Fractionation of plasma lipoprotein sub-classes
The effect of iodixanol concentration and tube-loading format, in the NVT65.2 is given in Figure 1. Panel 1 shows the banding of the major lipoproteins using the same tube format, centrifugation time and RCF as described in Application Sheet M07; the only difference is the rotor type (results from a TLN100 are shown in Figure 3 of Application Sheet M07).
In the NVT65.2 the gradient density profile causes the HDL to band more broadly (and the LDL more sharply) across the gradient fractions than in the TLN100. There is even some indication that the use of a uniform concentration of 12% iodixanol at 365,000g for 3 h in the NVT65.2 rotor may permit the resolution of the HDL into distinct subfractions; the three boxed fractions in Panel 1 clearly indicate a minor population of HDL particles denser than the major population.
Only by reducing the uniform iodixanol concentration to 9% in the NVT65.2 (Panel 2), does the relative linear distribution of the HDL and LDL approach that obtained with 12% iodixanol in the TLN100. Panel 3 shows the distribution of lipoproteins in a two layer tube-loading format of 12% and 9% iodixanol (equal volumes of each) with the plasma confined to the denser layer.
It might be expected that the gradient generated from the 12% iodixanol in the bottom half of the tube would have caused the HDL to band further up the tube when compared to the banding in a gradient generated from a uniform 9% iodixanol format (Panel 2).
The important consideration here is the amount of plasma proteins in
the system; plasma proteins contribute significantly to the total density of the gradient, particularly in
the lower half of the gradient. The 12%/9% format contains less than half of the plasma proteins present.