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.
Individual bilirubin pigments in the excreta were quantitated by newly developed methods. In meconium, bilirubin‐IX β predominated, whereas bilirubin‐IXγ and ‐IXδ remained undetectable. The daily excretion of bilirubin‐IXα plus ‐IXβ was 0.03–1.00 and 0.04–2.00 μmoles kg1 of birthweight in preterm and full‐term infants, respectively. The ratio of bilirubin‐IXα to ‐ IXβ in meconium was 0.25 ± 0.34, 0.32 ± 0.30 and 0.46 ± 0.55 in newborns of gestational ages below 30, from 31 to 36 and above 36 wk, respectively. The predominance of bilirubin‐IXβ disappeared within the first week in those with gestational age ± 31 wk but more slowly in the very preterm group. The ratio of monoconjugated to diconjugated bilirubin‐IXα was 4 to 5 in full‐term infants, whereas this ratio was only reached after 1 mo in preterm infants. The ratio of glucuronide or glucoside to xyloside varied widely, independent of gestational age. No correlation between faecal UCB‐IXα and β‐glucuronidase was observed. The daily coproporphyrin excretion fell from a median of 500 μg on day 1 to below 20 μg from day 7 onwards; this decrease correlated with that of bilirubin‐IXβ. The daily 3α‐hydroxylated bile acid loss in the excreta was two‐ to fivefold higher than in the adult; this, together with the higher neonatal serum levels (12–90 nmoles ml‐1), indicates an immature intestinal reabsorption and an enhanced bile acid synthesis. Conclusion: Both zinc coproporphyrin and bilirubin‐IXβ are characteristic compounds of human meconium, diconjugated bilirubin‐IXα is low or absent in meconium of very preterm infants, and faecal and serum bile acids are high.
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