Studies of the separation and purification of bilirubin conjugates from human bile

Abstract

The present work is concerned with the separation and purification of bilirubin monoglucuronide and diglucuronide from human bile, in a pure form so that they can be used as reference standards for the various clinical determinations of bilirubin conjugates in serum. The first step was to partially remove the lipid components of bile (ie. cholesterol and bile salts) by chromatography on a column packed with Amberlite XAD-2 resin, followed by ultrafiltration using a 500 molecular weight cut-off membrane filter. Then further separation was achieved on glass columns packed with silica gel or silicic acid. The behavior of bilirubin and its conjugates on Eastman Chromagram silica gel G thin layer chromatography sheets was studied at various stages of purification. The Amberlite XAD-2 removed from 23 to 48% of the bile salts present in samples of centrifuged bile. It also removed 10% of the conjugated bilirubin. Diafiltration was carried out at 45-65 psi., and approximately 250 ml. of diafiltrate was collected over a 24 hour period. Diafiltration carried out over 4 to 8 day periods resulted in losses of 50% of the total bilirubin and 75% of the direct reacting bilirubin present. When the bile was subjected to ultrafiltration, concentrating it to one fifth of its original volume, 71% of the cholesterol and 73% of the bile salts were passed out into the ultrafiltrate. During the ultrafiltration 18% of the total and 36% of the direct reacting bilirubin were hydrolyzed or oxidized, even though the process was carried out under red light and a nitrogen atmosphere. The five fold concentration gave a bile sample containing 96 mg.% direct reacting and 114 mg.% total bilirubin. The removal of the bile salts also had an effect on the thin layer chromatography of bilirubin and its conjugates. As the bile salt concentration was lowered the two conjugated pigments migrated considerably slower than they did in corresponding samples of centrifuged bile. By eluting the partially purified bile in a column packed with silica gel or silicic acid, using a solvent system of acetone, butanol, propionic acid and water (7:4:3:1), it was possible to attain a complete separation of the two conjugates from free bilirubin, With slower flow rates the silica gel or silicic acid columns produced a 75% reduction in bile salts and 25% reduction in cholesterol in the eluates as compared to the bile introduced onto the column. At faster flow rates the cholesterol and bilirubin conjugates were eluted at the same time. The product was a solution of approximately 2.0 mg.% bilirubin conjugates in the acetone, butanol, propionic acid and water mixture. The solution also contained about 10 mg.% cholesterol and approximately 0.0012 g. per 100 ml. bile salt. When the solution was stored in the dark at -15°c.,there was no loss in direct reacting bilirubin, even after three weeks. Attempts to evaporate the solvent and dissolve the solid residue in phosphate buffer or deionized water resulted in hydrolysis of the glucuronic acid from the bilirubin or oxidation to biliverdin. In the mixed solvent system the billrubin conjugates had an Amax. at 420 nm.,and in phosphate buffer the Amax was at 410 nm. The Malloy-Evelyn bilirubin assay on pure conjugated bilirubin gave a higher value for the direct reacting than for the total bilirubin. Recycling of the column eluates a second time through a silicic acid column did not result in sepration of the monoglucuronide from the diglucuronide.