Separation of apolipoprotein (apo) B subspecies in 125I-labeled very low-density lipoprotein (VLDL) by high performance liquid chromatography (HPLC) was compared with the separation of apo B by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE).
Delipidated VLDL-apo B isoproteins were fractionated by HPLC using a Shim-Pack Diol-300 column, and were detected at 280nm. The degree of radioactivity in the HPLC fractions was also assessed. Determination of the isoproteins showed a good linear increase in both the UV-detection and the radioactivity measurement according to the increase of applied protein. The percentage of B-48 in the total apo B (%B-48) was 26.4% by UV-detection, and 17.3% by the radioactivity measurement.
The percentage of apo B-48, separated by SDS-PAGE, was also determined by densitometry after staining with Coomassie Brilliant Blue R-250 as well as by measuring the radioactivity of each isoprotein. The chromogenic response curve of apo B-100 was not extrapolated to the ordinate. Measuring the radioactivity of %B-48 by cutting its dried gel gave a result of 17.6%, and this value was similar to that obtained by measuring the radioactivity by HPLC.
The immunoreactivity of the VLDL apolipoprotein fractions, separated by the HPLC method, was determined by an enzyme immunoassay using anti-human apo B-100 specific monoclonal and anti-apo B polyclonal antibodies. Immunoreactivity appeared at both R. T. 12.6min and R. T. 16.0min by the polyclonal anti-human apo B, and at R. T. 12.6min by the B-100 specific antibody, indicating that the protein at R. T. 12.6min was apo B-100, while the protein at R. T. 16.0min was apo B-48.
These results suggest that analyzing apo B isoproteins by HPLC is a reliable and useful method for the study of apo B metabolism in triglyceride-rich lipoproteins.
