A high-pressure liquid chromatographic-enzymatic assay for glycine and taurine conjugates of cholic, chenodeoxycholic and deoxycholic acid in serum

Abstract

An assay for glycine and taurine conjugates of cholic, chenodeoxycholic and deoxycholic acid in serum by a high pressure liquid chromatographic-enzymatic system is presented. The bile acids are extracted from serum by a reverse-phase liquid chromatographic process with an octadecylsilane column. The bile acid conjugates are separated on a μBondapak C₁₈ column with methanol/KH₂PO₄ buffer, 20 mmol/1, pH 5.3 as a mobile phase in less than 30 min at a flow rate of 1.4 ml/min. The bile acid fractions are measured by enzymatic fluorometry using a 3α-hydroxysteroid dehydrogenase-diaphorase system. Recoveries ranged from 82 to 96%, coefficients of variation were from 5 to 15%, and detection limits were from 0.03 to 0.08 μmol/l. Mean serum concentrations ranged from 0.10 to 0.39 μmol/1 in the fasting state and from 0.29 to 1.55 μmol/l postprandially in subjects with normal liver function.

Key-words:

• conjugated bile acids
• HPLC-enzymatic assay
• serum