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. 1977 Apr 15;164(1):237–249. doi: 10.1042/bj1640237

Characterization of the major diazo-positive pigments in bile of homozygous Gunn rats

Norbert Blanckaert *, Johan Fevery *, Karel P M Heirwegh *, Frans Compernolle †
PMCID: PMC1164779  PMID: 880230

Abstract

Bilinoid pigments in bile of homozygous Gunn rats (jj) were analysed either after formation of dipyrrolic ethyl anthranilate azo derivatives or as the unmodified parent tetrapyrroles. 1. T.l.c. of the azo derivatives revealed seven major unconjugated components which were structurally characterized by chemical tests, spectrophotometry and mass spectrometry. In addition, two minor components were identified as azodipyrrole (A+B)-glucoside and azodipyrrole (A+B)-β-d-glucuronide. 2. Extraction and t.l.c. of the tetrapyrrolic pigments showed 13 major yellow diazo-positive bands. Four of them, accounting for 59% of total diazo-positive material, were identified as unconjugated bilirubin-IXα, -IXβ, -IXγ and -IXδ. A fifth band (16%) was characterized as a mixture of two isomeric monohydroxyl derivatives and another band (8%) as a dihydroxyl derivative of bilirubin-IXα. 3. Although unconjugated bilirubin-IXα constitutes one-third of total diazo-positive material in bile of our strain of Gunn rats, the daily amount excreted represented only about 3–4% of daily bilirubin production. 4. Phototherapy caused a 2.2-fold increase in the biliary output of diazo-positive bilinoids, but did not affect markedly their composition. However, an additional diazo-negative pigment, accounting for one-third of total yellow colour, was observed but was not identified. Mass-spectral data on two dipyrrolic azopigments have been deposited as Supplementary Publication SUP 50076 (3 pages) with the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1977) 161, 1.

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Selected References

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  1. ARIAS I. M., LONDON I. M. Bilirubin glucuronide formation in vitro; demonstration of a defect in Gilbert's disease. Science. 1957 Sep 20;126(3273):563–564. doi: 10.1126/science.126.3273.563. [DOI] [PubMed] [Google Scholar]
  2. Arias I. M., Gartner L. M., Cohen M., Ezzer J. B., Levi A. J. Chronic nonhemolytic unconjugated hyperbilirubinemia with glucuronyl transferase deficiency. Clinical, biochemical, pharmacologic and genetic evidence for heterogeneity. Am J Med. 1969 Sep;47(3):395–409. doi: 10.1016/0002-9343(69)90224-1. [DOI] [PubMed] [Google Scholar]
  3. Berk P. D., Bloomer J. R., Howe R. B., Berlin N. I. Constitutional hepatic dysfunction (Gilbert's syndrome). A new definition based on kinetic studies with unconjugated radiobilirubin. Am J Med. 1970 Sep;49(3):296–305. doi: 10.1016/s0002-9343(70)80020-1. [DOI] [PubMed] [Google Scholar]
  4. Berk P. D., Scharschmidt B. F., Waggoner J. G., White S. C. The effect of repeated phlebotomy on bilirubin turnover, bilirubin clearance and unconjugated hyperbilirubinaemia in the Crigler-Najjar syndrome and the jaundiced Gunn rat: application of computers to experimental design. Clin Sci Mol Med. 1976 May;50(5):333–348. doi: 10.1042/cs0500333. [DOI] [PubMed] [Google Scholar]
  5. Berry C. S., Zarembo J. E., Ostrow J. D. Evidence for conversion of bilirubin to dihydroxyl derivatives in the gunn rat. Biochem Biophys Res Commun. 1972 Dec 4;49(5):1366–1375. doi: 10.1016/0006-291x(72)90617-1. [DOI] [PubMed] [Google Scholar]
  6. Black M., Billing B. H. Hepatic bilirubin udp-glucuronyl transferase activity in liver disease and gilbert's syndrome. N Engl J Med. 1969 Jun 5;280(23):1266–1271. doi: 10.1056/NEJM196906052802303. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Bonnet R., Stewart J. C. Photo-oxidation of bilirubin in hydroxylic solvents. J Chem Soc Perkin 1. 1975;(3):224–231. [PubMed] [Google Scholar]
  9. 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]
  10. CRIGLER J. F., Jr, NAJJAR V. A. Congenital familial nonhemolytic jaundice with kernicterus. Pediatrics. 1952 Aug;10(2):169–180. [PubMed] [Google Scholar]
  11. Compernolle F., Blanckaert N., Heirwegh K. P. Mass spectral study of derivatives of the four bilirubin-IX isomers. Biomed Mass Spectrom. 1976 Aug;3(4):155–160. doi: 10.1002/bms.1200030403. [DOI] [PubMed] [Google Scholar]
  12. Compernolle F., Jansen F. H., Heirwegh K. P. Mass-spectrometric study of the azopigments obtained from bile pigments with diazotized ethyl anthranilate. Biochem J. 1970 Dec;120(4):891–894. doi: 10.1042/bj1200891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. FOULK W. T., BUTT H. R., OWEN C. A., Jr, WHITCOMB F. F., Jr, MASON H. L. Constitutional hepatic dysfunction (Gilbert's disease): its natural history and related syndromes. Medicine (Baltimore) 1959 Feb;38(1):25–46. [PubMed] [Google Scholar]
  14. Fevery J., Leroy P., Heirwegh K. P. Enzymic transfer of glucose and xylose from uridine diphosphate glucose and uridine diphosphate xylose to bilirubin by untreated and digitonin-activated preparations from rat liver. Biochem J. 1972 Sep;129(3):619–633. doi: 10.1042/bj1290619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. Kapitulnik J., Blondheim S. H., Grunfeld A., Kaufmann N. A. Photodecomposition of bilirubin: ultrafiltrable derivatives. Clin Chim Acta. 1973 Aug 30;47(2):159–166. doi: 10.1016/0009-8981(73)90311-2. [DOI] [PubMed] [Google Scholar]
  19. Kapitulnik J., Kaufmann N. A., Goitein K., Cividalli G., Blondheim S. H. A pigment found in Crigler--Najjar syndrome and its similarity to an ultrafiltrable photoderivative of bilirubin. Clin Chim Acta. 1974 Dec 17;57(3):231–237. doi: 10.1016/0009-8981(74)90402-1. [DOI] [PubMed] [Google Scholar]
  20. Klaassen C. E., Roberts R. J., Plaa G. L. Maximal biliary excretion of bilirubin and sulfobromophthalein during various rates of infusion in rats of different weights and strains. Toxicol Appl Pharmacol. 1969 Jul;15(1):143–151. doi: 10.1016/0041-008x(69)90142-2. [DOI] [PubMed] [Google Scholar]
  21. Manitto P., Monti D. Acid-catalyzed addition of alcohols and thiols to bilirubin. Experientia. 1973 Feb 15;29(2):137–139. doi: 10.1007/BF01945431. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. McDonagh A. F. Photochemistry and photometabolism in bilirubin IXalpha. Birth Defects Orig Artic Ser. 1976;12(2):30–40. [PubMed] [Google Scholar]
  24. O'Carra P., Colleran E. Separation and identification of biliverdin isomers and isomer analysis of phycobilins and bilirubin. J Chromatogr. 1970 Aug 12;50(3):458–468. doi: 10.1016/s0021-9673(00)97973-1. [DOI] [PubMed] [Google Scholar]
  25. Ostrow J. D. Photocatabolism of labeled bilirubin in the congenitally jaundiced (Gunn) rat. J Clin Invest. 1971 Mar;50(3):707–718. doi: 10.1172/JCI106541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Petryka Z. J. Identification of isomers differing from 9, alpha, in the early labelled bilirubin of the bile. Proc Soc Exp Biol Med. 1966 Nov;123(2):464–466. doi: 10.3181/00379727-123-31515. [DOI] [PubMed] [Google Scholar]
  27. Robinson S. H. Production and excretion of bilirubin in Gunn rats treated with phenobarbital. Proc Soc Exp Biol Med. 1971 Oct;138(1):281–284. doi: 10.3181/00379727-138-35879. [DOI] [PubMed] [Google Scholar]
  28. Rüdiger W., Klose W., Vuillaume M., Barbier M. On the structure of pterobilin, the blue pigment of Pieris brassicae. Experientia. 1968 Oct 15;24(10):1000–1000. doi: 10.1007/BF02138705. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Stevenson I., Greenwood D., McEwen J. Hepatic UDP-glucuronyltransferase in Wistar and Gunn rats--in vitro activation by diethylnitrosamine. Biochem Biophys Res Commun. 1968 Sep 6;32(5):866–872. doi: 10.1016/0006-291x(68)90321-5. [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. Zakim D., Goldenberg J., Vessey D. A. Regulation of microsomal enzymes by phospholipids. VI. Abnormal enzyme-lipid interactions in liver microsomes from Gunn rats. Biochim Biophys Acta. 1973 Feb 28;297(2):497–502. doi: 10.1016/0304-4165(73)90097-4. [DOI] [PubMed] [Google Scholar]

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