Tagliaro F, Crivellente F, Manetto G, Puppi I, Deyl Z, Marigo M
Institute of Forensic Medicine, University of Verona, Italy.
Electrophoresis. 1998 Nov;19(16-17):3033-9. doi: 10.1002/elps.1150191640.
Carbohydrate deficient transferrin (CDT) is one of the most reliable markers of chronic alcohol abuse. It consists of a group of minor isoforms of human transferrin (the main iron transport serum protein) deficient in sialic acid groups (asialo, monosialo and disialo) with a pI > 5.7, while the main isotransferrin (tetrasialo) has a pI of 5.4. The aim of the present work was to develop a capillary electrophoretic method to determine CDT in serum, suitable for routine use as a confirmatory technique of the current screening methods based on immunoassays. Serum samples (0.5 mL) were saturated with iron by incubation with 10 mM FeCl3 (9 microL) and 500 mM NaHCO3 (12 microL) for 30 min, then diluted 1/10 in water and injected by positive pressure (0.5 psi for 10 s). Separation was performed with a capillary zone electrophoretic method using bare fused-silica capillaries (20 microm ID, 37 cm in length) and a buffer composed of 100 mM sodium tetraborate adjusted with boric acid to pH 8.3. Applied voltage was 10 kV and temperature 25 degrees C. Detection was by UV absorption at 200 nm wavelength. Under the described conditions, asialo-, monosialo-, disialo-, trisialo- and tetrasialo-transferrin were separated in human serum. The limit of detection (signal-to-noise ratio of 2) was about 0.3% for disialo-transferrin, and 0.4% of trisialo-transferrin, expressed as percentages of the terasialo-transferrin peak area. Relative standard deviations (RSD) of absolute migration times were < 1%, while RSD of relative migration times (on the basis of tetrasialo-transferrin) were < 0.1%. Intra-day and day-to-day peak quantitation precision studies showed RDS ranging from 4 to 9% and from 13 to 24% for disialo- and trisialo-transferrin, respectively. The results from 30 control subjects, including social drinkers, and 13 alcoholics showed disialo- and trisialo-transferrin significantly increased in patients by a factor of about 4.5 (P < 0.0001).
缺糖转铁蛋白（CDT）是慢性酒精滥用最可靠的标志物之一。它由一组人转铁蛋白（主要的铁转运血清蛋白）的次要异构体组成，这些异构体缺乏唾液酸基团（去唾液酸、单唾液酸和双唾液酸），其等电点（pI）>5.7，而主要的同型转铁蛋白（四唾液酸转铁蛋白）的pI为5.4。本研究的目的是开发一种毛细管电泳方法来测定血清中的CDT，适用于作为基于免疫测定的当前筛查方法的确认技术进行常规使用。血清样本（0.5 mL）通过与10 mM FeCl3（9 μL）和500 mM NaHCO3（12 μL）孵育30分钟使铁饱和，然后用水稀释1/10并通过正压（0.5 psi，持续10秒）进样。使用未涂覆的熔融石英毛细管（内径20 μm，长度37 cm）和由100 mM四硼酸钠用硼酸调节至pH 8.3的缓冲液，通过毛细管区带电泳法进行分离。施加电压为10 kV，温度为25℃。通过在200 nm波长处的紫外吸收进行检测。在所述条件下，去唾液酸转铁蛋白、单唾液酸转铁蛋白、双唾液酸转铁蛋白、三唾液酸转铁蛋白和四唾液酸转铁蛋白在人血清中得以分离。双唾液酸转铁蛋白的检测限（信噪比为2）约为0.3%，三唾液酸转铁蛋白为0.4%，以四唾液酸转铁蛋白峰面积的百分比表示。绝对迁移时间的相对标准偏差（RSD）<1%，而相对迁移时间（基于四唾液酸转铁蛋白） 的RSD<0.1%。日内和日间峰定量精密度研究表明，双唾液酸转铁蛋白和三唾液酸转铁蛋白的RSD分别为4%至9%和13%至24%。对包括社交饮酒者在内的30名对照受试者和13名酗酒者的检测结果显示，患者体内双唾液酸转铁蛋白和三唾液酸转铁蛋白显著增加，约为4.5倍（P < 0.0001）。
