VERY FAST ULTRACENTRIFUGATION OF SERUM-LIPOPROTEINS - INFLUENCE ON LIPOPROTEIN SEPARATION AND COMPOSITION

A very short run time and small sample volumes in the separation of li poproteins by preparative ultracentrifugation are needed for several i nvestigations. Recently, a very fast sequential separation method was described that needs only 100 min for one run in a centrifugal field o f 625 000 X g. We studied the influence of centrifugal fields of this dimension on lipoprotein separation and lipoprotein particle integrity using a Beekman Optima(TM) TLX ultracentrifuge with a TLA-120.2 rotor . Rotor speed (120/90/60/30.10(3) rev./min) and run time (100 min/3 h/ 6.7 h/27 h) were selected in such a way that the product of centrifuga l field and run time remained constant. The first conditions correspon d to the very fast ultracentrifugation (VFU) procedure with a centrifu gal field of 625 000 x g. Thirty different plasma samples covering a w ide range of lipid and protein concentrations were separated in the co urse of two centrifugal runs at densities of 1.006 and 1.063 kg/l whic h yielded very-low-density lipoproteins (VLDL), low-density lipoprotei ns (LDL), and the subnatant of low-density lipoproteins, including hig h-density lipoproteins (HDL) and concomitant sedimented plasma protein s. The major lipid components of the lipoproteins, triacylglycerols, f ree and esterified cholesterol, phospholipids and the apolipoproteins B and A-I, were estimated considering the masses of the tube contents after a slicing procedure. Measurements of lipids and proteins showed a very good recovery of better than 94% and 91%, respectively, and pre cision-within-series (coefficient of variation) of better than 4.2% an d 6.5%, respectively. The effects of the rotor speed on the lipoprotei n structure appeared to be weak. With increasing rotor speed, VLDL and LDL lipid constituents principally tended to decrease, whereas they i ncreased in the subnatant of the LDL-run. The mean lipoprotein mass co mposition, considering the mass percentage of each measured particle c onstituent, did not show significant alterations. Total protein decrea sed in VLDL and in LDL and increased in the subnatant of the LDL-run. As checked by an enzyme-linked immunosorbent assay (ELISA) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the pr otein effects were due to nearly complete disappearence of contaminati ng plasma proteins, especially albumin as the major contamination of V LDL and LDL. The apolipoproteins (ape) B-100, A-I, E and C-I to C-III remained nearly unaffected. The main advantages of VFU were the very s hort run time (cumulative flotation time is 3.4 h) and the elimination of albumin without repeated runs. The procedure was suitable for the assessment of lipid and protein constituents in lipoproteins from very small plasma samples (500 mu l).