Cholic acid

Cholic acid (CA), chenodeoxy Cholic acid (CDCA), deoxyCholic acid (DCA), lithoCholic acid (LCA), α muriCholic acid (αMCA), βMCA, ωMCA, ursodeoxyCholic acid (UDCA), hyodeoxyCholic acid (HDCA), murideoxyCholic acid (MDCA), taurine conjugated Cholic acid (T-CA), T-CDCA, T-DCA, T-LCA, T- αMCA, T- βMCA, T- ωMCA, T-UDCA, and T-HDCA were purchased from Steraloids (Newport, Rhode Island). T- ωMCA was given by Dr. Daniel Raftery’s laboratory at the University of Washington Northwest Metabolomics Research Center. For internal standards (ISs) , a total of 5 deuterated (d4) ISs were used. Among them the d4-Cholic acid (d4-CA) was purchased from Toronto Research Chemicals Inc (Ontario, Canada), the d4-deoxyCholic acid (d4-DCA) and d4-chenodeoxyCholic acid (d4-CDCA) were purchased from CDN Isotope Inc (Pointe-Claire, Quebec, Canada), the d4-glycine conjugated CDCA (d4-G-CDCA) was purchased from IsoSciences (Ambler, PA), and d4-lithoCholic acid (d4-LCA) was purchased from Steraloids (Newport, Rhode Island). One mg/mL stock solutions of the individual BAs (for standard curve) and IS were prepared in methanol and water (1:1). The 19 individual BA stock solutions were further diluted in 50% methanol to obtain 10 working standard solutions ranging from 0.05 to 10000 ng/mL). The 5 ISs were mixed to obtain a working IS solution in which the concentration of each IS was 10 µmol/mL.

Deoxycholic acid (DCA), chenoDeoxycholic acid (CDCA), cholic acid (CA), α-muricholic acid, β-muricholic acid, glycoDeoxycholic acid (GDCA), glycochenoDeoxycholic acid (GCDCA), and glycocholic acid (GCA) were obtained from Steraloids, Inc. Bile acid structures are shown in fig. S1. Bile acid A (37 ), 5-cholanic acid-3α,4β,7α-triol (7)(38 (link)), and AMPP (39 (link)) were synthesized according to published procedures. N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), 4-(dimethylamino)pyridine (DMAP), diethylamine, N,N-dimethylformamide, trisodium citrate, sodium hydroxide (NaOH), chloroform, Dulbecco’s modified Eagle’s medium, fetal calf serum, and penicillin G and streptomycin sulfate were obtained from Sigma–Aldrich. HPLC solvents (methanol and acetonitrile) were HPLC grade and were purchased from EMD Chemicals. Milli-Q ultrapure water was prepared in-house with a Milli-Q Integral Water Purification System.