AN EFFICIENT CHROMATOGRAPHIC SYSTEM FOR LIPOPROTEIN FRACTIONATION USING WHOLE PLASMA

We have validated a semi-automatic procedure for the efficient isolati on of plasma lipoproteins from 300 mu l of whole plasma (actual inject ion volume 200 mu l) by Fast Phase Liquid Chromatography (FPLC). Modif ied enzymatic assays were established to allow the determination of lo w concentrations (1-20 mg/dl) of triglycerides and cholesterol using t he Beckman CX-5 Autoanalyzer: The sum of the cholesterol contents in t he fractions corresponding to low density (LDL) and high density lipop rotein (HDL) can be demonstrated to be highly correlated to values obt ained with dextran sulfate/MgCl2 precipitation for HDLc (slope = 0.98, r(2) = 0.997) and ultracentrifugation (beta-quant) for LDLc (slope = 1.03, r(2) = 0.988). Using pure lipoprotein fractions isolated by ultr acentrifugation, linear ranges of detection for HDLc and HDL apoA-I we re 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 (r ange) fractional standard deviations for quadruplicate runs for 15 ind ividual plasma samples ranging widely in lipoprotein concentrations we re 0.97 (0.29-2.86%) for LDLc (range: 101.5-258.5 mg/dl), 3.67 (0.62-1 4.11%) for HDLc (range: 27.1-85.1 mg/dl) and 2.19 (0.16-6.56%) for VLD L-TG (range: 6.1-515.0 mg/dl).