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2 protocols using obeticholic acid

1

Ligand Screening for Nuclear Receptors

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Compound 1 (CITCO, (6-(4-chlorophenyl)imidazo [2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichloro-benzyl)oxime)),
rifampicin (rif), TCPOBOP, and PK11195 were obtained from Sigma-Aldrich
(St. Louis, Missouri, United States, now Merck), which is now known
as Merck (Darmstadt, Germany). Phenobarbital (Luminal 200 mg/mL injection)
was manufactured by Desitin Pharma spol. s.r.o. (Prague, Czech Republic).
Ligands for nuclear receptors (GW3965, thyroxin, obeticholic acid,
dexamethasone, fenofibrate, GW501516, rosiglitazone, 3-methylcholanthrene,
calcitriol, testosterone, and estradiol) were purchased from Sigma-Aldrich
(now Merck). The prototype ligands were used at 100 nM (dexamethasone,
calcitriol), 1 μM (thyroxin), or 10 μM concentrations.
The compounds were dissolved in DMSO, and the final concentration
of DMSO in the entire reaction mixture or cultivation media was 0.1%.
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2

Quantification of Bile Acids and Obeticholic Acid

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Enodogenous bile acid composition and the concentration of exogenous obeticholic acid was determined in portal bile. Bile acid standards and bile acid derivative obeticholic acid were purchased from Sigma-Aldrich Corporation (St Louis, MO) or Steraloids Inc (Newport, RI). Deuterated cholic acid (D-2452) and deuterated chenodeoxycholic acid (D-2772) were purchased from CDN Isotopes, Inc (Pointe-Claire, Quebec, Canada). High-performance liquid chromatography–grade chemicals were obtained from Fisher Scientific (Fair Lawn, NJ). Bile acids were extracted from portal serum and bile with 50% ice-cold methanol, followed by further extraction with acetonitrile (5% NH4OH). Ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS) was performed on the resultant supernatant using a modified method15 (link) and samples were analyzed on an Acquity UPLC system coupled to an LCT Premier mass spectrometer (Waters Corporation, Milford, MA). Analytes were quantified individually (3 technical reads per sample) against known bile acid and obeticholic standard curves. All quantifications were normalized relative to internal standards.
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