Optimization of an immobilized enzyme system for conjugated bile acids.

Abstract

The optimization of a reversed phase high performance liquid chromatography system incorporating an immobilized enzyme was investigated. The immobilized enzyme, cholylglycine hydrolase (CGH), was used to generate species that were detected using fluorescence. A separation of a standard containing 10 bile acids was achieved after signal to noise optimization of the system. The 10 conjugated bile acids were hydrolyzed after the separation by the immobilized CGH to yield free bile acids and the amino acids taurine and glycine. The amino acids were then reacted to give o-phthalaldehyde derivatives and detected by fluorescence. Emission and excitation wavelengths were set at 448 nm and 341 nm respectively. Bile samples from humans were also analyzed using the optimized system.