Materials and Methods

Preparation of Low-Density Lipoproteins {LDL). A starting volume of at least 2 L of either pooled blood bank plasma or serum from normolipidemic healthy volunteers was used. Plasma/serum was adjusted to solution density 1.21 g/mL by addition of solid potassium bromide and subjected to ultra-centrifugation at 168000g for 24 h at+ 4 C in a Prepspin 50 preparative ultracentrifuge (MSE, Crawley, West Sussex, England) utilizing a 6 X 100 mL rotor. The supernate was recovered by aspiration and adjusted to a solution density of f From the Arterial Biology Group, Department of Medical Biochem-istry and Medicine I, University of Goteborg, Sweden. Received June 11, 1979\revised manuscript received October31, 1979. This study was supported by grants from the Swedish Medical Research Council, No. 4531, the Swedish Oleo-Margarine Foundation for Nutritional Research, and the Swedish National Associationagainst Heart and Lung Diseases.

1.063 g/mL by dialysis against doubly distilled water for approximately 20 min and recentrifuged under the same conditions. The supernate was recovered with aspiration and subjected to three recentrifugations at density 1.063 g/mL under the same conditions. After recovery, the final supernate was overlayered with 0.05 M Tris buffer, pH 7.5, with a density of 1.006 g/mL (the ratio between lipoprotein solution and buffer was 1: 3). Ultracentrifugation was carried out at 120000g for 24 h at+ 4 C in a Superspeed 65 preparative ultracentrifuge (MSE, Crawley, West Sussex, England) uti-lizing an 8 X 35 mL rotor. The supernate (the upper half of the centrifuge tube) was removed by aspiration, and the clear yellow infranate was recovered. Theinfranate appeared as a single lipid staining band with/3 migration on agarose gel electrophoresis. The lipid composition agreed well with that reported for the human low-density lipoproteins (Oncley, 1963; Skipski et al., 1967).

Delipidization of LDL. LDL were dialyzed against doubly distilled water. Thewater was carefully deaerated before use, and the glassvessel used for dialysis was flushed with nitrogen before the water was added. The space not filled with water was filled with nitrogen and the vessel was sealed. Dialysis was carried out at+ 4 C for 6 h with 20 changes of a 40-fold excess of water. After dialysis the LDL were lyophilized. One hundred milliliters of chloroform (analytical grade, Merck, Darmstadt, West Germany) was added to the dry LDL, and the mixture was carefully shaken until a fine sus-pension was obtained. Two hundred milliliters of methanol was added, and the mixture was stored at-20 C for 30 min. The protein was recovered by low-speed centrifugation, and the organic solvent was removed by aspiration. The procedure was repeated twice, followed by four extractions with chloroform-methanol, 1: 1 (v/v), and one extraction with chloroform-methanol, 2: 1. In each step the chloroform was added before the methanol to the sample. Finally, the protein pellet