An improved method for isolation of unconjugated bilirubin from rat and dog bile.
Radiolabeled unconjugated bilirubin (UCB) is currently prepared by biosynthetic labeling of bilirubin in fistula bile from precursor-labeled delta-aminolevulinic acid (ALA) in rats or dogs. With existing methods, yields of labeled UCB from the bile are generally less than 50%. We here report modifications of the original method of Ostrow et al (Ostrow JD, Hammaker L, Schmid R. The preparation of crystalline bilirubin-C(14). J Clin Invest 1961;40:1442-52) that result in improvement of yields to 72% from both dog and rat bile. The modifications include the initial deproteination of bile with a reverse-phase C18 cartridge, removal of ethanol before alkaline hydro-lysis to avoid esterification of UCB, and adjustments for the high proportion of non-glucuronide UCB conjugates in dog bile not precipitated as lead salts. These improvements should save significantly on both costs and animal usage.
Webster
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C
Research Service, Veterans' Affairs Lakeside Medical Center; the Gastroenterology/Hepatology Division, Northwestern University, Chicago, IL, USA.
Tiribelli
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Ostrow
J D
JD
D000468:Alkalies / Q000737:chemistry; D000818:Animals; D001646:Bile / Q000737:chemistry*; D001654:Bile Pigments / Q000737:chemistry; D001663:Bilirubin / Q000032:analysis / Q000302:isolation & purification*; D002408:Catheters, Indwelling; D002725:Chloroform / Q000737:chemistry; D002845:Chromatography; D003460:Crystallization; D004285:Dogs; D006868:Hydrolysis; D008297:Male; D000432:Methanol / Q000737:chemistry; D051381:Rats; D015203:Reproducibility of Results; D013048:Specimen Handling / Q000379:methods
D000468:Alkalies; D001654:Bile Pigments; D002725:Chloroform; D001663:Bilirubin; D000432:Methanol