Journal of Lipid Research, Vol. 23, 14-27, January 1982
Copyright © 1982 by Lipid Research, Inc.

A quick and large-scale density gradient subfractionation method for low density lipoproteins

Diana M. Lee and Debbie Downs

Laboratory of Lipid and Lipoprotein Studies, Oklahoma Medical Research Foundation and the Department of Biochemistry and Molecular Biology, The University of Oklahoma, Oklahoma City, OK 73104

A quick density gradient-banding subfractionation method has been developed for d < 1.063 g/ml lipoproteins. Up to 324 ml of plasma can be resolved into five distinct layers by a single ultracentrifugation. The separation was achieved with a discontinuous density gradient formed between plasma and a layer of NaCl solution of d 1.080 g/ml in an angle-head rotor during centrifugation at 45,000 rpm for 26 hr at 5°C. VLDL and LDL₁ (layer 1, d < 1.020 g/ml) were at the top. Layer 2 (apparent d 1.025-1.028 g/ml), layer 3 (apparent d 1.032-1.043 g/ml) and layer 4 (apparent d 1.046-1.054 g/ml) were subfractions of normal LDL₂. Layer 5 (d > 1.071 g/ml) contained HDL and plasma proteins. A second step centrifugation separates VLDL from LDL₁. When opaque tubes are used, additional centrifugation is needed to separate layer 4 from layer 5. The subfractionation method was reproducible and was verified by analytical ultracentrifugation, chemical analyses, agarose electrophoresis, and electron microscopy. This method has been applied to plasma of normal males and females of the same age group. The chemical composition of a given subfraction from subjects of the same category was constant. However, compositional differences were found between normal males and females. The triglyceride content was higher in layer 2 and the cholesteryl ester content was lower in layer 4 for normal females than for males. Quantitatively, cholesterol concentration was significantly higher in layer 2 for normal males than for females. Layer 4 and layer 5 were the only fractions containing Lp(a). Applicability of the subfractionation method to studies of dyslipoproteinemia was demonstrated with plasma from patients with type III and type IV hyperlipoproteinemias. Marked differences were found in VLDL and LDL₁, both qualitatively and quantitatively, between the two types of patients and between the type III patient and normal subjects. A primarily quantitative difference was found in VLDL between the type IV patient and normal subjects. This isolation method yields concentrated subfractions that reveal the heterogeneity of LDL₂ in one spin, and offers quick isolation of narrow density ranges of LDL species, thereby providing better defined molecular entities for structural and/or metabolic studies.—Lee, D. M., and D. Downs. A quick and large-scale density gradient subfractionation method for low density lipoproteins.

Supplementary key words very low density lipoproteins • narrow density range • hyperlipoproteinemia • Lp(a) • electron microscopy • agarose electrophoresis

Submitted on January 22, 1981
Revised on August 12, 1981

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