Partial specific volume and preferential hydration of low density lipoprotein subfractions.

We have determined the partial specific volume (v-) for five low density lipoprotein (LDL) subfractions (n = 5-7) and evaluated preferential hydration (n = 2) for LDL subfraction 3 in normolipoproteinemic subjects in order to characterize these highly atherogenic components of the human plasma lipoprotein spectra. Values for v- at 1 g were determined by sixth place density measurements of the solvent and lipoprotein solutions and carbon, hydrogen and nitrogen (CHN) absolute mass of the lipoprotein concentrations. Mean values for v- were 0.9757 +/- 0.0019, 0.9701 +/- 0.0007, 0.9674 +/- 0.0016, 0.9616 +/- 0.0016 and 0.9550 +/- 0.0025 ml/g for subfractions 1, 2, 3, 4 and 5, respectively. However, molecular densities (sigma) obtained from rho (rho) = 1/v- for respective LDL subfractions were 1.0249, 1.0308, 1.0337, 1.0399 and 1.0471 g/ml, respectively. The preferential hydration of lipoprotein subfraction 3 (n = 2) in NaCl/H2O solutions was 2.9-4.8 wt percent, whereas values were much lower (0.3-0.6 wt percent) in NaCl/NaBr/H2O solvent system. Unhydrated densities for LDL subfraction 3 (n = 2) at 1 g (sixth-place density meter) were 1.0287 and 1.0269 g/ml, whereas at 200,000 X g (used in D2O flotation eta F degrees vs rho determinations) both values were 1.0308 g/ml, indicating that these similar LDL fractions have 23 and 53% higher compressibility than the solvent at 200,000 X g force. It was observed that the linearity of eta F degrees vs rho may not be valid for solvents NaCl/NaBr/H2O of density as high as 1.4744 g/ml. Thus, flotation velocity data using extreme salt concentrations (1.4744 g/ml and higher) may be viewed with caution.

Analytic ultracentrifuge calibration and determination of lipoprotein-specific refractive increments.

Accurate quantification of the major classes and subfractions of human serum lipoproteins is an important analytical need in the characterization and evaluation of therapy of lipid and lipoprotein abnormalities. For calibrating the analytic ultracentrifuge (AnUC), we routinely use a Beckman calibration wedge cell with parallel scribed lines 1 cm apart. Such a cell gives a rectangular pattern in the schlieren diagram, which determines magnification and also provides an area corresponding to an invariant refractive increment. We have independently validated this wedge calibration cell using a special boundary-forming cell in which 1.174% sucrose is overlayered with distilled water. Comparing wedge cell area with extrapolated zero time boundary area refractive increment gives agreement to within less than 1%, corresponding to a refractive increment error of +/- 0.00002 delta n. Complete calibration for AnUC analysis of lipoproteins also requires accurate determination of the specific refractive increments (SRI) of the major lipoprotein classes, namely low density lipoprotein (LDL) and high density lipoprotein (HDL). These are measured in the density in which they are analyzed, i.e., 1.061 g/ml for LDL and 1.200 g/ml for HDL. Five fresh serum samples were fractionated for total LDL and total HDL and their SRI determined. Total lipoprotein mass was determined using precise CHN elemental analysis and compositional analyses. The results yielded corrected SRI of 0.00142 and 0.00135 delta n/g/100 ml for LDL and HDL. Thus, our current values using 0.00154 and 0.00149 delta n/g/100 ml underestimate LDL and HDL by 9% and 11%. Corrections of all previous LDL and HDL AnUC data can be made using appropriate factors of 1.087 and 1.106.