The Distribution and Chemical Composition of Ultracentrifugally Separated Lipoproteins

In the past few years several methods have been developed for the analysis of serum lipoproteins. Lindgren, Elliott, and Gofman (1) have utilized the relatively low density of the lipoproteins to separate them from the other serum proteins by ultracentrifugal flotation. Quantitation was subsequently performed by refractometric methods in the analytical ultracentrifuge. Separations of lipoproteins have also been made by Cohn fractionation in cold ethanol, and the quantities of lipoprotein have been estimated from the lipid. content of the fractions (2, 3). Widely used at the present time is the method of zone electrophoresis with quantitation either by staining (4) or by chemical analysis of eluates from the supporting medium (5, 6). Each of these methods has serious limitations. Analytical ultracentrifugal techniques (7, 8) require the possession of expensive equipment. The quantitation of data is subject to considerable error and gives no information regarding the chemical composition of the lipoproteins. Cohn fractionation requires facilities for operation at 5° C. It permits accurate determination of the lipid components of the alpha and beta lipoproteins, but with this technique it is impossible to subfractionate these groups. With certain abnormal sera the method is unreliable (9). Determination of electrophoretically separated fractions by staining techniques or by chemical analysis of eluates is subject to appreciable error. Both Cohn fractionation and electrophoretic techniques fail to separate lipoproteins from other serum proteins, thus making impossible the study of the protein moiety. The combination of preparative ultracentrifugation with chemical analysis of the separated fractions would seem to be a procedure by which both the distribution and composition of lipoproteins could be determined simply and accurately. Such a procedure was described by Turner, Snavely, Goldwater, Randolph, Sprague, and Unglaub (10). Under their conditions of ultracentrifugation, however, separation of discrete lipoproteins did not occur and their results are difficult to interpret. In the present study, lipoproteins have been separated from serum by repeated ultracentrifugations after progressively raising the solvent density. The separated fractions have been analyzed for total cholesterol, lipid phosphorus, and protein. The method has proved reliable and is, in our opinion, the simplest available procedure for the accurate quantitation of serum lipoproteins.