RESUMO
A simple and reliable method has been developed for the determination of high-density lipoprotein (HDL) turnover in humans. In this method, complex formation of [14C]methylavidin with biotinyl-HDL3 and subsequent precipitation of excess [14C]methylavidin with biotinyl-silica-gel is utilized for the detection of as little as 0.1 microgram of biotinyl-HDL3 (by protein)/ml of serum with high precision and reproducibility, at 7.9 +/- 0.9% (n = 7) of the peptidyl lysine modified. In serial dilutions and quadruplicate determinations the intra- and interassay variations were less than 4.7% and 5.2%, respectively (n = 5). Recovery of biotinyl-HDL3 averaged 92 +/- 5.7% throughout the working range of the assay (n = 4). Variations in the levels of HDL or very-low-density lipoprotein (VLDL) did not interfere with the measurement of biotinyl-HDL3 in serum. Also, results were not affected by storage of serum samples at -80 degrees C for up to 4 weeks. After reinjection of autologous biotinyl-HDL3 (0.12 mg protein/kg body wt.) into five normolipidemic male volunteers, typical decay curves were obtained. The mean half-life of biotinyl-HDL3 was 5.1 +/- 0.5 days, no different from that reported for radiolabeled HDL or radiolabeled HDL apolipoproteins. Routine immunobinding analysis did not reveal formation of antibodies with specificity towards HDL 4 weeks after the reinjection studies. From this it appears that biotinyl-HDL3 is a suitable probe and a safe and reliable alternative for the determination of HDL turnover in humans when application of radiolabeled HDL is not desirable.
