Reverse-phase h.p.l.c. separation, quantification and preparation of bilirubin and its conjugates from native bile. Quantitative analysis of the intact tetrapyrroles based on h.p.l.c. of their ethyl anthranilate azo derivatives.
Reverse-phase h.p.l.c. separation, quantification and preparation of bilirubin and its conjugates from native bile. Quantitative analysis of the intact tetrapyrroles based on h.p.l.c. of their ethyl anthranilate azo derivatives.
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DOI：
10.1042/bj2250787
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发表时间：
1985-02
期刊：
影响因子：
--
通讯作者：
W. Spivak;M. Carey
中科院分区：
文献类型：
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作者：
W. Spivak;M. Carey
我们描述了一种简便、灵敏的反相高效液相色谱法。胆汁胆色素的分析分离和胆汁中非结合胆红素（UCB）及其单葡萄糖醛酸苷（BMG）和二葡萄糖醛酸苷（BDG）结合物的直接定量方法。该方法可以“放大”，用于从富含色素的胆汁中制备分离纯BDG和BMG。在制备步骤中采用Altex ultrasound ODS柱，在分离和分析步骤中采用沃茨mu-Bondapak C18柱。胆汁色素用乙酸铵缓冲液（pH 4.5）和60-100%（v/v）甲醇的20 min线性梯度洗脱，制备性分离的流速为2.0 ml/min，分析性分离的流速为1.0 ml/min。胆汁色素按极性降序洗脱（葡糖苷酸大于葡萄糖大于木糖结合物大于UCB），并通过t.l.c.进行化学鉴别。它们各自的邻氨基苯甲酸乙酯偶氮衍生物。通过使用与h.p.l.c.范围相关的标准曲线对UCB进行定量。积分峰面积与纯结晶UCB的浓度。一种纯的结晶邻氨基苯甲酸乙酯偶氮衍生物的UCB（AZO。UCB）作为单一h.p.l.c.用于定量BMG和BDG的参考标准。我们证明：胆汁胆色素的分离和定量是快速的（大约25分钟）;通过使用5微升胆汁，可以“在线”测定范围为1-500 μ M的胆色素浓度，而无需样品预处理;可以以毫克的量间接获得胆红素缀合物，而不会被胆汁的其它组分降解或污染。H.p.l.c.对一系列哺乳动物胆汁的分析表明，胆汁中UCB的浓度范围一般为1 - 17 μ M。这些数值大大低于t.l.c.先前估计的数值。BMG是许多啮齿类动物（豚鼠、仓鼠、小鼠、草原犬）胆汁中主要的（如果不是唯一的）胆红素结合物，这些啮齿类动物是色素和胆固醇胆结石形成的实验模型。其他动物（人、猴、小马、猫、大鼠和狗）胆汁中的缀合葡糖红蛋白在化学上更多样化，包括葡萄糖醛酸、木糖和葡萄糖的单-、二-和混合的非缀合物，其比例为每个物种提供不同的模式。
We describe a facile and sensitive reverse-phase h.p.l.c. method for analytical separation of biliary bile pigments and direct quantification of unconjugated bilirubin (UCB) and its monoglucuronide (BMG) and diglucuronide (BDG) conjugates in bile. The method can be 'scaled up' for preparative isolation of pure BDG and BMG from pigment-enriched biles. We employed an Altex ultrasphere ODS column in the preparative steps and a Waters mu-Bondapak C18 column in the separatory and analytical procedures. Bile pigments were eluted with ammonium acetate buffer, pH 4.5, and a 20 min linear gradient of 60-100% (v/v) methanol at a flow rate of 2.0 ml/min for the preparative separations and 1.0 ml/min for the analytical separations. Bile pigments were eluted in order of decreasing polarity (glucuronide greater than glucose greater than xylose conjugates greater than UCB) and were chemically identified by t.l.c. of their respective ethyl anthranilate azo derivatives. Quantification of UCB was carried out by using a standard curve relating a range of h.p.l.c. integrated peak areas to concentrations of pure crystalline UCB. A pure crystalline ethyl anthranilate azo derivative of UCB (AZO . UCB) was employed as a single h.p.l.c. reference standard for quantification of BMG and BDG. We demonstrate that: separation and quantification of biliary bile pigments are rapid (approximately 25 min); bile pigment concentrations ranging from 1-500 microM can be determined 'on line' by using 5 microliters of bile without sample pretreatment; bilirubin conjugates can be obtained preparatively in milligram quantities without degradation or contamination by other components of bile. H.p.l.c. analyses of a series of mammalian biles show that biliary UCB concentrations generally range from 1 to 17 microM. These values are considerably lower than those estimated previously by t.l.c. BMG is the predominant, if not exclusive, bilirubin conjugate in the biles of a number of rodents (guinea pig, hamster, mouse, prairie dog) that are experimental models of both pigment and cholesterol gallstone formation. Conjugated bilirubins in the biles of other animals (human, monkey, pony, cat, rat and dog) are chemically more diverse and include mono-, di- and mixed disconjugates of glucuronic acid, xylose and glucose in proportions that give distinct patterns for each species.