Bilirubin diglucuronide formation in intact rats and in isolated Gunn rat liver

Jayanta Roy-Chowdhury, N. R. Chowdhury, U. Gartner, Allan W. Wolkoff, I. M. Arias

Research output: Contribution to journalArticle

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Abstract

Bilirubin diglucuronide (BDG) may be formed in vitro my microsomal UDP glucuronosyl transferase (EC 2.4.1.17)-mediated transfer of a second mole of glucuronic acid from UDP-glucuronic acid, or by dismutation of bilirubin monoglucuronide (BMG) to BDG and unconjugated bilirubin, catalyzed by an enzyme (EC 2.4.1.95) that is concentrated in plasma membrane-enriched fractions of rat liver. To evaluate the role of these two enzymatic mechanisms in vivo, [3H]bilirubin mono-[14C]glucuronide was biosynthesized, purified by thin-layer chromatography, and tracer doses were infused intravenously in homozygous Gunn (UDP glucuronyl transferase-deficient) rats or Wistar rats. Bilirubin conjugates in bile were separated by high-pressure liquid chromatography and 3H and 14C were quantitated. In Gunn rats, the 14C:3H ratio in BDG excreted in bile was twice the ratio in injected BMG. In Wistar rats the 14C:3H ratio in biliary BDG was 1.25 ± 0.06 (mean ± SEM) times the ratio in injected BMG. When double labeled BMG was injected in Wistar rats after injection of excess unlabeled unconjugated bilirubin (1.7 μmol), the 14C:3H ratio in BDG excreted in bile was identical to the ratio in injected BMG. Analysis of isomeric composition of bilirubin conjugates after alkaline hydrolysis or alkaline methanolysis indicated that the bile pigments retained the IX(α) configuration during these experiments. The results indicate that both enzymatic dismutation and UDP glucuronyl transferase function in vivo in BDG formation, and that dismutation is inhibited by a high intrahepatic concentration of unconjugated bilirubin. This hypothesis was supported by infusion of [3H]bilirubin-monoglucuronide in isolated perfused homozygous Gunn rat liver after depletion of intrahepatic bilirubin by perfusion with bovine serum albumin (2.5%), and after bilirubin repletion following perfusion with 0.34 mM bilirubin. From 20 to 25% of injected radioactivity was recovered in BDG in bile in the bilirubin-depleted state; only 8-10% of radioactivity was in BDG in bile after bilirubin repletion. After infusion of [3H]bilirubin di-[14C]glucuronide in homozygous Gunn rats, 5-7% of the injected pigment was excreted in bile as BMG. The 14C:3H ratio in the injected BDG was 10% greater than the 14C:3H ratio in BMG excreted in bile. These results indicate that in vivo, dismutation rather than partial hydrolysis, is responsible for BMG formation. Incubation of [3H]bilirubin, BDG and a rat liver plasma membrane preparation resulted in formation of BMG (3.3 nmol/min per mg protein) indicating that dismutation is also reversible in vitro.

Original languageEnglish (US)
Pages (from-to)595-603
Number of pages9
JournalJournal of Clinical Investigation
Volume69
Issue number3
StatePublished - 1982

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Gunn Rats
Bilirubin
Liver
Bile
Wistar Rats
Glucuronosyltransferase
bilirubin glucuronoside glucuronosyltransferase
Glucuronides
bilirubin diglucuronide
Radioactivity
Hydrolysis
Perfusion
Uridine Diphosphate Glucuronic Acid
Cell Membrane
Bile Pigments
bilirubin glucuronate
Glucuronic Acid
Uridine Diphosphate
Thin Layer Chromatography
Bovine Serum Albumin

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Bilirubin diglucuronide formation in intact rats and in isolated Gunn rat liver. / Roy-Chowdhury, Jayanta; Chowdhury, N. R.; Gartner, U.; Wolkoff, Allan W.; Arias, I. M.

In: Journal of Clinical Investigation, Vol. 69, No. 3, 1982, p. 595-603.

Research output: Contribution to journalArticle

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abstract = "Bilirubin diglucuronide (BDG) may be formed in vitro my microsomal UDP glucuronosyl transferase (EC 2.4.1.17)-mediated transfer of a second mole of glucuronic acid from UDP-glucuronic acid, or by dismutation of bilirubin monoglucuronide (BMG) to BDG and unconjugated bilirubin, catalyzed by an enzyme (EC 2.4.1.95) that is concentrated in plasma membrane-enriched fractions of rat liver. To evaluate the role of these two enzymatic mechanisms in vivo, [3H]bilirubin mono-[14C]glucuronide was biosynthesized, purified by thin-layer chromatography, and tracer doses were infused intravenously in homozygous Gunn (UDP glucuronyl transferase-deficient) rats or Wistar rats. Bilirubin conjugates in bile were separated by high-pressure liquid chromatography and 3H and 14C were quantitated. In Gunn rats, the 14C:3H ratio in BDG excreted in bile was twice the ratio in injected BMG. In Wistar rats the 14C:3H ratio in biliary BDG was 1.25 ± 0.06 (mean ± SEM) times the ratio in injected BMG. When double labeled BMG was injected in Wistar rats after injection of excess unlabeled unconjugated bilirubin (1.7 μmol), the 14C:3H ratio in BDG excreted in bile was identical to the ratio in injected BMG. Analysis of isomeric composition of bilirubin conjugates after alkaline hydrolysis or alkaline methanolysis indicated that the bile pigments retained the IX(α) configuration during these experiments. The results indicate that both enzymatic dismutation and UDP glucuronyl transferase function in vivo in BDG formation, and that dismutation is inhibited by a high intrahepatic concentration of unconjugated bilirubin. This hypothesis was supported by infusion of [3H]bilirubin-monoglucuronide in isolated perfused homozygous Gunn rat liver after depletion of intrahepatic bilirubin by perfusion with bovine serum albumin (2.5{\%}), and after bilirubin repletion following perfusion with 0.34 mM bilirubin. From 20 to 25{\%} of injected radioactivity was recovered in BDG in bile in the bilirubin-depleted state; only 8-10{\%} of radioactivity was in BDG in bile after bilirubin repletion. After infusion of [3H]bilirubin di-[14C]glucuronide in homozygous Gunn rats, 5-7{\%} of the injected pigment was excreted in bile as BMG. The 14C:3H ratio in the injected BDG was 10{\%} greater than the 14C:3H ratio in BMG excreted in bile. These results indicate that in vivo, dismutation rather than partial hydrolysis, is responsible for BMG formation. Incubation of [3H]bilirubin, BDG and a rat liver plasma membrane preparation resulted in formation of BMG (3.3 nmol/min per mg protein) indicating that dismutation is also reversible in vitro.",
author = "Jayanta Roy-Chowdhury and Chowdhury, {N. R.} and U. Gartner and Wolkoff, {Allan W.} and Arias, {I. M.}",
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AU - Roy-Chowdhury, Jayanta

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AU - Wolkoff, Allan W.

AU - Arias, I. M.

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N2 - Bilirubin diglucuronide (BDG) may be formed in vitro my microsomal UDP glucuronosyl transferase (EC 2.4.1.17)-mediated transfer of a second mole of glucuronic acid from UDP-glucuronic acid, or by dismutation of bilirubin monoglucuronide (BMG) to BDG and unconjugated bilirubin, catalyzed by an enzyme (EC 2.4.1.95) that is concentrated in plasma membrane-enriched fractions of rat liver. To evaluate the role of these two enzymatic mechanisms in vivo, [3H]bilirubin mono-[14C]glucuronide was biosynthesized, purified by thin-layer chromatography, and tracer doses were infused intravenously in homozygous Gunn (UDP glucuronyl transferase-deficient) rats or Wistar rats. Bilirubin conjugates in bile were separated by high-pressure liquid chromatography and 3H and 14C were quantitated. In Gunn rats, the 14C:3H ratio in BDG excreted in bile was twice the ratio in injected BMG. In Wistar rats the 14C:3H ratio in biliary BDG was 1.25 ± 0.06 (mean ± SEM) times the ratio in injected BMG. When double labeled BMG was injected in Wistar rats after injection of excess unlabeled unconjugated bilirubin (1.7 μmol), the 14C:3H ratio in BDG excreted in bile was identical to the ratio in injected BMG. Analysis of isomeric composition of bilirubin conjugates after alkaline hydrolysis or alkaline methanolysis indicated that the bile pigments retained the IX(α) configuration during these experiments. The results indicate that both enzymatic dismutation and UDP glucuronyl transferase function in vivo in BDG formation, and that dismutation is inhibited by a high intrahepatic concentration of unconjugated bilirubin. This hypothesis was supported by infusion of [3H]bilirubin-monoglucuronide in isolated perfused homozygous Gunn rat liver after depletion of intrahepatic bilirubin by perfusion with bovine serum albumin (2.5%), and after bilirubin repletion following perfusion with 0.34 mM bilirubin. From 20 to 25% of injected radioactivity was recovered in BDG in bile in the bilirubin-depleted state; only 8-10% of radioactivity was in BDG in bile after bilirubin repletion. After infusion of [3H]bilirubin di-[14C]glucuronide in homozygous Gunn rats, 5-7% of the injected pigment was excreted in bile as BMG. The 14C:3H ratio in the injected BDG was 10% greater than the 14C:3H ratio in BMG excreted in bile. These results indicate that in vivo, dismutation rather than partial hydrolysis, is responsible for BMG formation. Incubation of [3H]bilirubin, BDG and a rat liver plasma membrane preparation resulted in formation of BMG (3.3 nmol/min per mg protein) indicating that dismutation is also reversible in vitro.

AB - Bilirubin diglucuronide (BDG) may be formed in vitro my microsomal UDP glucuronosyl transferase (EC 2.4.1.17)-mediated transfer of a second mole of glucuronic acid from UDP-glucuronic acid, or by dismutation of bilirubin monoglucuronide (BMG) to BDG and unconjugated bilirubin, catalyzed by an enzyme (EC 2.4.1.95) that is concentrated in plasma membrane-enriched fractions of rat liver. To evaluate the role of these two enzymatic mechanisms in vivo, [3H]bilirubin mono-[14C]glucuronide was biosynthesized, purified by thin-layer chromatography, and tracer doses were infused intravenously in homozygous Gunn (UDP glucuronyl transferase-deficient) rats or Wistar rats. Bilirubin conjugates in bile were separated by high-pressure liquid chromatography and 3H and 14C were quantitated. In Gunn rats, the 14C:3H ratio in BDG excreted in bile was twice the ratio in injected BMG. In Wistar rats the 14C:3H ratio in biliary BDG was 1.25 ± 0.06 (mean ± SEM) times the ratio in injected BMG. When double labeled BMG was injected in Wistar rats after injection of excess unlabeled unconjugated bilirubin (1.7 μmol), the 14C:3H ratio in BDG excreted in bile was identical to the ratio in injected BMG. Analysis of isomeric composition of bilirubin conjugates after alkaline hydrolysis or alkaline methanolysis indicated that the bile pigments retained the IX(α) configuration during these experiments. The results indicate that both enzymatic dismutation and UDP glucuronyl transferase function in vivo in BDG formation, and that dismutation is inhibited by a high intrahepatic concentration of unconjugated bilirubin. This hypothesis was supported by infusion of [3H]bilirubin-monoglucuronide in isolated perfused homozygous Gunn rat liver after depletion of intrahepatic bilirubin by perfusion with bovine serum albumin (2.5%), and after bilirubin repletion following perfusion with 0.34 mM bilirubin. From 20 to 25% of injected radioactivity was recovered in BDG in bile in the bilirubin-depleted state; only 8-10% of radioactivity was in BDG in bile after bilirubin repletion. After infusion of [3H]bilirubin di-[14C]glucuronide in homozygous Gunn rats, 5-7% of the injected pigment was excreted in bile as BMG. The 14C:3H ratio in the injected BDG was 10% greater than the 14C:3H ratio in BMG excreted in bile. These results indicate that in vivo, dismutation rather than partial hydrolysis, is responsible for BMG formation. Incubation of [3H]bilirubin, BDG and a rat liver plasma membrane preparation resulted in formation of BMG (3.3 nmol/min per mg protein) indicating that dismutation is also reversible in vitro.

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