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Clinical Chemistry 39: 960-964, 1993;
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Clinical Chemistry, Vol 39, 960-964, Copyright © 1993 by American Association for Clinical Chemistry

Sequential ultracentrifugation micromethod for separation of serum lipoproteins and assays of lipids, apolipoproteins, and lipoprotein particles

T Brousseau, V Clavey, JM Bard and JC Fruchart
SERLIA et U. 325 INSERM, Institut Pasteur, Lille, France.

We describe a fast sequential separation of very-low-density, low- density, and high-density lipoproteins from 400 microL of serum, using the Beckman TL100 ultracentrifuge. The cumulative centrifugation time is 9.5 h. The purity of lipoprotein fractions was verified by a gel- filtration procedure. The major contaminant is the serum albumin, which can be eliminated by a second centrifugation at the same density. Enzymatic measurement of lipids shows good recovery (> 91%) and weak within-sample variation (< 7%). In comparison with a density-gradient procedure, the deleterious effects on the lipoprotein structure appear to be limited, as shown by the low concentrations of apolipoprotein (apo) E and apo A-I in the fraction > 1.21 kg/L. Furthermore, the micro- ultracentrifugation also gives a better recovery rate. Finally, we have studied the distribution of lipids, apolipoproteins, and lipoprotein particles (LpA-I:A-II, LpB:C-III, LpB:E) in each fraction separated from 10 serum samples from healthy subjects.


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