A study of the heparin-manganese chloride method for determination of plasma α-lipoprotein cholesterol concentration

Abstract

To assess the limits of the heparin-MnCl₂ precipitation method for quantitation of α-lipoprotein cholesterol (C-HDL), effects of varying final MnCl₂ and heparin concentrations were studied, and the precipitation method was compared to preparative ultracentrifugation. In 65 parallel plasma aliquots, C-HDL$\left( {\bar X \pm SE} \right)$ determined by ultracentrifugation (54.3±1.8 mg/dl) correlated significantly (r=0.98, P<0.001) with the precipitation method (56.0±1.9 mg/dl). C-HDL by ultracentrifugal and precipitation methods were also similar in 16 subjects with triglycerides ranging from 150 to 312 mg/dl (41.4±2.6, 43.4±2.8, r=0.97, P<.001). A constant amount of cholesterol in the supernatant was measured over a final MnCl₂ range of 0.046–0.23M, and cholesterol values in the supernatant at final MnCl₂ concentrations of 0.046, 0.05, and 0.055M did not differ from each other, P>0.1. However, cholesterol levels in the supernatant at final MnCl₂ concentration of 0.042M differed from those at concentrations of 0.046, 0.05, and 0.055M, P<0.05, and the amount of supernatant cholesterol increased as the final MnCl₂ concentration was reduced from 0.042 to 0.02M. A constant amount of cholesterol in the supernatant was measured over a heparin concentration range of 92–734 USP units/ml. The final MnCl₂ and heparin concentrations of 0.046M and 184 USP units/ml, which are incorporated in widely used procedures, gave C-HDL values for the precipitation method which were in close agreement with the ultracentrifugal method. There is no evidence for a heparin-Mn⁺⁺ precipitation of HDL and systematic underestimation of HDL by the precipitation method. However, the final MnCl₂ concentration is very near the minimum required for accurate measurement of C-HDL. To preclude incomplete precipitation of low and very low density lipoproteins by insufficient manganese concentration, an increase of the manganese concentration should be considered.