High-performance liquid chromatography of bilirubin conjugates in bile: effect of beta-glucuronidase on the bile pigments.

Abstract

A simple, specific, and technically easy high-performance liquid chromatography (HPLC) method for the separation and quantification of unconjugated bilirubin, bilirubin monoglucoside-monoglucuronide, bilirubin diglucuronide, and bilirubin monoglucuronide has been developed. The method was used to determine the bilirubin compounds of bile obtained endoscopically from the common bile duct in 43 patients with gallstone disease and in 6 subjects without gallstones or liver disease. The bile samples were also assessed for the presence of beta-glucuronidase-producing bacteria. The amount of unconjugated bilirubin was significantly higher (p less than 0.01) in bile containing bacteria producing beta-glucuronidase than in bile without such bacterial strains. In six 'normal' bile samples the following quantities of bilirubin conjugates and unconjugated bilirubin were found (median and range): bilirubin monoglucoside-monoglucuronide (mixed conjugate), 61 (27-80) mumol/l; bilirubin diglucuronide, 632 (512-861) mumol/l; bilirubin monoglucuronide, 113 (70-175) mumol/l; and unconjugated bilirubin, 3 (1-7) mumol/l. These results are in good agreement with those obtained with other HPLC methods. The concentrations of unconjugated bilirubin were lower than those found when using conventional diazo methods and thin-layer chromatography. HPLC proved to be a useful tool in gallstone pathogenesis studies. Our results support bacterial glucuronidase as a possible pathogenic factor in pigment gallstone disease.