Sequential ultracentrifugation micromethod for separation of serum lipoproteins and assays of lipids, apolipoproteins, and lipoprotein particles.
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.
SERLIA et U. 325 INSERM, Institut Pasteur, Lille, France.
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Brousseau
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Clavey
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Bard
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Fruchart
D000328:Adult; D000368:Aged; D016632:Apolipoprotein A-I / Q000032:analysis; D001053:Apolipoproteins / Q000097:blood*; D001057:Apolipoproteins E / Q000097:blood; D002499:Centrifugation, Density Gradient; D006801:Humans; D008055:Lipids / Q000097:blood*; D008074:Lipoproteins / Q000097:blood*; D008075:Lipoproteins, HDL / Q000097:blood; D008077:Lipoproteins, LDL / Q000097:blood; D008079:Lipoproteins, VLDL / Q000097:blood; D008875:Middle Aged; D012016:Reference Values; D014461:Ultracentrifugation / Q000379:methods* / Q000706:statistics & numerical data
D016632:Apolipoprotein A-I; D001053:Apolipoproteins; D001057:Apolipoproteins E; D008055:Lipids; D008074:Lipoproteins; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL