Separation of Plasma Lipoproteins in Self-Generated Gradients of Iodixanol

Abstract

The major classes of plasma lipoprotein, very low density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL), are characterized on the basis of differences in density and charge ( Table 1 ). Centrifugation is the 'gold-standard' for the analysis of plasma lipo-protein classes and beta quantitation, and other analytical procedures (1-4). Lipoprotein classes are separated by flotation of plasma or serum in a series of centrifugation steps in which the density of the plasma is increased sequentially by addition of potassium bromide. Alternatively, plasma is layered beneath a discontinuous gradient and centrifuged to separate the lipoprotein classes in a single step. However, it is impractical to use these methods in a routine analytical or clinical laboratory because of the long centrifugation steps required. It is also necessary to remove the high salt concentrations used before further analysis (e.g., agarose gel electrophoresis or determination of the cholesterol and/or triglyceride levels)can be carried out. The current method used for the assay of LDL and HDL levels in the chemical pathology laboratory involves determination of total plasma cholesterol followed by selective precipitation of HDL and determination of the cholesterol remaining in the supernatant (1). From the data obtained, the LDL and HDL cholesterol levels are indirectly calculated. It is generally accepted that this method is limited and that the results are compromised by modest elevation in levels of plasma triglyceride, such as frequently occurs in clinical samples (5). Table 1 Plasma Lipoproteins Lipoprotein Protein % PL % Cho1 % TAG % Mobility % Density % Meanvalues in serum (mg/dL) Chylomicrons 2 4 5 89 Start <1.000 <10 VLDL 10 16 17 57 pre-β <1.006 50-200 LDL 29 24 47 6 β 1.019-1.063 200-300 HDL 49 29 19 5 α 1.063-1.210 200-300 The mean % composition of the major classis of lipoproteins is given: PL, phospholipids; chol, cholesterol and cholesterol ester; TAG, triglyceride. The mobility from the origin is pre-β; β; a see Fig. 1 for example. The density is the band at which the lipoproteins float from salt gradients.