Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1982 Mar;69(3):595–603. doi: 10.1172/JCI110486

Bilirubin Diglucuronide Formation in Intact Rats and in Isolated Gunn Rat Liver

J Roy Chowdhury 1, N Roy Chowdhury 1, Ulrich Gärtner 1, Allan W Wolkoff 1, Irwin M Arias 1
PMCID: PMC371016  PMID: 6801091

Abstract

Bilirubin diglucuronide (BDG) may be formed in vitro by 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 IXα 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.

Full text

PDF
595

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BILLING B. H., COLE P. G., LATHE G. H. The excretion of bilirubin as a diglucuronide giving the direct van den Bergh reaction. Biochem J. 1957 Apr;65(4):774–784. doi: 10.1042/bj0650774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barrett P. V., Mullins F. X., Berlin N. I. Studies on the biosynthesis production of bilirubin-C14: an improved method utilizing delta-aminolevulinic acid-4-C14 in dogs. J Lab Clin Med. 1966 Dec;68(6):905–912. [PubMed] [Google Scholar]
  3. Blanckaert N. Analysis of bilirubin and bilirubin mono- and di-conjugates. Determination of their relative amounts in biological samples. Biochem J. 1980 Jan 1;185(1):115–128. doi: 10.1042/bj1850115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blanckaert N., Compernolle F., Leroy P., Van Houtte R., Fevery J., Heirwegh K. P. The fate of bilirubin-IXalpha glucuronide in cholestasis and during storage in vitro. Intramolecular rearrangement to positional isomers of glucuronic acid. Biochem J. 1978 Apr 1;171(1):203–214. doi: 10.1042/bj1710203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blanckaert N., Gollan J., Schmid R. Bilirubin diglucuronide synthesis by a UDP-glucuronic acid-dependent enzyme system in rat liver microsomes. Proc Natl Acad Sci U S A. 1979 Apr;76(4):2037–2041. doi: 10.1073/pnas.76.4.2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blanckaert N., Gollan J., Schmid R. Mechanism of bilirubin diglucuronide formation in intact rats: bilirubin diglucuronide formation in vivo. J Clin Invest. 1980 Jun;65(6):1332–1342. doi: 10.1172/JCI109797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chowdhury J. R., Chowdhury N. R., Bhargava M. M., Arias I. M. Purification and partial characterization of rat liver bilirubin glucuronoside glucuronosyltransferase. J Biol Chem. 1979 Sep 10;254(17):8336–8339. [PubMed] [Google Scholar]
  8. Chowdhury J. R., Jansen P. L., Fischberg E. B., Daniller A., Arias I. M. Hepatic conversion of bilirubin monoglucuronide to diglucuronide in uridine diphosphate-glucuronyl transferase-deficient man and rat by bilirubin glucuronoside glucuronosyltransferase. J Clin Invest. 1978 Jul;62(1):191–196. doi: 10.1172/JCI109105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fevery J., Blanckaert N., Heirwegh K. P., Préaux A. M., Berthelot P. Unconjugated bilirubin and an increased proportion of bilirubin monoconjugates in the bile of patients with Gilbert's syndrome and Crigler-Najjar disease. J Clin Invest. 1977 Nov;60(5):970–979. doi: 10.1172/JCI108877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fevery J., Leroy P., Van de Vijver M., Heirwegh K. P. Structures of bilirubin conjugates synthesized in vitro from bilirubin and uridine diphosphate glucuronic acid, uridine diphosphate glucose or uridine diphosphate xylose by preparations from rat liver. Biochem J. 1972 Sep;129(3):635–644. doi: 10.1042/bj1290635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fevery J., Van Damme B., Michiels R., De Groote J., Heirwegh K. P. Bilirubin conjugates in bile of man and rat in the normal state and in liver disease. J Clin Invest. 1972 Sep;51(9):2482–2492. doi: 10.1172/JCI107062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gordon E. R., Dadoun M., Goresky C. A., Chan T. H., Perlin A. S. The isolation of an azobilirubin beta-D-monoglucoside from dog gall-bladder bile. Biochem J. 1974 Oct;143(1):97–105. doi: 10.1042/bj1430097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heirwegh K. P., Fevery J., Michiels R., van Hees G. P., Compernolle F. Separation by thin-layer chromatography and structure elucidation of bilirubin conjugates isolated from dog bile. Biochem J. 1975 Feb;145(2):185–199. doi: 10.1042/bj1450185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Heirwegh K. P., Van Hees G. P., Leroy P., Van Roy F. P., Jansen F. H. Heterogeneity of bile pigment conjugates as revealed by chromatography of their ethyl anthranilate azopigments. Biochem J. 1970 Dec;120(4):877–890. doi: 10.1042/bj1200877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Heirwegh K. P., Van de Vijver M., Fevery J. Assay and properties of dititonin-activated bilirubin uridine diphosphate glucuronyltransferase from rat liver. Biochem J. 1972 Sep;129(3):605–618. doi: 10.1042/bj1290605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Howe R. B., Berk P. D., Bloomer J. R., Berlin N. I. Preparation and properties of specifically labeled radiochemically stable 3H-bilirubin. J Lab Clin Med. 1970 Mar;75(3):499–502. [PubMed] [Google Scholar]
  17. Jansen P. L., Chowdhury J. R., Fischberg E. B., Arias I. M. Enzymatic conversion of bilirubin monoglucuronide to diglucuronide by rat liver plasma membranes. J Biol Chem. 1977 Apr 25;252(8):2710–2716. [PubMed] [Google Scholar]
  18. Jansen P. L., Tangerman A. Separation and characterization of bilirubin conjugates by high-performance liquid chromatography. J Chromatogr. 1980 Apr 11;182(1):100–104. doi: 10.1016/s0378-4347(00)81656-4. [DOI] [PubMed] [Google Scholar]
  19. Jansen P. L. The isomerisation of bilirubin monoglucuronide. Clin Chim Acta. 1973 Dec 12;49(2):233–240. doi: 10.1016/0009-8981(73)90296-9. [DOI] [PubMed] [Google Scholar]
  20. McDonagh A. F., Assisi F. Commercial bilirubin: A trinity of isomers. FEBS Lett. 1971 Nov 1;18(2):315–317. doi: 10.1016/0014-5793(71)80475-1. [DOI] [PubMed] [Google Scholar]
  21. Onishi S., Itoh S., Kawade N., Isobe K., Sugiyama S. An accurate and sensitive analysis by high-pressure liquid chromatography of conjugated and unconjugated bilirubin IX-alpha in various biological fluids. Biochem J. 1980 Jan 1;185(1):281–284. doi: 10.1042/bj1850281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Touster O., Aronson N. N., Jr, Dulaney J. T., Hendrickson H. Isolation of rat liver plasma membranes. Use of nucleotide pyrophosphatase and phosphodiesterase I as marker enzymes. J Cell Biol. 1970 Dec;47(3):604–618. doi: 10.1083/jcb.47.3.604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Van Roy F. P., Meuwissen J. A., De Meuter F., Heirwegh K. P. Determination of bilirubin in liver homogenates and serum with diazotized p-iodoaniline. Clin Chim Acta. 1971 Jan;31(1):109–118. doi: 10.1016/0009-8981(71)90367-6. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES