LOW-DENSITY-LIPOPROTEIN FOR OXIDATION AND METABOLIC STUDIES - ISOLATION FROM SMALL VOLUMES OF PLASMA USING A TABLETOP ULTRACENTRIFUGE

A rapid method is described for the isolation of small volumes of plas ma low density lipoprotein (LDL) free of plasma protein contaminants u sing the TL-100 Tabletop Ultracentrifuge (Beekman). The isolation of L DL was achieved by a 25 min discontinuous gradient density centrifugat ion between the density range of 1.006 and 1.21 g/ml, recovery of LDL by tube slicing followed by a 90 min flotation step (d = 1.12 g/ml). T he purity of LDL and apolipoprotein B-100 (apo B-100) were monitored b y agarose electrophoresis, sodium dodecyl sulfate-polyacrylamide gel e lectrophoresis (SDS-PAGE), radial immunodiffusion and micropreparative fast protein liquid chromatography (FPLC). The ability of LDL oxidati on was assessed by following absorbance at 234 nm after addition of co pper ions. The functional integrity of the isolated LDL was checked by clearance kinetics after injection of [I-125]-labelled LDL in estroge n-treated rats. The additional purification step led to LDL fractions free of protein contamination and left apo B-100, alpha-tocopherol and beta-carotene intact. The LDL prepared in this way was free of albumi n, as evident from analytic tests and from its enhanced oxidative modi fication by copper ions. Used for analytical purposes, this method all ows LDL preparations from plasma volumes up to 570 mu l. This method i s also convenient for metabolic studies in small animals, especially t hose relating to the determination of kinetic parameters of LDL in whi ch LDL-apo B-100 has to be specifically radiolabelled.