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 ∈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 ∈total 28.4×103lmol−1cm−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 Pb2+ but not by Ba2+, Ca2+ or Zn2+. 3. At pH7.0 and 37°C the conjugated bilirubin was oxidized at a rate of 1%/h without hydrolysis, whereas 84% was hydrolysed by β-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 [14C]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 [14C]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.
Isolation and properties of conjugated bilirubin from bile
