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

The measurement of apolipoprotein B (apoB) in purified lipoproteins by immu nological assays is subject to criticism because of denatured epitopes or i mmunoreactivity differences between purified lipoproteins and standard. Che mical methods have therefore been developed, such as the selective precipit ation 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 chrom atography/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 < 1.06 3 g/mL) were purified by ultracentrifugation. Apolipoprotein B-100 was sele ctively precipitated by isopropanol. The leucine content of the pellet was then determined by gas chromatography/mass spectrometry, using norleucine a s internal standard. Knowledge of the number of leucine molecules in one ap oB-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/m L from apoB in lipoproteins d < 1.063 g/mL, respectively. The isopropanol p recipitate was verified as pure apoB (>97%) in lipoprotein fractions isolat ed from normo- and hyperlipidemic plasma and the method appeared reproducib le. The combination of isopropanol precipitation and the GC/MS method appea rs therefore to be a precise and reliable method for kinetic and epidemiolo gical studies.