An efficient chromatographic system for lipoprotein fractionation using whole plasma

Abstract

We have validated a semi-automatic procedure for the efficient isolation of plasma lipoproteins from 300 μl of whole plasma (actual injection volume 200 μl) by Fast Phase Liquid Chromatography (FPLC). Modified enzymatic assays were established to allow the determination of low concentrations (1–20 mg/dl) of triglycerides and cholesterol using the Beckman CX-5 Autoanalyzer. The sum of the cholesterol contents in the fractions corresponding to low density (LDL) and high density lipoprotein (HDL) can be demonstrated to be highly correlated to values obtained with dextran sulfate/MgCl2 precipitation for HDLc (slope = 0.98, r2 = 0.997) and ultracentrifugation (beta-quant) for LDLc (slope = 1.03, r2 = 0.988). Using pure lipoprotein fractions isolated by ultracentrifugation, linear ranges of detection for HDLc and HDL apoA-I were performed at 18–95 mg/dl and 59–262 mg/dl, respectively. The ranges for LDLc were 41–435 mg/dl and 21–280 mg/dl for LDL apoB. The mean (range) fractional standard deviations for quadruplicate runs for 15 individual plasma samples ranging widely in lipoprotein concentrations were 0.97 (0.29–2.86%) for LDLc (range: 101.5–258.5 mg/dl), 3.67 (0.62–14.11%) for HDLc (range: 27.1–85.1 mg/dl) and 2.19 (0.16–6.56%) for VLDL-TG (range: 6.1–515.0 mg/dl).—Innis-Whitehouse, W., X. Li, W. V. Brown, and N-A. Le. An efficient chromatographic system for lipoprotein fractionation using whole plasma.

Keywords

• fast phase liquid chromatography (FPLC)
• lipid distribution
• apolipoprotein distribution