Bile acid standards

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Bile acid standards are reference materials used to calibrate and validate analytical instruments for the measurement of bile acids in biological samples. They provide a known concentration of specific bile acid compounds to ensure accurate and reliable quantification.

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Bile acids (22 citations)

7 protocols using bile acid standards

Comprehensive Bile Acid Quantification

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Bile acid standards (Steraloids, USA), six stable isotopes labeled standards (C/D/NIsotopes, Canada/Steraloids), ammonium acetate (analytical grade, Sigma–Aldrich, St Louis, MO, USA), methanol (Optima LC-MS), acetonitrile (Optima LC-MS), isopropanol (Optima LC-MS), glacial acetic acid and formic acid (Optima LC-MS) were all purchased from ThermoFisher Scientific (Fairlawn, NJ, USA). The following equipment was used: ACQUITYUPLC-XevoTQ-S liquid-mass spectrometer (WatersCorp., Milford, MA, USA); Mill-Q ultrapure water system (Millipore, Billerica, MA, USA); homogenizer (BB24; NextAdvance, Averill Park, NY, USA); microcentrifuge (Microfuge20R; Beckman Coulter, Indianapolis, IN, USA); and lyophilizer (Labconco, Kansas City, MO, USA).

Song W., Sun L.Y, & Zhu Z.J. (2021). Effects of Previous Kasai Surgery on Gut Microbiota and Bile Acid in Biliary Atresia With End-Stage Liver Disease. Frontiers in Medicine, 8, 704328.

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Colonic Bile Acid Extraction and Quantification

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Colonic bile acid extraction was performed as previously described with proper modification [20 (link)]. Briefly, colonic content (20 mg) was homogenized in 400 μL methanol and centrifuged at 12,000× g and 4 °C for 15 min. The supernatant was collected and evaporated in a centrifuge (Eppendorf, Hamburg, Germany) to remove the solvent. The obtained solute was resolved in 400 μL methanol and centrifuged under the same conditions before use. Bile acid standards (Sigma-Aldrich, St. Louis, MO, USA), including deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), tauroursodeoxycholic acid (TUDCA), hyodeoxycholic (HDCA), lithocholic acid (LCA), β-muricholic acid (β-MCA), taurocholic acid (TCA), and cholic acid (CA), were used as internal standards. A UPLC-MS system (Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze the prepared samples. The instrumental settings were set and data processing was performed as previously described [21 (link)].

Chen X., Chen Y., Stanton C., Ross R.P., Zhao J., Chen W, & Yang B. (2023). Dose–Response Efficacy and Mechanisms of Orally Administered Bifidobacterium breve CCFM683 on IMQ-Induced Psoriasis in Mice. Nutrients, 15(8), 1952.

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Isotopic Labeling for Bile Acid Analysis

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Sodium 3-(trimethylsilyl) [2,2,3,3-²H₄] propionate (TSP), D₂O (99.9% in D), and PCB 126 were ordered from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). Bile acid standards and deuterated internal standards were obtained from Sigma-Aldrich (St Louis, MO, USA) and Cayman Chemical (Ann Arbor, MI, USA).

Tian Y., Rimal B., Gui W., Koo I., Smith P.B., Yokoyama S, & Patterson A.D. (2022). Early Life Polychlorinated Biphenyl 126 Exposure Disrupts Gut Microbiota and Metabolic Homeostasis in Mice Fed with High-Fat Diet in Adulthood. Metabolites, 12(10), 894.

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Quantitative Metabolomic Analysis Protocol

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HPLC-grade methanol, acetonitrile, and chloroform were obtained from Merck (Darmstadt, Germany). HPLC-grade ammonium formate and acetate were procured from Shanghai Aladdin Reagent Co., Ltd. (Shanghai, China). The energy metabolite standards were purchased from Shanghai Anpel Laboratory Technologies (Shanghai, China) Inc. (Shanghai, China). Phospholipid standards were obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Bile acid standards were bought from Sigma-Aldrich (St. Louis, MO, USA). Deionized water was produced using a Direct-Q water purification system (Millipore, El Paso, TX, USA). The target compound information is demonstrated in Table S1.

Zhou T., Yang K., Huang J., Fu W., Yan C, & Wang Y. (2023). Effect of Short-Chain Fatty Acids and Polyunsaturated Fatty Acids on Metabolites in H460 Lung Cancer Cells. Molecules, 28(5), 2357.

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Metabolomic Profiling of Breast Tissues

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Metabolomic profiling of human breast tissues was performed using both an untargeted discovery approach and a targeted approach for validation and absolute quantification. Untargeted metabolic profiling of known and unknown metabolites in the discovery set included 67 human breast tumors and 65 tumor-adjacent noncancerous tissues and was performed by Metabolon Inc, as described previously (11 (link),17 (link)). Additional metabolic profiling with absolute quantification of the four bile acids, deoxycholate, chenodeoxycholate, glycodeoxycholate, and glycochenodeoxycholate was performed at the Alkek Center for Molecular Discovery of Baylor College of Medicine using selective reaction monitoring and the Agilent 6490 Triple Quadrupole Mass Spectrometer system. For absolute quantification of bile acids in tissue extracts from 20 tumor-adjacent noncancerous tissue pairs, a random subset of the 67 tumors described above, we prepared serial dilutions of bile acid standards (all Sigma-Aldrich) to generate the calibration curve.

Tang W., Putluri V., Ambati C.R., Dorsey T.H., Putluri N, & Ambs S. (2019). Liver- and Microbiome-derived Bile Acids Accumulate in Human Breast Tumors and Inhibit Growth and Improve Patient Survival. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(19), 5972-5983.

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Bile Acid and Lysophospholipid Standards

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Methanol was purchased from Merck (Darmstadt, Germany) and water was purified in-house using a Milli-Q Advantage A10 system from Merck Millipore (Billerica, MA, USA). Ammonium acetate and acetic acids were supplied by Sigma-Aldrich (St. Louis, MO, USA). Bile acid standards were purchased from Sigma-Aldrich (St. Louis, MO, USA), Toronto Research Chemicals (North York, Canada), Cayman Chemicals (Ann Arbor, MI, USA), and Steraloids (Newport, RI, USA). Lysophospholipid standards were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Refer to Tables S1 and S7 for details about standards.

Christinat N., Valsesia A, & Masoodi M. (2020). Untargeted Profiling of Bile Acids and Lysophospholipids Identifies the Lipid Signature Associated with Glycemic Outcome in an Obese Non-Diabetic Clinical Cohort. Biomolecules, 10(7), 1049.

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Pharmacological Evaluation of CDCA and Pravastatin

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CDCA 250 mg tablets (Chenodal; lot #004949) were kindly supplied by Travere Therapeutics (San Diego, CA). Pravastatin 80 mg tablets were obtained from Accord Healthcare (Durham, NC). Metformin 500 mg tablets were obtained from Granules Pharmaceuticals (Chantilly, VA). Placebo tablets were manufactured at the University of Maryland Good Manufacturing Practice facility using PROSOLV EASYtab SP (JRS PHARMA, Weissenborn, Germany). The FGF19 human enzyme‐linked immunosorbent assay (ELISA) kit was obtained from Invitrogen (Waltham, MA). Liquid‐chromatography tandem mass spectrometry (LC–MS/MS) grade solvents were purchased from Fisher Scientific (Pittsburgh, PA). Pravastatin was obtained from United States Pharmacopeia (Rockville, MD). Pravastatin‐d₃ was obtained from Toronto Research Chemicals (North York, ON, Canada). Bile acid standards and stable isotope labeled Bile acid standards were purchased from Sigma Aldrich (St. Louis, MO), Toronto Research Chemicals (North York, ON, Canada), Steraloids (Newport, RI), Cambridge Isotope Laboratories (Tewksbury, MA), or CDN Isotopes (Pointe‐Claire, QC, Canada). ISOLUTE PLD+ phospholipid depletion columns were purchased from Biotage (Uppsala, Sweden).

Metry M., Krug S.A., Karra V.K., Kane M.A., Fink J.C., Shu Y., Wang H, & Polli J.E. (2022). Differential effects of metformin‐mediated BSEP repression on pravastatin and bile acid pharmacokinetics in humans: A randomized controlled trial. Clinical and Translational Science, 15(10), 2468-2478.

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