Studies on the Characterization of Human Serum Lipoproteins Separated by Ultracentrifugation in a Density Gradient

Abstract

Ultracentrifugation in a density gradient is an adequate one-step procedure for the separation of human serum lipoproteins, although 5 to 10 per cent of the cholesterol in the S_f 3–9 lipoprotein fraction isolated by this method represents contamination with high density lipoproteins. The density gradient procedure may be combined with ultracentrifugation at homogeneous densities in order to purify the S_f 3–9 lipoprotein fraction and subfractionate the S_f 10–400 lipoproteins. Purification and subfractionation are necessary for the characterization of serum lipoprotein distributions. A total fatty acid ester analysis (hydroxamic acid method) supplements cholesterol and phospholipid analyses on serum and serum lipoprotein fractions. Fatty acid ester:cholesterol ratios as well as cholesterol:phospholipid ratios may be used to characterize lipoprotein fractions and serum lipoprotein distributions. Serum lipid concentrations and the cholesterol: phospholipid and fatty acid ester:cholesterol ratios reflect the concentration and composition of the constituent lipoproteins in serum. While the analysis of total serum lipids indicates the general nature of the lipoprotein pattern, the specific details of the lipoprotein pattern are obtained only by fractionation. Hyperthyroid subjects exhibit moderate hypocholesterolemia, a significantly lowered S_f 3–9 lipoprotein fraction and a moderately lowered high density lipoprotein fraction. These lipoprotein fractions have a normal composition. Hypercholesterolemic subjects, whether hypothyroid, euthyroid or hyperthyroid, all have the same abnormal lipoprotein pattern. This is characterized by elevated serum cholesterol, phospholipid and fatty acid esters, an elevated cholesterol: phospholipid ratio, and a normal fatty acid ester:cholesterol ratio. The chylomicron and high density lipoprotein fractions have a normal concentration; the high density lipoprotein fraction may have a slightly elevated fatty acid ester content. The S_f 3–9 lipoprotein fraction has a normal composition but is significantly elevated in concentration. The S_f 10–400 lipoprotein fraction is moderately elevated and probably concentrated in the S_f 10–20 distribution range. In hypothyroid subjects, hypercholesterolemia can be corrected by the administration of thyroid hormones. Elevated S_f 10–400 and S_f 3–9 lipoprotein fractions are lowered and maintained at normal levels during therapy. No significant alteration in the high density lipoprotein fraction occurs, an observation consistent with the hypothesis proposed for lipoprotein metabolic relationships. Hypercholesterolemia in euthyroid subjects is not adequately controlled by thyroid hormones. The alterations in cholesterol metabolism proposed as causal factors in hypercholesterolemia are summarized as (1) a primary hepatic overproduction of cholesterol (euthyroid hypercholesterolemia), and (2) underutilization of cholesterol (hypothyroid hypercholesterolemia). While thyroid hormones may increase the cholesterol to bile acid conversion, it is suggested that a bile acid feed-back mechanism prevents the adequate control of hypercholesterolemia observed in euthyroid subjects.