Isolation and properties of conjugated bilirubin from bile
J. Donald Ostrow,Nancy H. Murphy +1 more
TLDR
The evidence favours the existence of a true bilirubin mono-glucuronide that is not a complex, and may be modified for isolation of conjugates from human bile with 15-22% yield, and for preparation of unconjugated bilirUBin from rat or human biles with yields of 55-62%.Abstract:
1. A simple, rapid solvent partition method is described for isolation of conjugated bilirubin, free of unconjugated bilirubin, bile salts, phospholipids and cholesterol, from rat bile. Yields are 40-58%. The product is a phosphate-buffered solution containing approx. 0.4mg of bilirubin/ml, principally as mono- and di-glucuronide conjugates. The method may be modified for isolation of conjugates from human bile with 15-22% yield, and for preparation of unconjugated bilirubin from rat or human bile with yields of 55-62%. 2. The conjugated pigment has red-brown fluorescence and an absorption maximum at 450nm with in(mM) 59.8cm(-1). Diazotization by the Malloy-Evelyn method gives a direct Van den Bergh reaction (in water) 12% greater than the total reaction (in methanol), with in(total) 28.4x10(3)lmol(-1)cm(-1) at 550nm. After desalting by elution from Sephadex LH-20 in 50% (v/v) ethanol, the product gave water-soluble mustard-yellow crystalline needles. Such desalted conjugates were precipitated by Pb(2+) but not by Ba(2+), Ca(2+) or Zn(2+). 3. At pH7.0 and 37 degrees C the conjugated bilirubin was oxidized at a rate of 1%/h without hydrolysis, whereas 84% was hydrolysed by beta-glucuronidase or aqueous alkali. 4. Mono- and di-glucuronides were separated by elution from Sephadex LH-20 in 95% (v/v) ethanol or by extraction with chloroform at pH3.2-3.4. The monoconjugated bilirubin did not become labelled during incubation with unconjugated [(14)C]bilirubin, and chromatographed as a single spot without dissociating into unconjugated bilirubin and diglucuronide as would be expected of a complex. 5. After intravenous injection of mono- or di-conjugated [(14)C]bilirubin into normal or Gunn rats, 79-91% was excreted in bile and 2-7% in urine over 2h. In these experiments injected diglucuronide was not hydrolysed whereas 30-41% of injected monoglucuronide was converted into diglucuronide by the normal but not by the Gunn rats. The evidence favours the existence of a true bilirubin mono-glucuronide that is not a complex.read more
Citations
More filters
Journal ArticleDOI
Identification of a defect in the UGT1A1 gene promoter and its association with hyperbilirubinemia.
Junko Sugatani,Kasumi Yamakawa,Kouich Yoshinari,Takashi Machida,Hitoshi Takagi,Masatomo Mori,Satoru Kakizaki,Tatsuya Sueyoshi,Masahiko Negishi,Masao Miwa +9 more
TL;DR: The results indicate that homozygosity and compound heterozygosity for mutations in the UGT1A1 gene promoter (T-3263G and A[TA](7)TAA) and/or exon 1 of the gene (G211A) could explain the hyperbilirubinemia seen in the majority of individuals with Gilbert's syndrome.
Journal ArticleDOI
Transcriptional Regulation of Human UGT1A1 Gene Expression: Activated Glucocorticoid Receptor Enhances constitutive Androstane Receptor/Pregnane X Receptor-Mediated UDP-Glucuronosyltransferase 1A1 Regulation with Glucocorticoid Receptor-Interacting Protein 1
Junko Sugatani,Shinichi Nishitani,Kasumi Yamakawa,Kouichi Yoshinari,Tatsuya Sueyoshi,Masahiko Negishi,Masao Miwa +6 more
TL;DR: In this paper, the authors investigated the molecular mechanism of UGT1A1 induction by glucocorticoids at submicromolar concentrations and PXR activators and the functional cross-talk between the glucoc Corticoid receptor (GR) and CAR/PXR.
Journal ArticleDOI
Structure and binding of unconjugated bilirubin: relevance for physiological and pathophysiological function.
Journal ArticleDOI
Bacteria and gallstones. Etiological significance.
Masahisa Tabata,Fumio Nakayama +1 more
TL;DR: In this article, the authors found that Escherichia coli, Bacteroides and Clostridium often found in the biliary tract may contribute to the formation of bile pigment calcium stones by producing beta-glucuronidase and deconjugating bilirubin diglucuronide to form free unconjugated bilirus, which in turn combines with calcium, leading to stone formation.
References
More filters
Journal ArticleDOI
A simple method for the isolation and purification of total lipides from animal tissues.
TL;DR: In this paper, the authors described a simplified version of the method and reported the results of a study of its application to different tissues, including the efficiency of the washing procedure in terms of the removal from tissue lipides of some non-lipide substances of special biochemical interest.
Journal ArticleDOI
Statistical Methods for the Behavioral Sciences.
David Gold,Allen L. Edwards +1 more
Journal ArticleDOI
Determination of bilirubin glucuronide and assay of glucuronyltransferase with bilirubin as acceptor
F. P. Van Roy,K. P. M. Heirwegh +1 more
TL;DR: Chromatographic analysis suggests that bilirubin monoglucuronide is the product of the enzyme systems studied, and this method has been used in the development of assays for UDP-glucuronyltransferase.
Journal ArticleDOI
The excretion of bilirubin as a diglucuronide giving the direct van den Bergh reaction.
Journal ArticleDOI
The preparation of crystalline bilirubin-C14.
Related Papers (5)
Determination of bilirubin glucuronide and assay of glucuronyltransferase with bilirubin as acceptor
F. P. Van Roy,K. P. M. Heirwegh +1 more