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

To assess the limits of the heparin MnCl2 precipitation method for quantitation of alpha-lipoprotein cholesterol (C-HDL), effects of varying final McCl2 and heparin concentrations were studied, and the precipitation method was compared to preparative ultracentrifugation. In 65 parallel plasma aliquots, C-HDL (X+/-SE) determined by ultracentrifugation (54.3+/-1.8 mg/dl) correlated significantly (r=0.98, P less than 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 less than .001). A constant amount of cholesterol in the supernatant was measured over a final McCl2 range of 0.046-0.23 M, and cholesterol values in the supernatant at final McCl2 concentrations of 0.046, 0.05, and 0.055 M did not differ from each other, P greater than 0.1. However, cholesterol levels in the supernatant at final MnCl2 concentration of 0.042 M differed from those at concentrations of 0.046, 0.05, and 0.055 M, P less than 0.05 and the amount of supernatant cholesterol increased as the final McCl2 concentration was reduced from 0.042 to 0.02 M. 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 0.046 M 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.