[Fractional determination of bile acids bound with protein in serum by high-performance liquid chromatography using...

A simple and rapid technique for the fractional determination of bile acids bound with protein in the serum was developed by using high performance liquid chromatography with a dual-column switching system. The serum samples were directly injected onto a first column (hydroxyapatite), which was initially flushed with 1 mM phosphate buffer. Serum proteins were strongly retained on a column of hydroxyapatite, but free bile acids were not retained. The bile acids were adsorbed on a second column (Serumout-25) and eluted onto a column of immobilized 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) with an elution solvent (CH3CN-MeOH-30 mM ammonium acetate, 30:30:40, v/v/v). Reduced nicotinamideadenine dinucleotide was produced on the immobilized 3 alpha-HSD column and then determined fluorometrically. Subsequently, the hydroxyapatite column was flushed with 20 mM phosphate buffer. Bile acids bound with albumin were eluted and condensed on Serumout-25. The phosphate buffer (400 mM) was finally used for the elution of bile acids bound with globulin from the hydroxyapatite column. Each condensed bile acid was eluted onto the immobilized 3 alpha-HSD column as described above.