尿液结合性胆红素的纯化

Purification of conjugated bilirubin from human urine]

OBJECTIVES: To develop a simple, practical method with fine yield and purity for separating conjugated bilirubin(Bc) from a sustainable and facile material in order that it could serve as a calibrator for total and direct bilirubin determination. METHODS: Isolating Bc from urine of patients with obstructive jaundice by CHCl3: CH3CH2OH(1:1, V/V) followed by CNBr activated Sepharose 4B-HAS affinity chromatography, we analyzed the products by the use of Doumas J-G diazo method and HPLC, respectively. RESULTS: Determined by J-G diazo method, the crude product extracted by CHCl3: CH3CH2OH(1:1, V/V) yields 90.2 +/- 5.4%(n = 3) of urine Bc, the ratio of Bc to total dry weight being 67.9 +/- 6.4%(n = 3). The final product purified by affinity chromatography with the ratio of separation of 1.5, 1.737 and analyzed by HPLC has the yield of 46.9% and purity of 94.7%(BDG of 76.2%, BMG of 18.5%). CONCLUSION: The above data demonstrate that urine conjugated bilirubin can be successfully purified by CHCl3: CH3CH2OH extraction followed by Sephrose-HSA affinity chromatography with sufficient purity and output.