β-Glucuronidase in common duct bile, methodological aspects, variation of pH optima and relation to gallstones

Abstract

β-Glucuronidase of human or bacterial origin may deconjugate bilirubin diglucuronide, causing pigment gallstones. Intrinsic interference by biliary compounds must be minimized for accurate assay of β-glucuronidase.

We report a modified ion-pair extraction of interfering substances by tetrahexylammonium chloride (THAC) in ethyl acetate in the presence of albumin, and a microtitre plate assay for biliary β-glucuronidase activity in bile with the substrate β-nitrophenol-glucuronide. Adding albumin improved the recovery of β-glucuronidase activity to 99.8% (CV 1.9%), and 92.2% of the bilirubin in bile samples was extracted in one step. Competitive inhibition was overcome by increasing the substrate concentration. In endoscopically obtained common duct bile from 44 patients, five different β-glucuronidase activity peaks were identified, at pH 3.9, 4.8, 5.3, 5.8 and 7.2. The pH profiles were classified into one bacterial pattern and five patterns for presumed human β-glucuronidase. Of the latter patterns, four displayed dual activity peaks. In a second sample, obtained at follow up in four patients, their original pH profile was maintained.

In conclusion, using the modified purification and assay system, we found functionally diverse subcategories of human β-glucuronidase with respect to activity at variable pH. Our results indicate that several pH optima have to be taken into consideration in order to clarify the role of human biliary β-glucuronidase in the pathogenesis of pigment gallstones. Bacterial β-glucuronidase activity was associated with duodenal diverticula (p<0.05) and common duct stones (p<0.05).

Key Words:

• β-glucuronidase
• common duct bile
• choledocholithiasis
• duodenal diverticula
• endoscopy
• pH optimum
• tetrahexylammonium chloride