Isolation and properties of conjugated bilirubin from bile.
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 in(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 in(total) 28.4x10(3)lmol(-1)cm(-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 Pb(2+) but not by Ba(2+), Ca(2+) or Zn(2+). 3. At pH7.0 and 37 degrees C the conjugated bilirubin was oxidized at a rate of 1%/h without hydrolysis, whereas 84% was hydrolysed by beta-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 [(14)C]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 [(14)C]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.
D000818:Animals; D001464:Barium; D001646:Bile / Q000032:analysis*; D001663:Bilirubin / Q000302:isolation & purification* / Q000378:metabolism / Q000652:urine; D002021:Buffers; D002118:Calcium; D002247:Carbon Isotopes; D011232:Chemical Precipitation; D002845:Chromatography; D003979:Diazonium Compounds; D005453:Fluorescence; D005965:Glucuronates / Q000378:metabolism; D005966:Glucuronidase; D006801:Humans; D007275:Injections, Intravenous; D007854:Lead; D008297:Male; D008722:Methods; D010084:Oxidation-Reduction; D010710:Phosphates; D051381:Rats; D012995:Solubility; D013057:Spectrum Analysis; D015032:Zinc
D002021:Buffers; D002247:Carbon Isotopes; D003979:Diazonium Compounds; D005965:Glucuronates; D010710:Phosphates; D001464:Barium; D007854:Lead; D005966:Glucuronidase; D015032:Zinc; D001663:Bilirubin; D002118:Calcium