Isolation of human serum HDL1 by zonal ultracentrifugation

• Main Authors: G Schmitz, G Assmann
• Format: Article
• Language: English
• Published: Elsevier 1982-08-01
• Series: Journal of Lipid Research
• Online Access: http://www.sciencedirect.com/science/article/pii/S0022227520380937

High density lipoprotein subfraction-1 (HDL(1)) 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 HDL(1) in normal and hypercholesterolemic sera were studied using zonal ultracentrifugation. To permit separation of the HDL(1) from VLDL, LDL, and Lp(a), the apoB-containing lipoproteins were first precipitated from serum using the phosphotungstic acid/magnesium chloride (PTA/MgCl(2)) method after which the supernatant fraction was subjected to zonal ultracentrifugation. It could be demonstrated that following PTA/MgCl(2) precipitation HDL(1) floats as a single peak at d 1.08-1.09 g/ml (NaBr) and is sufficiently separated from high density lipoprotein-2 (HDL(2)) and high density lipoprotein-3 (HDL(3)). The HDL(2)/HDL(3) subfraction pattern was not affected by the precipitation method. As previously described, in vitro incubation of serum leads to the LCAT-dependent interconversion of HDL(3) or HDL(2). Using the technique described here, it was discovered that a simultaneous elevation of HDL(1) occurred. This increase in HDL(1) 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 HDL(1) peak could be detected, but in patients with familial hypercholesterolemia (apoB, E receptor deficiency) HDL(1) 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), HDL(1) was undetectable. Analysis of the HDL fractions in serum of a patient with abetalipoproteinemia revealed that following in vitro incubation there was formation of HDL(1) despite the lack of apoprotein B-containing lipoproteins. These data support the concept that HDL(1) formation occurs during LCAT-mediated HDL(3)/HDL(2) interconversion in vitro.-Schmitz, G., and G. Assmann. Isolation of human serum HDL(1) by zonal ultracentrifugation.