Subfractionation and characterization of native and incubation enlarged human plasma high density lipoprotein particles by high performance gel filtration

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Abstract
Incubation of human plasma in vitro at 37 C results in an increase of the mean particle size of the high density lipoproteins (HDL) accompanied by an almost complete disappearance of the original particles present prior to incubation. A rapid high performance gel filtration technique has been developed in order to study the chemical composition of subfractions of native and incubation enlarged HDL particles as a function of particle size. Subfractionation of HDL isolated by preparative ultracentrifugation from 3 normal human plasmas incubated in vitro at 0 and 37 C for 24 hr have been performed using a 150 cm long TSK-G 3000 SW column. The separation time was less than 65 min. The curves obtained at high performance gel filtration of HDL, by monitoring the effluents from the column at 280 nm, agreed well both in positions of peak maxima and relative peak intensities with the particle distribution patterns observed at polyacrylamide gradient gel electrophoresis of the corresponding HDL preparations run in parallel. The different HDL particle subfractions of the effluents from the gel filtration column have been characterized by quantification of free and esterified cholesterol, total phospholipids and apolipoprotein A-I and A-II. The incubation enlarged HDL particles, subfractionated by the high performance gel filtration technique, were found to have a composition which differed from that of native HDL particles of corresponding size. Incubation enlarged HDL had a generally higher and almost constant relative cholesteryl ester content over the whole particle range compared to native HDL in which a continuous increase in relative cholesteryl ester content could be observed when going from large to small particles. The molar ratio of phospholipids to free cholesterol was higher in small native HDL particles than in the corresponding large ones. The relation between apolipoprotein A-I and A-II remained nearly constant between small and large HDL particles in each subfractionation experiment. The results demonstrate that the high performance gel filtration technique is a rapid and reproducible means for studying the composition of subfractions of HDL particle populations.

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