Lipoprotein apolipoprotein synthesis by human hepatoma cells in culture.

Lipoprotein synthesis was demonstrated by double diffusion with low density lipoprotein antibody, and by 3H-labeled amino acid incorporation into proteins of the d less than 1.063 g/ml centrifugally isolated lipoprotein fraction. Radioactive label was incorporated predominantly into apolipoprotein B (60%), apolipoprotein A-I (20%) and apolipoprotein C (12%), as determined by Sepharose column chromatography and polyacrylamide gel electrophoresis. Incorporation of radioactive label into apolipoprotein B was inhibited by the presence of albumin in the medium, and was restored to control levels with the addition of 1 mM oleic acid, indicating that cell synthesis of apolipoproteins could be modified by culture conditions. The human hepatoma cell line, Hep G2, provides a potential in vitro model for the study of regulation of human hepatic lipoprotein and apolipoprotein synthesis.

Lipid interference in steroid radioimmunoassay.

To study lipid interference in steroid radioimmunoassays in which dextran-coated charcoal is used as the separating agent, we tested triolein and phosphatidylcholine as model hydrophobic and amphipathic lipids, respectively. Addition of either caused distortion of the standard curve to an extent that was inversely related to the polarity of the steroid molecule. Both lipids form a dispersion that entraps steroid molecules. When we increased the charcoal concentration, the effect of phosphatidylcholine addition was eliminated for assays of both polar and nonpolar steroids. In contrast, the effect from triacylglycerol was not corrected, particularly in assays of nonpolar steroids. We also studied mixtures of lipids mimicking the mixture of lipids extracted from plasma of normolipemic and hyperlipemic indviduals. The degree of lipemia that can be tolerated differs from assay to assay, and primarily varies directly with the polarity of the steroid being assayed.

Mechanisms of interference of nonesterified fatty acids in radioimmunoassays of steroids.

Addition of nonesterified fatty acids caused an apparent increase in unbound steroid present in supernatants in radioimmunoassays for steroids utilizing dextran-coated charcoal. Nonesterified fatty acid interference occurred at the initial binding interaction between steroid and its antiserum, and also at the step separating bound from unbound steroid. It was determined that nonesterified fatty acids, which had been added to radioimmunoassays, formed micelles and trapped steroids. The extent of the entrapment was inversely related to the number of polar groups in the steroid molecule, that is, hydrophobic steroids more easily interacted with the nonesterified fatty acid micelles. However, if the charcoal concentration were increased, the nonesterified fatty acid effect was eliminated for assays of polar steroids and greatly reduced for non-polar steroid assays.