VERY-FAST ULTRACENTRIFUGATION OF HUMAN PLASMA-LIPOPROTEINS - INFLUENCE OF THE CENTRIFUGAL FIELD ON LIPOPROTEIN COMPOSITION

A short run-time in separation of lipoproteins by preparative ultracen trifugation is desirable for several reasons. Recently, a method was d escribed that needs only 100 min for one run in a centrifugal field of 625,000 x g. It is assumed that lipoprotein separation depends on the rotor speed but this has not been systematically studied in centrifug al fields of this order. We performed such a study. Rotor speeds of 12 0, 90, 60 and 30 x 10(3) rev./min and run-times of 100 min, 3 h, 6.7 h and 27 h were selected in such a way that the product of centrifugal field and run-time remained constant. The first conditions correspond to the 'very fast ultracentrifugation' (VFU) procedure with a centrifu gal field of 625,000 x g. The Optima(TM) tabletop ultracentrifuge, the rotor TLA-120.2 and thickwall open tubes for 1 ml were used. Thirteen different plasma samples covering a wide range of cholesterol and tri glyceride concentrations were separated into VLDL, LDL and HDL in the course of two centrifugal runs at densities of 1.006 and 1.063. The co nstituents of the lipoproteins were calculated considering the mass of the tube contents after slicing. Recoveries of cholesterol, triglycer ides and protein were 97, 98, and 90, respectively. The influence of t he rotor speed on the apparent composition of the lipoproteins was sma ll. With increasing rotor speed, VLDL-cholesterol became higher (by 14 %, P < 0.001), VLDL-triglyceride became lower (by 6%, P < 0.012), LDL- cholesterol became lower (by 9%, P < 0.000). The effects on LDL-trigly ceride and on HDL-cholesterol and HDL-triglyceride, did not reach stat istical significance. Protein in VLDL and in LDL decreased and increas ed in 'HDL' (the subnatant of the LDL run). As checked by SDS-PAGE the protein effects were due to complete disappearance of albumin from VL DL and LDL while the apolipoproteins B-100, E and C-I to C-III remaine d unaffected. It is concluded that the main advantages of VFU are the short run-time and the disappearance of albumin from VLDL and LDL. The other compositional changes need to be further investigated.