Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1980 Jan 1;185(1):115–128. doi: 10.1042/bj1850115

Analysis of bilirubin and bilirubin mono- and di-conjugates. Determination of their relative amounts in biological samples.

N Blanckaert
PMCID: PMC1161276  PMID: 7378044

Abstract

1. A novel method for determination of the relative amounts of unconjugated bilirubin and sugar mono- and di-conjugates of bilirubin in biological samples, including serum, is described and illustrated by its application to the analysis of bilinoids in rat bile. 2. The method is based on specific conversion of the carbohydrate conjugates of bilirubin into the corresponding mono- or di-methyl esters by base-catalysed transesterification in methanol. Under the selected reaction conditions, unconjugated biliru-in remains intact and no dipyrrole exchange in the bilinoids is detectable; transesterification of bilirubin mono- or di-glucuronide is virtually complete (approx. 99%), and sponification is negligible (less than 1%); recovery of the pigments is approx. 95%. 3. The reaction products bilirubin and its methyl esters are separated by t.l.c. and determined spectrophotometrically; the two isomeric bilirubin-IX alpha monomethyl esters are separated and therefore can be determined individually. 4. Reference bilirubin mono- and di-methyl esters have been synthesized and characterized, and the two isomers of bilirubin-IX alpha monomethyl ester and bilirubin dimethyl ester were obtained individually, in crystalline form. 5. With this new method, virtually all bilinoids (over 99%) in normal rat bile have been found to be conjugated, with diconjugates (71%) predominating. A significantly increased proportion of monoconjugates is present in bile collected from heterozygous Gunn rats or from normal rats that were refused with large amounts of bilirubin.

Full text

PDF
118

Images in this article

119Fig. 2.
on p.119

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. BRODERSEN R., VIND I. A specific method for quantitative determination of unconjugated bilirubin and mesobilirubin. Scand J Clin Lab Invest. 1963;15:225–232. doi: 10.3109/00365516309079737. [DOI] [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. Boonyapisit S. T., Trotman B. W., Ostrow J. D., Olivieri P. J., Gallo D. Measurement of conjugated and unconjugated bilirubin in bile. II. A new thin-layer chromatographic method. J Lab Clin Med. 1976 Nov;88(5):857–863. [PubMed] [Google Scholar]
  8. Bratlid D., Winsnes A. Determination of conjugated and unconjugated bilirubin by methods based on direct spectrophotometry and chloroform-extraction. A reappraisal. Scand J Clin Lab Invest. 1971 Sep;28(1):41–48. doi: 10.3109/00365517109090661. [DOI] [PubMed] [Google Scholar]
  9. COLE P. G., LATHE G. H., BILLING B. H. Separation of the bile pigments of serum, bile and urine. Biochem J. 1954 Jul;57(3):514–518. doi: 10.1042/bj0570514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Compernolle F., Van Hees G. P., Fevery J., Heirwegh K. P. Mass-spectrometric structure elucidation of dog bile azopigments as the acyl glycosides of glucopyranose and xylopyranose. Biochem J. 1971 Dec;125(3):811–819. doi: 10.1042/bj1250811. [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 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]
  14. 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]
  15. Gordon E. R., Chan T. H., Samodai K., Goresky C. A. The isolation and further characterization of the bilirubin tetrapyrroles in bile-containing human duodenal juice and dog gall-bladder bile. Biochem J. 1977 Oct 1;167(1):1–8. doi: 10.1042/bj1670001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. JACOBS S. L., HENRY R. J., SEGALOVE M. STUDIES ON THE DETERMINATION OF BILE PIGMENTS. V. COMPARISON OF SOME METHODS FOR DETERMINATION OF TOTAL, FREE, AND CONJUGATED BILIRUBIN IN SERUM. Clin Chem. 1964 May;10:433–439. [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Kuenzle C. C. Bilirubin conjugates of human bile. Nuclear-magnetic-resonance, infrared and optical spectra of model compounds. Biochem J. 1970 Sep;119(3):395–409. doi: 10.1042/bj1190395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lightner D. A., Wooldridge T. A., McDonagh A. F. Configurational isomerization of bilirubin and the mechanism of jaundice phototherapy. Biochem Biophys Res Commun. 1979 Jan 30;86(2):235–243. doi: 10.1016/0006-291x(79)90857-x. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. McDonagh A. F. Thermal and photochemical reactions of bilirubin IX-alpha. Ann N Y Acad Sci. 1975 Apr 15;244:553–569. doi: 10.1111/j.1749-6632.1975.tb41554.x. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. Thompson R. P., Hofmann A. F. Separation of bilirubin and its conjugates by thin layer chromatography. Clin Chim Acta. 1971 Dec;35(2):517–519. doi: 10.1016/0009-8981(71)90234-8. [DOI] [PubMed] [Google Scholar]
  32. Van Roy F. P., Heirwegh K. P. Determination of bilirubin glucuronide and assay of glucuronyltransferase with bilirubin as acceptor. Biochem J. 1968 Apr;107(4):507–518. doi: 10.1042/bj1070507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. WEBER A. P., SCHALM L. Quantitative separation and determination of bilirubin and conjugated bilirubin in human serum. Clin Chim Acta. 1962 Nov;7:805–810. doi: 10.1016/0009-8981(62)90063-3. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES