Plasma High-Density Lipoprotein Cholesterol Concentrations Determined by Heparin-Manganese Chloride Method or by Ultracentrifugation

Abstract

The association between premature atherosclerosis and increased concentrations of low density lipoprotein (LDL) is well known. Miller G.J. et al proposed that a reduction of plasma high density lipoprotein (HDL) concentration may accelerate the development of atherosclerosis by impairing the clearance of cholesterol from the arterial wall. Cholesterol-HDL (C-HDL) has generally been measured using either the heparin-manganese precipitation method of Burstein and Samaille or as the lipoprotein cholesterol after ultracentrifugal fractionation of HDL (1.063<d<1.210g/ml). The purpose of the present study was to compare these two methods. The following conditions were used to assess the effects of changes in final MnCl₂ and heparin concentrations on the precipitation of LDL and very low density lipoprotein (VLDL):
1. Mn²⁺ concentration was varied while heparin concentration was held constant.
2. Heparin concentration was varied while Mn²⁺ concentration was held constant.
After the heparin-manganese chloride precipitation of plasma, cholesterol quantitation, agarose gel electrophoresis and immunodiffusion studies using rabbit anti-lipoprotein B serum of the supernatant were carried out. A constant amount of cholesterol in the supernatant was measured over a final MnCl₂ concentration greater than 35mM and over a heparin concentration range of 46-945 units/ml. After observing no contamination of β- and pre β-lipoprotein by agarose gel electrophoresis and the absence of lipoprotein B by immundiffusion in the supernatant, optimum final concentration of heparin and MnCl₂ was determined. Final heparin and MnCl₂ concentration used in our study were 197 units/ml and 87mM, respectively. Lipoprotein fractions of d>1.063g/ml were isolated by ultracentrifugation. The density of plasma was adjusted to d=1.063g/ml with KBr solution and centrifuged in a Type 40.3 rotor on the Beckman L5-50 preparative ultracentrifuge for 44 hours at 105, 000 x g, 10°C. Lipoprotein fractions of d>1.063g/ml were recovered using a tube slicer to cut the cellulose nitrate tube. C-HDL quantitation by precipitation and ultracentrifugation was compared in 30 samples. Ultracentrifugally quantitated C-HDL (X±S.D.) was 48.5±11.8mg/dl, while by precipitation in parallel aliquots was 42.6±8.5mg/dl. C-HDL measured by ultracentrifugation and precipitation methods were significantly correlated, r=0.85, p>0.001. The cholesterol contents of the ultracentrifuged fraction in most samples were higher than those of the heparin/Mn²⁺ supernatant, presumably as a result of the contribution of the Lp(a)-lipoprotein and lipoprotein B in HDL. Ishikawa, T. T. et al reported that an additional problem with the heparin-MnCl₂ precipitation method appears when the plasma triglycerides are elevated (usually>300mg/dl) and a “floating precipitate” appears in the supernatant after an addition of heparin and manganese, providing an overestimation of C-HDL by the precipitation method as compared to ultracentrifugation. We confirmed this finding in a patient whose plasma triglyceride and cholesterol were 307mg/dl and 231mg/dl, respectively. After precipitation of plasma by heparin-MnCl₂, the supernatant contained β and pre-β lipoprotein other than α lipoprotein in agarose gel electrophoresis. C-HDL by precipitation method and by ultracentrifugation were 44mg/dl and 27mg/dl, respectively. When heparin-manganese precipitation method is used for the determination of plasma high-density lipoprotein cholesterol concentration, these problems should be considered.