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Journal of Lipid Research, Vol. 23, 903-910, August 1982
Copyright © 1982 by Lipid Research, Inc.

Isolation of human serum HDL by zonal ultracentrifugation

G. Schmitz and G. Assmann

Zentrallaboratorium der Medizinischen Einrichtungen der Westfälischen Wilhelms-Universität, Domagkstrasse 3, 4400 Münster, and Institut für Arterioskleroseforschung an der Universität Münster

High density lipoprotein subfraction-1 (HDL₁) is thought to interact with the high-affinity apoprotein B, E receptors of peripheral cells and may act as a modulator of LDL binding and uptake. In the present study the concentration and composition of HDL₁ in normal and hypercholesterolemic sera were studied using zonal ultracentrifugation. To permit separation of the HDL₁ from VLDL, LDL, and Lp(a), the apoB-containing lipoproteins were first precipitated from serum using the phosphotungstic acid/magnesium chloride (PTA/MgCl₂) method after which the supernatant fraction was subjected to zonal ultracentrifugation. It could be demonstrated that following PTA/MgCl₂ precipitation HDL₁ floats as a single peak at d 1.08-1.09 g/ml (NaBr) and is sufficiently separated from high density lipoprotein-2 (HDL₂) and high density lipoprotein-3 (HDL₃). The HDL₂/HDL₃ subfraction pattern was not affected by the precipitation method. As previously described, in vitro incubation of serum leads to the LCAT-dependent interconversion of HDL₃ or HDL₂. Using the technique described here, it was discovered that a simultaneous elevation of HDL₁ occurred. This increase in HDL₁ concentration could not be observed when LCAT was inhibited by heat inactivation or addition of Ellman's reagent. In normal fresh serum only a small HDL₁ peak could be detected, but in patients with familial hypercholesterolemia (apoB, E receptor deficiency) HDL₁ was elevated five to tenfold compared to normal values and further increased in concentration upon incubation of serum. On the other hand, in sera of patients with familial HDL deficiency (Tangier disease), HDL₁ was undetectable. Analysis of the HDL fractions in serum of a patient with abetalipoproteinemia revealed that following in vitro incubation there was formation of HDL₁ despite the lack of apoprotein B-containing lipoproteins. These data support the concept that HDL₁ formation occurs during LCAT-mediated HDL₃/HDL₂ interconversion in vitro.—Schmitz, G., and G. Assmann. Isolation of human serum HDL₁ by zonal ultracentrifugation.

Supplementary key words LCAT • HDL₂ • HDL₃ • apoE

Submitted on September 9, 1981
Revised on April 16, 1982

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