THE PREPARATION OF CRYSTALLINE BILIRUBIN-C14*

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Abstract
Bilirubin-C14 was prepared by administering glycine-2-C14 to rats with stimulated erythropoiesis, and subsequently isolating the radioactive pigment from fistula bile collected during the "early labeling period". Peak radioactivity of the excreted bilirubin occurred between 6 and 18 hours following administration of the isotopic glycine. Crystalline bilirubin was obtained from the bile in yields in excess of 40% by means of a new isolation procedure involving precipitation of bilirubin glucuronide with lead acetate, hydrolysis of the glucuronide with alkali, extraction of proteins with acetic acid and crystallization of the pigment from chloroform-methanol. Specific activity of the crystalline pigment ranged from 0.66 to 1.0 mc per mmole. Radio-chemical purity was demonstrated by spectral and elemental analysis, by recrystallization to constant specific activity, and by countercurrent distribution. Parallel studies revealed that bilirubin, dissolved in benzene, chloroform, or in chloroform containing up to 50% methanol was stable in the dark but rapidly deteriorated on illumination. In alkaline solution, bilirubin was unstable even in the dark. The breakdown products formed included yellow, diazo-negative pigments and dipyrrolic propentdyopent.