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通过结合选择性沉淀和质谱法测定血浆脂蛋白中载脂蛋白B的浓度。

Measurement of apolipoprotein B concentration in plasma lipoproteins by combining selective precipitation and mass spectrometry.

作者信息

Beghin L, Duhal N, Poulain P, Hauw P, Lacroix B, Lecerf J M, Bonte J P, Fruchart J C, Luc G

机构信息

Department of Atherosclerosis, Institut Pasteur de Lille, France.

出版信息

J Lipid Res. 2000 Jul;41(7):1172-6.

Abstract

The measurement of apolipoprotein B (apoB) in purified lipoproteins by immunological assays is subject to criticism because of denatured epitopes or immunoreactivity differences between purified lipoproteins and standard. Chemical methods have therefore been developed, such as the selective precipitation of apoB followed by quantification of the precipitate. In this study, we present the measurement of apoB concentration in lipoproteins purified by ultracentrifugation by combining isopropanol precipitation and gas chromatography/mass spectrometry. Very low density lipoprotein (VLDL; d < 1.006 g/mL); VLDL plus intermediate density lipoprotein (VLDL + IDL; d < 1.019 g/mL); and VLDL, IDL, and low density lipoprotein (VLDL + IDL + LDL; d < 1.063 g/mL) were purified by ultracentrifugation. Apolipoprotein B-100 was selectively precipitated by isopropanol. The leucine content of the pellet was then determined by gas chromatography/mass spectrometry, using norleucine as internal standard. Knowledge of the number of leucine molecules in one apoB-100 molecule makes it possible to calculate the plasma concentration of apoB in the various lipoprotein fractions. ApoB in IDL (d 1.006-1.019 g/mL) and LDL (d 1.019-1.063 g/mL) were then determined by subtracting VLDL-apoB from apoB in lipoproteins d < 1.019 and apoB in lipoproteins d < 1.019 g/mL from apoB in lipoproteins d < 1.063 g/mL, respectively. The isopropanol precipitate was verified as pure apoB (>97%) in lipoprotein fractions isolated from normo- and hyperlipidemic plasma and the method appeared reproducible. The combination of isopropanol precipitation and the GC/MS method appears therefore to be a precise and reliable method for kinetic and epidemiological studies.

摘要

通过免疫测定法对纯化脂蛋白中的载脂蛋白B(apoB)进行测量受到质疑,因为存在变性表位或纯化脂蛋白与标准品之间的免疫反应性差异。因此,已开发出化学方法,例如apoB的选择性沉淀,随后对沉淀物进行定量。在本研究中,我们通过结合异丙醇沉淀和气相色谱/质谱法,对超速离心纯化的脂蛋白中的apoB浓度进行了测量。通过超速离心纯化极低密度脂蛋白(VLDL;d < 1.006 g/mL)、VLDL加中间密度脂蛋白(VLDL + IDL;d < 1.019 g/mL)以及VLDL、IDL和低密度脂蛋白(VLDL + IDL + LDL;d < 1.063 g/mL)。用异丙醇选择性沉淀载脂蛋白B-100。然后以正亮氨酸作为内标,通过气相色谱/质谱法测定沉淀物中的亮氨酸含量。了解一个apoB-100分子中亮氨酸分子的数量,就可以计算出各种脂蛋白组分中apoB的血浆浓度。然后分别通过从d < 1.019的脂蛋白中的apoB减去VLDL-apoB以及从d < 1.063 g/mL的脂蛋白中的apoB减去d < 1.019 g/mL的脂蛋白中的apoB,来测定IDL(d 1.006 - 1.019 g/mL)和LDL(d 1.019 - 1.063 g/mL)中的apoB。从正常和高脂血症血浆中分离的脂蛋白组分中的异丙醇沉淀物经验证为纯apoB(>97%),且该方法具有可重复性。因此,异丙醇沉淀与气相色谱/质谱法的结合似乎是一种用于动力学和流行病学研究的精确且可靠的方法。

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