Fractionation of human serum lipoproteins and simultaneous enzymatic determination of cholesterol and triglycerides
Fractionation of human serum lipoproteins and simultaneous enzymatic determination of cholesterol and triglycerides
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DOI：
10.1016/j.aca.2009.06.060
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发表时间：
2009-11-03
影响因子：
6.2
通讯作者：
Schoenmakers, Peter J.
中科院分区：
文献类型：
--
作者：
Qureshi, Rashid Nazir;Kok, Wim Th.;Schoenmakers, Peter J.
建立了一种基于非对称流场-流分级（AF 4）的分离人血清中不同类型脂蛋白的方法。方法优化的重点是表征最大脂蛋白组分（LDL和VLDL）的可能性，这通常不可能使用常规分析中应用的分子排阻色谱法。不同的通道几何形状和流动程序进行了测试和比较。与传统的长通道相比，使用短分级通道在相同的分级能力下显示出较少的样品稀释度。用指数衰减和线性错流程序获得不同的尺寸选择性。紫外吸收信号的光散射信号的比率被用来验证保留时间和分级particle.An实验装置的大小之间的关系开发的同时测定胆固醇和甘油三酯分布的脂蛋白馏分，基于酶促反应，然后在500 nm处的紫外检测。比较了PTFE盘管和编织管式反应器。用针织管反应器观察到改进的峰锐度和灵敏度。在优化实验条件后，获得了令人满意的线性和精密度（胆固醇的RSD为2-3%，甘油三酯的RSD为5-6%）。最后，血清样本，从健康志愿者和败血症患者的样本的合并样本，与开发的方法进行了分析。脂蛋白分馏和胆固醇和甘油三酯分布可能与样本的临床背景相关。(C)2009 Elsevier B. V.保留所有权利。
A method based on Asymmetric Flow Field-Flow Fractionation (AF4) was developed to separate different types of lipoproteins from human serum. The emphasis in the method optimization was on the possibilities to characterize the largest lipoprotein fractions (LDL and VLDL), which is usually not possible with the size-exclusion chromatography methods applied in routine analysis. Different channel geometries and flow programs were tested and compared. The use of a short fractionation channel was shown to give less sample dilution at the same fractionation power compared to a conventional, long channel. Different size selectivities were obtained with an exponential decay and a linear cross flow program. The ratio of the UV absorption signal to the light scattering signal was used to validate the relation between retention time and size of the fractionated particles.An experimental setup was developed for the simultaneous determination of the cholesterol and triglycerides distribution over the lipoprotein fractions, based on enzymatic reactions followed by UV detection at 500 nm. Coiled and knitted PTFE tubing reactors were compared. An improved peak sharpness and sensitivity were observed with the knitted tubing reactor. After optimization of the experimental conditions a satisfactory linearity and precision (2-3% rsd for cholesterol and 5-6% rsd for triglycerides) were obtained. Finally, serum samples, a pooled sample from healthy volunteers and samples of sepsis patients, were analyzed with the method developed. Lipoprotein fractionation and cholesterol and triglyceride distributions could be correlated with the clinical background of the samples. (C) 2009 Elsevier B.V. All rights reserved.