A comprehensive evaluation of the heparin–manganese precipitation procedure for estimating high density lipoprotein cholesterol

Abstract

The accurate quantitation of high density lipoproteins has recently assumed greater importance in view of studies suggesting their negative correlation with coronary heart disease. High density lipoproteins may be estimated by measuring cholesterol in the plasma fraction of d > 1.063 g/ml. A more practical approach is the specific precipitation of apolipoprotein B (apoB)-containing lipoproteins by sulfated polysaccharides and divalent cations, heparin-Mn(2+) being the most commonly used combination. The present heparin-Mn(2+) procedure was found to be reasonably specific and not often subject to large errors; however, 9% (primarily hypertriglyceridemic samples) of the 966 plasma samples treated with heparin-Mn(2+) had obvious supernatant turbidity, indicating incomplete sedimentation of apoB-associated lipoproteins. Furthermore, 48% of the nonturbid supernates contained more than 1 mg/dl (mean 2.5 mg/dl) of apoB-associated cholesterol when measured by a radial immunodiffusion procedure, indicating slight overestimation of HDL cholesterol. Determination of the extent of the unprecipitated apoB-associated lipoproteins by sensitive radioimmunoassay and of the amount of precipitated high density lipoprotein by radial immunodiffusion assay of apolipoproteins A-I and A-II at various heparin and Mn(2+) concentrations indicated that the usual heparin level (approximately 1.3 mg/ml) was adequate. However, a twofold increase in Mn(2+) concentration to 0.092 M improved precipitation of the apoB-associated lipoproteins without excessive precipitation of high density lipoprotein from plasma. This increased Mn(2+) level also provided improved sedimentation of the apoB-associated lipoproteins from hypertriglyceridemic plasma. Additional observations suggested that, for convenience, the heparin and Mn(2+) can be added simultaneously as a combined reagent, that samples can be incubated for 10 minutes at room temperature before centrifugation, and that turbid supernates from hypertriglyceridemic samples can usually be made free of apoB-associated lipoproteins by centrifugation at 12,000 g for 10 minutes.