Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1980 Jun;65(6):1332–1342. doi: 10.1172/JCI109797

Mechanism of Bilirubin Diglucuronide Formation in Intact Rats

BILIRUBIN DIGLUCURONIDE FORMATION IN VIVO

Norbert Blanckaert 1,2, John Gollan 1,2, Rudi Schmid 1,2
PMCID: PMC371471  PMID: 6773983

Abstract

Although it is well established that bilirubin monoglucuronide is formed in the liver from bilirubin by a microsomal bilirubin uridine diphosphate (UDP)-glucuronosyltransferase, the subcellular site of conversion of monoglucuronide to diglucuronide and the molecular mechanism involved in diglucuronide synthesis have not been identified. Based on in vitro studies, it has been proposed that two fundamentally different enzyme systems may be involved in diglucuronide synthesis in rat liver: (a) a microsomal UDP-glucuronosyltransferase system requiring UDP-glucuronic acid as sugar donor or (b) a transglucuronidation mechanism that involves transfer of a glucuronosyl residue from one monoglucuronide molecule to another, catalyzed by a liver plasma membrane enzyme. To clarify the mechanism by which bilirubin monoglucuronide is converted in vivo to diglucuronide, three different experimental approaches were used. First, normal rats were injected with either equal amounts of bilirubin-IIIα [14C]monoglucuronide and unlabeled bilirubin-XIIIα monoglucuronide, or bilirubin-XIIIα [14C]monoglucuronide and unlabeled bilirubin-IIIα monoglucuronide. Analysis of radiolabeled diglucuronide excreted in bile showed that [14C]glucuronosyl residues were not transferred between monoglucuronide molecules. Second, in normal rats infused intravenously with dual-labeled [3H]bilirubin [14C]monoglucuronide, no transfer or exchange of the [14C]glucuronosyl group between injected and endogenously produced bilirubin monoglucuronide could be detected in the excreted bilirubin diglucuronide. Third, in homozygous Gunn rats, injected 14C-labeled or unlabeled bilirubin mono- or diglucuronides were excreted in bile unchanged (except that diglucuronide was hydrolyzed to a minor degree). This indicates that Gunn rats, which lack bilirubin UDP-glucuronosyltransferase activity, are unable to convert injected monoglucuronide to diglucuronide. Collectively, these findings establish that a transglucuronidation mechanism is not operational in vivo and support the concept that bilirubin diglucuronide is formed by a microsomal UDP-glucuronosyltransferase system.

Full text

PDF
1332

Selected References

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

  1. Acocella G., Tenconi L. T., Armas-Merino R., Raia S., Billing B. H. Does deconjugation of bilirubin glucuronide occur in obstructive jaundice? Lancet. 1968 Jan 13;1(7533):68–69. doi: 10.1016/s0140-6736(68)90069-x. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Blanckaert N., Fevery J., Heirwegh K. P., Compernolle F. Characterization of the major diazo-positive pigments in bile of homozygous Gunn rats. Biochem J. 1977 Apr 15;164(1):237–249. doi: 10.1042/bj1640237. [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., Heirwegh K. P., Compernolle F. Synthesis and separation by thin-layer chromatography of bilirubin-IX isomers. Their identification as tetrapyrroles and dipyrrolic ethyl anthranilate azo derivatives. Biochem J. 1976 May 1;155(2):405–417. doi: 10.1042/bj1550405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blanckaert N., Heirwegh K. P., Zaman Z. Comparison of the biliary excretion of the four isomers of bilirubin-IX in Wistar and homozygous Gunn rats. Biochem J. 1977 Apr 15;164(1):229–236. doi: 10.1042/bj1640229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. CARBONE J. V., GRODSKY G. M. Constitutional nonhemolytic hyperbilirubinemia in the rat: defect of bilirubin conjugation. Proc Soc Exp Biol Med. 1957 Mar;94(3):461–463. doi: 10.3181/00379727-94-22979. [DOI] [PubMed] [Google Scholar]
  9. Campbell M. T., Dutton G. J. The formation and distribution of bilirubin monoglucuronide and diglucuronide in rat liver slices. Biochem J. 1979 Jun 1;179(3):473–477. doi: 10.1042/bj1790473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Compernolle F., Van Hees G. P., Blanckaert N., Heirwegh K. P. Glucuronic acid conjugates of bilirubin-IXalpha in normal bile compared with post-obstructive bile. Transformation of the 1-O-acylglucuronide into 2-, 3-, and 4-O-acylglucuronides. Biochem J. 1978 Apr 1;171(1):185–201. doi: 10.1042/bj1710185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Fevery J., Van de Vijver M., Michiels R., Heirwegh K. P. Comparison in different species of biliary bilirubin-IX alpha conjugates with the activities of hepatic and renal bilirubin-IX alpha-uridine diphosphate glycosyltransferases. Biochem J. 1977 Jun 15;164(3):737–746. doi: 10.1042/bj1640737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gollan J. L., Huang S. N., Billing B., Sherlock S. Prolonged survival in three brothers with severe type 2 Crigler-Najjar syndrome. Ultrastructural and metabolic studies. Gastroenterology. 1975 Jun;68(6):1543–1555. [PubMed] [Google Scholar]
  15. Gordon E. R., Goresky C. A., Chang T. H., Perlin A. S. The isolation and characterization of bilirubin diglucuronide, the major bilirubin conjugate in dog and human bile. Biochem J. 1976 Jun 1;155(3):477–486. doi: 10.1042/bj1550477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goresky C. A., Gordon E. R., Shaffer E. A., Paré P., Carassavas D., Aronoff A. Definition of a conjugation of dysfunction in Gilbert's syndrome: studies of the handling of bilirubin loads and of the pattern of bilirubin conjugates secreted in bile. Clin Sci Mol Med. 1978 Jul;55(1):63–71. doi: 10.1042/cs0550063. [DOI] [PubMed] [Google Scholar]
  17. Heirwegh K. P., Fevery J., Meuwissen J. A., De Groote J., Compernolle F., Desmet V., Van Roy F. P. Recent advances in the separation and analysis of diazo-positive bile pigments. Methods Biochem Anal. 1974;22:205–250. doi: 10.1002/9780470110423.ch5. [DOI] [PubMed] [Google Scholar]
  18. Heirwegh K. P., Meuwissen J. A., Fevery J. Critique of the assay and significance of bilirubin conjugation. Adv Clin Chem. 1973;16:239–288. doi: 10.1016/s0065-2423(08)60347-9. [DOI] [PubMed] [Google Scholar]
  19. Jansen F. H., Billing B. H. The identification of monoconjugates of bilirubin in bile as amide derivatives. Biochem J. 1971 Dec;125(3):917–919. doi: 10.1042/bj1250917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Noir B. A. Bilirubin conjugates in bile of man, rat and dog. Semi-quantitative analysis of bile composition by thin-layer chromatography. Biochem J. 1976 May 1;155(2):365–373. doi: 10.1042/bj1550365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. OSTROW J. D., HAMMAKER L., SCHMID R. The preparation of crystalline bilirubin-C14. J Clin Invest. 1961 Aug;40:1442–1452. doi: 10.1172/JCI104375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ostrow J. D., Boonyapisit S. T. Inaccuracies in measurement of conjugated and unconjugated bilirubin in bile with ethyl anthranilate diazo and solvent-partition methods. Biochem J. 1978 Jul 1;173(1):263–267. doi: 10.1042/bj1730263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ostrow J. D., Murphy N. H. Isolation and properties of conjugated bilirubin from bile. Biochem J. 1970 Nov;120(2):311–327. doi: 10.1042/bj1200311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. SCHALMEN L., WEBER A. P. [Bilirubin pigments in the blood and bile after loading with bilirubin in man]. Ned Tijdschr Geneeskd. 1962 May 26;106:1079–1084. [PubMed] [Google Scholar]
  27. SCHMID R., AXELROD J., HAMMAKER L., SWARM R. L. Congenital jaundice in rats, due to a defect in glucuronide formation. J Clin Invest. 1958 Aug;37(8):1123–1130. doi: 10.1172/JCI103702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. SCHMID R., HAMMAKER L. METABOLISM AND DISPOSITION OF C14-BILIRUBIN IN CONGENITAL NONHEMOLYTIC JAUNDICE. J Clin Invest. 1963 Nov;42:1720–1734. doi: 10.1172/JCI104858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schmid R. Bilirubin metabolism: state of the art. Gastroenterology. 1978 Jun;74(6):1307–1312. [PubMed] [Google Scholar]

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

RESOURCES