The fate of bilirubin-IXα glucuronide in cholestasis and during storage in vitro. Intramolecular rearrangement to positional isomers of glucuronic acid

Abstract

1. In aqueous solution above pH7 bilirubin-IXalpha 1-O-acylglucuronide rapidly isomerizes to the non-C-1 glucuronides by sequential migration of the bilirubin acyl group from position 1 to positions 2, 3 and 4 of the sugar moiety. The transformations are enhanced by increasing the pH. Compared with the rates at 37 degrees C the transformations are rather slow at 0 degrees C. Virtually complete inhibition is observed at values below pH6. The isomerization at 25 degrees C and pH 7.4 is not affected by the presence in the solutions of a molar excess of human serum albumin. 2. Isomerization in bile kept at 37 degrees C at pH7.7-7.8 is probably non-enzymic, as the rates of change are similar to those observed under comparable conditions for aqueous solutions of glucuronides of bilirubin-1Xalpha and of azodipyrrole. 3. Analysis without delay of normal biles of man and rats collected at 0 degrees C over a maximum period of 10 min shows that the bilirubin-IXalpha mono- and di-glucuronides consist exclusively of the 1-O-acyl isomers. 4. The mixtures of the four positional isomers of bilirubin-IXalpha glucuronide found in freshly collected biles of man and rats with cholestasis probably originate from initially synthesized 1-O-acylglucuronide by the same mechanism of sequential migration as has been observed in aqueous solutions of conjugated bilirubin-IXalpha.

Bilirubin Glucuronidation Revisited: Proper Assay Conditions to Estimate Enzyme Kinetics with Recombinant UGT1A1

“…Equally as problematic is the instability of bilirubin glucuronides, especially the monoglucuronides (Jansen, 1973;Blanckaert et al, 1978;Adachi et al, 1985). In aqueous solutions, the monoglucuronides rapidly disproportionate to diglucuronide and parent bilirubin (Adachi et al, 1985).…”

“…For this purpose, a simple and robust bilirubin glucuronidation assay is critical. However, the instability of bilirubin (Doumas et al, 1973) and its glucuronides (Jansen, 1973;Blanckaert et al, 1978;Adachi et al, 1985) makes in vitro bilirubin glucuronidation assays technically challenging. Furthermore, because the glucuronidation of bilirubin involves a sequential reaction that produces two monoglucuronides and a diglucuronide, establishment of initial rate conditions can be difficult, if not given particular attention.…”

Unconjugated bilirubin induces apoptosis in colon cancer cells by triggering mitochondrial depolarization

“…Bilirubin is secreted into bile as the 1-O-acylglucuronide conjugate, 28,82 which partially undergoes acyl migration in the gallbladder and intestine to form 2-, 3-and 4-O-acyl isomers. 83,84 Deconjugation of bilirubin 1-O-acylglucuronide, but not the other positional isomers, 84 occurs within the intestinal lumen both by spontaneous nonenzymic hydrolysis 29 and through the action of mucosal and bacterial ␤-glucuronidases 30,31 present primarily in the distal small bowel and colon, where luminal bilirubin levels are the highest. 32,85 Plasma unconjugated (but not conjugated) bilirubin also enters the gut via passive diffusion across the intestinal mucosa.…”

Synthesis of biliverdin and bilirubin glucuronides

“…The resulting anomeric methyl esters were separated by TLC with CHCla-MeOH (9: 1,2 developments). Whereas the lower moving anomer was identical with the azopigment derived from natural bilirubin P-glucuronides, the cw-anomer differed in RF-value from the previously [3,4] …”