Journal of Lipid Research, Vol. 11, 7-22, January 1970
Copyright © 1970 by Lipid Research, Inc.

Isolation of plasma lipoproteins by zonal ultracentrifugation in the B14 and B15 titanium rotors

Henry G. Wilcox and Murray Heimberg

Department of Pharmacology, Vanderbilt University, School of Medicine, Nashville, Tennessee 37203

Lipoproteins were isolated from plasma of man, dog, rabbit, rat, and chicken by ultracentrifugation in continuous density gradients using the B14 titanium and B15 titanium zonal rotors. Both the VLDL and the LDL of human plasma were separated easily from the HDL and from the other more plentiful plasma proteins by centrifugation for only 1 or 2 hr in the B14 or B15 rotor, respectively. Satisfactory separation of the HDL from the more dense plasma proteins was not achieved with these rotors. The human LDL achieved isopycnic equilibrium (d 1.04) on prolonged periods (> 24 hr) of centrifugation in a sucrose-KBr density gradient. The pattern of distribution of cholesterol and phospholipid throughout the density gradient coincided with the pattern of distribution of the lipoprotein-protein measured spectrophotometrically or chemically. The concentration of cholesterol and phospholipid in the lipoproteins isolated by zonal ultracentrifugation agreed with analyses reported for lipoproteins isolated by sequential centrifugation in solutions of increasing density. The lipoproteins isolated by zonal ultracentrifugation were characterized further by their electrophoretic behavior. The fractions which were identified as the LDL (d 1.04-1.05) from all species migrated on paper as a ßbeta;-globulin; the LDL from plasma of dogs contained an additional component which has been designated as an ₂-globulin. The fractions which were identified as the HDL from all species migrated as an ₁-globulin.

Reaction of human LDL with either rabbit antihuman ßbeta;-lipoprotein or rabbit antihuman serum resulted in a single immunodiffusion band. The S_{f, 1.063} of the human LDL was calculated to be 6.0. When plasma from humans or rabbits was centrifuged in the B15 rotor, the HDL was not visible as a distinct peak and was not separable from the bulk of the more dense plasma proteins; when plasma from dogs or chickens was centrifuged under identical conditions, the HDL was clearly detectable. Even though the mean density of the HDL from dogs or chickens was not different from that of man or rabbits, the visibility of this lipoprotein in dogs and chickens was probably due to its high concentration in the plasma of these species. When plasma from the rat was centrifuged under similar conditions, the HDL was also clearly in evidence. Although rat plasma contained a relatively small concentration of HDL, the lipoprotein had a lower mean density than did the HDL of the other species and was therefore more easily separable from the dense plasma proteins.

The procedure of zonal ultracentrifugation for the isolation of lipoproteins by flotation is simultaneously preparative and analytical and should find useful application in the investigation of the soluble lipoproteins from plasma and tissues.

Supplementary key words human • dog • chicken • rabbit • rat • continuous density gradient • lipoprotein protein • lipoprotein lipids

Submitted on October 7, 1968
Revised on June 30, 1969
Accepted on August 25, 1969

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