Quantification of sinking pre beta lipoprotein in human plasma

Two low density protein carriers of plasma cholesterol (beta-lipoprotein and sinking pre-beta-lipoprotein -- a genetic variant of beta-lipoprotein with pre-beta electrophoretic mobility) were isolated from human plasma by ultracentrifugation and gel filtration. They were subjected to agarose gel electrophoresis, followed by staining with Sudan Black B, and their concentration was determined by optical densitometry. The staining of the low lipoproteins was proportional to their total cholesterol the low lipoproteins was proportional to their total cholesterol content over a range of 2-50 mg/dl for sinking pre-beta-lipoprotein (SPB) and 50-250 mg/dl for beta-lipoprotein. Mixing experiments of the two purified lipoprotein preparations indicated that the cholesterol in the SPB could be estimated by the formula: SPB cholesterol = TBC x OD of SPB/(OD of SPB + OD of beta cholesterol), where OD is optical density and the TBC was either the total cholesterol content of mixtures of purified beta- and SPB-lipoproteins, or was determined as the total plasma low density lipoprotein cholesterol content measured by ultracentrifugation and polyanionic precipitation of beta- and SPB-lipoproteins from plasma. The SPB cholesterol values calculated by the above formula deviated from the true value (amount added) by a mean of 6% when beta-lipoprotein cholesterol was present at concentrations of 100, 165, and 216 mg/dl. The deviation did not correlate with the concentration of beta-lipoprotein cholesterol. The coefficient of variation for the determination of SPB lipoprotein by the scanning procedure wa 5-10% for SPB cholesterol concentrations of 6-30 mg/dl. SPB concentrations of 1-2 mg/dl cholesterol could be detected, but the method was unsatisfactory for quantification below 4 mg/dl cholesterol. By providing an estimate of the distribution of cholesterol between the subfractions of low density lipoprotein, the method should prove helpful in assessing risk of atherosclerosis.