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

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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–IXα, 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–IXβ, in agreement with nearly exclusive cleavage of heme at the α–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 bttirubin conjugates was comparable to that in healthy adults, but the proportion of bilirubin diglucuronides (52 ± 8%) was decreased. The concentration of unconjugated bilirubin–IXα showed a fair positive correlation with that of bilirubin monoglucuronide and was increased in half of the patients with Gilbert's syndrome.

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