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
✍ Scribed by Johan Fevery; Norbert Blanckaert; Pol Leroy; Roger Michiels; Karel P. M. Heirwegh
- Publisher
- John Wiley and Sons
- Year
- 2007
- Tongue
- English
- Weight
- 725 KB
- Volume
- 3
- Category
- Article
- ISSN
- 0270-9139
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✦ Synopsis
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 NaC1. 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 2 1.3% unconjugated bilirubin-IXa, 69 f 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 f 3%. Most samples (6 of 8) contained trace amounts (0.6 +-0.6%) of unconjugated bilirubin-IXP, in agreement with nearly exclusive cleavage of heme at the a-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 = lo), the concentration of bilirubin conjugates was comparable to that in healthy adults, but the proportion of bilirubin diglucuronides (52 f 8%) was decreased. The concentration of unconjugated bilirubin-IXa showed a fair positive correlation with that of bilirubin monoglucuronide and was increased in half of the patients with Gilbert's syndrome.
Various carbohydrate conjugates of bilirubin-IXa occur in nature. The mono-and diglucuronides predominate in bile but glucose and xylose conjugates are also present (1). Their importance depends mainly on the species examined (l), whereas the ratio of mono-to diglucuronide varies with pathophysiological conditions (2-5). In addition, small amounts of non-a isomers of bilirubin-IX have been identified in bile of pig (6), Gunn rat (7), man, and dog (8).
Analysis of dipyrrolic azo derivatives has mostly been