Abstract
Precipitation methods for the assay of HDL-cholesterol were examined using four different reagents, such as heparin-Mn++, heparin-Ca++, dextran sulfate-Mg++ and sodium phosphotungstate-Mg++.
For the isolation of HDL by former three reagents, the assay system was consisted of 0.1ml of test serum and 2ml of the reagent was used. The mixture was allowed to stand for 30min. at 4°C before centrifugation at 1, 500 G for 15min. at 4°C. In sodium phosphotungstate-Mg++, 0.5ml of test serum was added to 0.05ml of the reagent and the mixture was allowed to stand for 5min. at room temperature before centrifugation.
Cholesterol levels in the supernate were determined by enzymatic method.
On the basis of these analysis, the optimum condition for polyanion and cation in the precipitation reagents were instituted as follows:
Heparin-Mn++ (48.10mg/dl, 0.0600M, NaCl: 0.125M) heparin-Ca++ (47.62mg/dl, 0.0238M), dextran sulfate-Mg++ (1.5%, 0.1125M) and sodium phosphotungstate-Mg++ (0.4%, 0.0500M, pH 6.20).
We estimated the capability of four precipitation methods for the isolation of HDL. For this purpose, we used Ouchterlony double diffusion method.
The correlation coefficient between ultracentrifugation method (UC) and heparin-Mn++ method was 0.882, that between UC and heparin-Ca++ method was 0.972 and that between UC and sodium phosphotungstate-Mg++ method was 0.889. In the case of dextran sulfate-Mg++ method, the correlation coefficient between UC was 0.831-0.839, showing a difference when a reagent from different lot number was employed.
Thus, it is postulated that heparin-Ca++ method for the assay of HDL in the routine work could be the best among various precipitation methods currently available.
