Bile Acids, Phospholipids, and Cholesterol Analysis

For bile acid analysis [30 (link)], 1 μL of bile sample was diluted with 99 μL of Milli-Q^® water and then incubated with the reagent solution (6 mg NAD, 0.5 M hydrazine hydrate buffer, 0.05 M Na-pyrophosphate) for 4 min. The mix was then incubated with the solution (0.03 M Tris-EDTA; 0.3 U/mL 3-alpha-OH steroid dehydrogenase), and the bile acid concentration was determined by spectrophotometry (excitation of 340/330 nm/emission of 440/420 nm, CLARIOstar Plus microplate reader, BMG Labtech, Ortenberg, Germany). For phospholipid (PL) analysis, 1 μL of bile sample was diluted with 49 μL of Milli-Q^® water and then incubated with the reagent solution (100 mM MOPS, pH 8; 0.55 mM HVA; 20 mM CaCl₂; 11 U/mL phospholipase-D; 1.66 U/mL peroxidase; 0.1% Triton X-100) for 4 min. The mix was then incubated with a start reagent (1 M MOPS, pH 8, 50 U/mL choline oxidase), and the PL concentration was determined by spectrophotometry (excitation 340/330 nm/emission 440/420 nm using a CLARIOstar Plus microplate reader, BMG Labtech, Ortenberg, Germany). For cholesterol analysis, 1 μL of bile sample was diluted with 29 μL of Milli-Q^® water and then incubated with the reagent solution (100 mM MOPS, pH 8, 0.25 mM HVA; 0.1% Triton X-100) for 4 min. The mix was then incubated with a start reagent (0.1 M MOPS, pH 8, 0.06 U/mL cholesterol oxidase, 0.15 U/mL cholesterol esterase, 0.45 U/mL peroxidase, 0.06 mM taurocholate), and the cholesterol concentration was determined by spectrophotometry (excitation of 330/340 nm/emission of 420/440 nm, CLARIOstar Plus microplate reader, BMG Labtech, Ortenberg, Germany).