generate a correlation plot.

Circulating glycine levels were measured in serum samples from patients with and without sCAD by investigators blinded to the study groups as we previously described [8 ,30 (link)]. Briefly, liquid chromatography (LC)-mass spectrometry (MS)/MS was used to measure serum amino acids using isotope-labeled amino acids (MSK-CAA-1, Cambridge Isotopes Laboratory) as internal standard. The LC-MS/MS analysis was performed using a Shimadzu LC-20AD HPLC system (Shimadzu Corporation) coupled in-line to an AB Sciex QTrap 5500 system (Applied Biosystems) equipped with an electrospray ionization source. Chromatographic separation was conducted on an Agilent Poroshell 120 EC-C18 column (50 mm × 2.1 mm I.D., 2.7 μm; Agilent) at a flow rate of 0.4 mL/min. The mobile phase consisted of water (A) and acetonitrile containing 0.1% (v/v) formic acid (B). The mass spectrometer was operated in positive ionization mode with multiple reaction monitoring (MRM). Analyst version 1.6.2 software (Applied Biosystems) was used for data acquisition and analysis. In mice, blood glucose levels were measured using glucometer and test strips (Contour Next). Plasma samples were analyzed for total cholesterol (TC) and triglycerides (TG) using the Wako Cholesterol E kit (999-02601, FUJIFILM medical system) and LabAssay Triglyceride kit (290-63701, FUJIFILM medical system).