‘ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG note on methodology I zyx Rapid preparative isolation of concentrated low density lipoproteins and of lipoprotein-deficient serum using vertical rotor gradient ultracentrifugation zyxwvutsr Yves Poumay’ and Marie-France Ronveaux-Dupal’ U n i t i de Cytologie, Facultis Universitaires Notre-Dame zyxwvutsr de la Paix, 61 rue de Bruxelles, 5000 NanzucBelgium Summary In order to study cellular metabolism of low density lipoproteins (LDL), ultracentrifugal methods have been used to isolate the lipoproteins. The use of vertical rotor ultracentrifuga- tion very quickly produces small quantities of diluted lipopro- teins per gradient, as well as small volumes of lipoprotein- deficient serum. We present modifications to this method in order to prepare routinely more concentrated LDL and a suffi- cient volume of lipoprotein-deficient serum in a relatively short time with minimal cost and handling. -Pournay, zyxwvut Y., and M-F. Ronveaux-Dupal. Rapid preparative isolation of concentrated low density lipoproteins and of lipoprotein-deficient serum using vertical rotor gradient u1tracentrifugation.J Lipid Res. 1985. 26: 1476-1480. Supplementary key words VLDL LDI, - HDL Since the first in vitro studies on binding and degrada- tion oflow density lipoproteins (LDL) by cultured cells (l), many authors have isolated lipoproteins in order to study their metabolism in different tissues. For analytical or preparative purposes, serum lipoproteins have usually been isolated by ultracentrifugation. In addition to the standard sequential flotation method (2), density gradient ultracentrifugation procedures have been developed for theseparation of lipoproteinfractions by a single spin (3-5). However, with these latter methods, the volume of plasma is limited to a maximum of about 24 ml per spin and a centrifugation time of at least 24 or 48 hr is required in order to obtain a sufficient separation of lipoproteins for preparative purposes (6). In order to circumvent these limitations, Chung et al. (7) developed a rapid, single spin, ultracentrifugal method suitable for preparative and quantitative isolation, employing a single discontinuous density gradient in a vertical rotor. In spite of the rapidity of the separation, there is only a small quantity of LDL isolated per tube, and the preparation is very dilute and needs to be concentrated. We present here some modifications to this method in ordertoprepare large volumes of concentrated LDL. Furthermore, our preparation produces a lipoprotein- deficient serum (LPDS) useful for incubation with cul- tured cells. MATERIALS AND METHODS Freshly collected serum (80 ml) from about 20 healthy volunteers was obtained from the local Red Cross Center. The serum was pooled and used immediately for lipopro- tein isolation. The serum was adjusted to a density of 1.25 g/ml with solid KBr and transferred into two 40-ml cen- trifugationtubes(Beckman,QuickSeal, 25 x 89 mm). Centrifugation was performed in a Beckman vertical VTi 50 rotor for 16 hr at 10°C and 50,000 rpm in a Beckman L5-65 ultracentrifuge equipped with a slow acceleration accessory. The brake was cut off at the end of the run at 2,000 rpm. The top of each tube was then gently sliced with a scalpel and the upper yellow-orange, slightly opalescent layer was cautiously removed using a narrow Pasteur pipette. This layer, corresponding to lipoproteins, was separated by a colorless zone from the rest of serum proteins. Lipoproteins from the two tubes were pooled (10 ml) and the density of this solution was adjusted to 1.30 g/ml with solid KBr as described (7). Thirty ml of 0.9% NaCl was added to a Quick Seal centrifugation tube and the lipoprotein solution was layered under the saline with a syringe and narrow plastic tubing to fill the tube, which was then sealed. Centrifugation was performed as de- scribed above for 180 min. At the end of the run, VLDL, LDL, and HDL appeared well separated and the gradient was fractionated using Pasteur pipettes (5). In our first series, a constant volume of 1 ml was removed in order to study the gradient. For preparative purposes, fractions were removed on the basis of the different lipoprotein bands.Fractions of gradient were analysed for density, protein, and total cholesterol content (8, 9). Distribution and size of lipoproteins was determined by negative stain- ing electron microscopy using a 2% potassium phospho- tungstate solution, pH 6.3 (10). Electrophoretic migration of intact lipoprotein fractionswas studied in 0.8% agarose Abbreviations: VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; LPDS, lipoprotein-deficient serum; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electro- phoresis. ‘Y. P. is a Fellow of the Institut pour I’Encouragement de la Recherche Scirntifique dans 1’Industrie et 1’Agriculture (I.R.S.I.A.). ‘To whom correspondence should be sent. zyxw 14.76 Journal of Lipid Research Volume 26, 1985 Note on Methodology by guest, on July 21, 2015 www.jlr.org Downloaded from