Guo Y, Chen M, Tu Y, Luo H, Li S, Yan D, Niu M
School of Basic Medical Sciences, WCUMS, Chengdu 610041, China.
Hua Xi Yi Ke Da Xue Xue Bao. 2001 Dec;32(4):579-81.
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.
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.
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%).
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.
开发一种简单、实用且产率和纯度良好的方法，用于从可持续且便捷的材料中分离结合胆红素（Bc），使其可作为总胆红素和直接胆红素测定的校准物。
通过用三氯甲烷：乙醇（1:1，V/V）从梗阻性黄疸患者尿液中分离Bc，然后进行溴化氰活化的琼脂糖4B - 人血清白蛋白亲和层析，我们分别使用杜马斯J - G重氮法和高效液相色谱法分析产物。
用J - G重氮法测定，经三氯甲烷：乙醇（1:1，V/V）提取的粗产物产生尿液Bc的产率为90.2±5.4%（n = 3），Bc与总干重的比例为67.9±6.4%（n = 3）。经亲和层析纯化的最终产物，分离比为1.5、1.737，并通过高效液相色谱法分析，产率为46.9%，纯度为94.7%（结合二葡萄糖醛酸胆红素为76.2%，结合单葡萄糖醛酸胆红素为18.5%）。
上述数据表明，尿液结合胆红素可通过三氯甲烷：乙醇提取，随后进行琼脂糖 - 人血清白蛋白亲和层析成功纯化，具有足够的纯度和产量。
