Improved HPLC method for carbohydrate-deficient transferrin in serum
Improved HPLC method for carbohydrate-deficient transferrin in serum
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DOI：
10.1373/clinchem.2003.023341
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发表时间：
2003-11-01
影响因子：
9.3
通讯作者：
Jeppsson, JO
中科院分区：
文献类型：
--
作者：
Helander, A;Husa, A;Jeppsson, JO
背景资料：需要一种血清中转铁蛋白糖型的参考方法，用于酒精标记物碳水化合物缺乏转铁蛋白（CDT）的常规免疫学方法可以追溯。我们描述了一种改进的HPLC法测定转铁蛋白glycoforms.Methods：转铁蛋白是铁饱和的血清与铁氮基三乙酸，和脂蛋白沉淀与硫酸葡聚糖和氯化钙。在SOURCE 15 Q阴离子交换柱上使用盐梯度洗脱进行糖型的分离。定量依赖于铁-转铁蛋白复合物在470 nm处的选择性吸光度。每个糖型的相对量计算为曲线下的面积的百分比，使用基线integration.Results：HPLC系统提供了可重复的分离和定量的去唾液酸，单唾液酸，二唾液酸，三唾液酸，四唾液酸，pentasialo，和hexasialotransferrin糖型。最重要的是，二唾液酸转铁蛋白和三唾液酸转铁蛋白几乎基线分离。二唾液酸转铁蛋白的测定内和测定间CV为6%，三唾液酸转铁蛋白，单唾液酸转铁蛋白在20%时检测到。
Background: There is need for a reference method for transferrin glycoforms in serum to which routine immunologic methods for the alcohol marker carbohydrate-deficient transferrin (CDT) can be traceable. We describe an improved HPLC method for transferrin glycoforms.Methods: Transferrin was iron-saturated by mixing the serum with ferric nitrilotriacetic acid, and lipoproteins were precipitated with dextran sulfate and calcium chloride. Separation of glycoforms was performed on a SOURCE 15Q anion-exchange column using salt gradient elution. Quantification relied on selective absorbance of the iron-transferrin complex at 470 run. The relative amount of each glycoform was calculated as a percentage of the area under the curve, using baseline integration.Results: The HPLC system provided reproducible separation and quantification of the asialo-, monosialo-, disialo-, trisialo-, tetrasialo-, pentasialo-, and hexasialotransferrin glycoforms. Most importantly, disialo- and trisialotransferrin were almost baseline separated. The intra- and interassay CV for disialotransferrin were 6%) trisialotransferrin, monosialotransferrin was detected at