Analysis of bilirubins in biological fluids by extraction and thin-layer chromatography of the intact tetrapyrroles: application to bile of patients with Gilbert's syndrome, hemolysis, or cholelithiasis.

Abstract

A method was developed to extract quantitatively the bilirubins from bile, urine, serum, stool, and preparations from liver with a chloroform-ethanol mixture at pH 1.8 in the presence of ascorbic acid and NaCl. Extracted pigment was submitted to thin-layer chromatography, and the separated bilirubins were either immediately eluted and determined spectrophotometrically or individually converted to ethyl anthranilate azo derivatives for thin-layer chromatographic analysis of each isolated pigment band. Bilirubins in duodenal bile of eight healthy adults comprised 1.5 +/- 1.3% unconjugated bilirubin-IX alpha, 69 +/- 6% bilirubin diglucuronide, and 16 +/- 4% bilirubin monoglucuronides. Mixed diconjugates containing one glucuronosyl moiety and either one xylosyl or one glucosyl group amounted to 10 +/- 3%. Most samples (6 of 8) contained trace amounts (0.6 +/- 0.6%) of unconjugated bilirubin-IX beta, in agreement with nearly exclusive cleavage of heme at the alpha-meso position. The composition of the bilirubins in bile was normal in 6 patients with cholesterol gallstones, 4 with chronic hepatitis, and 3 with hemolysis. In duodenal bile of individuals with Gilbert's syndrome (n = 10), the concentration of bilirubin conjugates was comparable to that in healthy adults, but the proportion of bilirubin diglucuronides (52 +/- 8%) was decreased. The concentration of unconjugated bilirubin-IX alpha showed a fair positive correlation with that of bilirubin monoglucuronide and was increased in half of the patients with Gilbert's syndrome.

• Authors: J Fevery, N Blanckaert, P Leroy, R Michiels, K P Heirwegh
• Journal: Hepatology (Baltimore, Md.) (Hepatology) 1983 Mar-Apr Vol. 3 Issue 2 Pg. 177-83 ISSN: 0270-9139 [Print] United States
• PMID: 6832709 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Bilirubin

Topics

• Bile (analysis, metabolism)
• Bilirubin (isolation & purification)
• Cholelithiasis (metabolism)
• Chromatography, Thin Layer
• Feces (analysis)
• Gilbert Disease (metabolism)
• Hematologic Diseases (metabolism)
• Hepatitis (metabolism)
• Humans
• Hyperbilirubinemia, Hereditary (metabolism)