bilirubin-diglucuronide and Cholelithiasis

Bacterial beta-glucuronidase causes deconjugation of bilirubin diglucuronide resulting in the precipitation of calcium bilirubinate, which contributes to biliary sludge and stone formation. This process is attributed to enzyme activity produced by the aerobic enterobacteriaceae such as Escherichia coli and Klebsiella sp. The presence of Clostridium sp. was detected in 48 of 56 intrahepatic stones by using polymerase chain reaction techniques and cultured Clostridium perfringens from 14 of 18 unblocked biliary stents. Such bacteria are reported to produce beta-glucuronidase activity. The aim of this study was to determine the proportion of biliary bacteria isolated from pigment stones and stents that produce beta-glucuronidase and to compare the enzyme activity expressed by the different bacteria in human bile.. A total of 202 bacteria were isolated from blocked and unblocked biliary stents and pigment ductal stones recovered from patients. Of these, 61 bacteria expressed beta-glucuronidase activity in brain heart infusion broth. These 61 bacteria were subsequently grown in human bile under aerobic or anaerobic conditions to the early stationary phase and assayed for beta-glucuronidase activity by using rho-nitrophenyl beta-D glucuronide as substrate. Results were normalized and reported as units of enzyme activity per milligram protein of the bacteria.. C. perfringens produced beta-glucuronidase enzyme activity that was 34-fold higher than that for E coli, Staphylococcus, Corynebacterium sp., Bacillus sp., Enterococcus sp., Acinetobacter sp., Streptococcus sp., and Klebsiella sp.. C. perfringens with its higher enzyme activity is more important in the deconjugation of bilirubin diglucuronide than E coli and Klebsiella sp.

Topics: Bacteria, Anaerobic; Bile; Bile Pigments; Bilirubin; Cholelithiasis; Clostridium perfringens; Escherichia coli; Glucuronidase; Humans; In Vitro Techniques; Klebsiella; Stents

Pigment gallstones contain considerable amounts of unconjugated bilirubin (UCB) in the form of calcium bilirubinate and/or bilirubin polymers. Since more than 98% of bile pigments are excreted as conjugates of bilirubin, the source of this UCB needs to be identified. By using a rapid h.p.l.c. method, we compared the non-enzymic hydrolysis of bilirubin monoglucuronide (BMG) and bilirubin diglucuronide (BDG) to UCB in model bile and in native guinea-pig bile. Model biles containing 50 microM solutions of pure BMG and BDG were individually incubated in 25 mM-sodium taurocholate (NaTC) and 0.4 M-imidazole/5 mM-ascorbate buffer (TC-BUF) at 37 degrees C. Over an 8 h period, BMG hydrolysis produced 4-6 times more UCB than BDG hydrolysis. At pH 7.4, 25% of the BMG was converted into UCB, whereas only 4.5% of BDG was converted into UCB. Hydrolysis rates for both BMG and BDG followed the pH order 7.8 greater than 7.6 approximately equal to 7.4 greater than 7.1 Incubation with Ca2+ (6.2 mM) at pH 7.4 in TC-BUF resulted in precipitated bile pigment which, at 100 X magnification, appeared similar to precipitates seen in the bile of patients with pigment gallstones. At pH 7.4, lecithin (crude phosphatidylcholine) (4.2 mM) was a potent inhibitor of hydrolysis of BMG and BDG. The addition of a concentration of cholesterol equimolar with that of lecithin eliminated this inhibitory effect. Guinea-pig gallbladder bile incubated with glucaro-1,4-lactone (an inhibitor of beta-glucuronidase) underwent hydrolysis similar to the model bile systems. The non-enzymic hydrolysis of bile pigments, especially BMG, may be an important mechanism of bile-pigment precipitation and, ultimately, of gallstone formation.

Topics: Animals; Bile; Bilirubin; Calcium; Cholelithiasis; Cholesterol; Chromatography, High Pressure Liquid; Guinea Pigs; Hydrolysis; Models, Biological; Phosphatidylcholines