A one-step separation of human serum high density lipoproteins 2 and 3 by rate-zonal density gradient ultracentrifugation in a swinging bucket rotor

Abstract

A method was developed for the separation of the high density lipoprotein subclasses HDL2 and HDL3 from human serum. Six serum samples are fractionated in a single-step ultracentrifugal procedure using the Beckman (SW-40) swinging bucket rotor. The method is based on a difference in flotation rate of the high density lipoprotein subclasses. Separation of HDL2 and HDL3 is accomplished by a discontinuous NaBr density gradient applied on top of 2 ml of serum brought to a density of 1.40 g/ml. After centrifugation, high density lipoprotein subclass profiles were obtained using a specially designed gradient fractionator. Contamination of the isolated high density lipoprotein subclasses by serum albumin or by apolipoprotein B-containing lipoproteins was minimal while only a slight overlap between the HDL2 and HDL3 profiles was observed. Chemical and immunochemical analyses of the high density lipoprotein subclasses isolated by the present method were in close agreement with the results obtained by rate-zonal density gradient ultracentrifugation in zonal rotors (Patsch, et al. 1980. J. Biol. Chem. 255: 3178-3185). The major advantage of the method presented in this paper as compared with the zonal rotor method is the possibility to analyze as many as six serum samples simultaneously.