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.