Enzymatic modification of plasma low density lipoproteins in rabbits: a potential treatment for hypercholesterolemia

Proc. Natl. Acad. Sci. USA

Vol. 90, pp. 3476-3480, April 1993 Applied Biological Sciences

Enzymatic modification of plasma low density lipoproteins in rabbits: A potential treatment for hypercholesterolemia REGINE LABEQUE*t, CLAUDY J. P. MULLON*t, JOAO PAULO M. FERREIRA*, ROBERT S. LEES§, AND ROBERT LANGER*§¶ *Department of Chemical Engineering, Massachusetts Institute of Technology, Division of Health Sciences and Technology, Cambridge, MA 02139

Cambridge, MA 02139; and §Harvard-Massachusetts Institute of Technology,

Communicated by Judah Folkman, January 4, 1993 (received for review February 6, 1992)

Coronary heart disease accounts for more deaths annually than any other disease, including all forms of cancer combined (1). Epidemiologic studies have established that the higher the total plasma cholesterol and low density lipoprotein (LDL) cholesterol levels, the greater the risk of coronary heart disease (2, 3). Plasma cholesterol may be reduced by drugs, diet, and direct removal of LDL from the blood. However, drug therapy may have side effects which limit its use. For example, drugs that decrease cholesterol synthesis, such as lovastatin, may cause liver and muscle injuries and fetal abnormalities (4). Moreover, many familial hypercholesterolemic patients are resistant to diet and drug therapy. Plasmapheresis, the direct removal of the patient's highcholesterol plasma and replacement with donor albumin, although a successful therapy (5, 6), is expensive and removes a wide range of circulating proteins in addition to LDL. Alternative extracorporeal techniques have been developed to improve LDL specificity and to lower cost. Removal of LDL can be accomplished with columns of heparin-agarose (7), dextran sulfate-cellulose (8), or antibody-agarose (9-12) or by LDL precipitation at acidic pH (13). However, the limited capacity of the adsorbents makes these techniques cumbersome and very expensive. We report here that a particular phospholipase modifies plasma LDL, so that it may be removed by an individual's own metabolic processes. A small extracorporeal filter containing immobilized phospholipase A2 (PLA2; EC 3.1.1.4) caused a 13-28% decrease in plasma cholesterol concentration in cholesterolfed rabbits after a 90-min treatment.

soybean type II-S phosphatidylcholine were from Sigma. Na125I (17.4 Ci/mg in 0.1 M NaOH; 1 Ci = 37 GBq) was from New England Nuclear. Rabbit Chow containing 0.5% cholesterol was purchased from Purina. PLA2. PLA2 from Crotalus atrox venom (Miami Serpentarium, Salt Lake City, UT) was purified as reported (14). The protein concentration was determined by the method of Lowry et al. (15) with bovine serum albumin as a standard. PLA2 was immobilized to Affi-Gel 15 according to the manufacturer's protocol. The enzymatic activity of soluble and immobilized PLA2 was measured by the method of Nieuwenhuizen et al. (16), and 1 unit of enzyme was defined as the amount that hydrolyzes 1 ,umol of phosphatidylcholine per minute at pH 8.9 and 25°C. One gram of the agarose support had 60-70 units of enzymatic activity. Immobilized PLA2 was stable, with no detectable loss of activity, even after 2-3 months of storage at 4°C. For control studies, PLA2 was inactivated at 160°C for 15 min. Radioiodination of PLA2. C. atrox PLA2 was radiolabeled with the lodo-Beads technique (Pierce). After 5 min of incubation of Na'251 solution (1 mCi) with 4-6 beads, 2-3 mg of enzyme in 100 mM phosphate buffer (pH 7.4) was added and the reaction proceeded for 15 min. The mixture was passed through a gel-filtration column (Econo-Pac 10 DG, Bio-Rad) and the protein fractions pooled were dialyzed against water. Final labeling efficiencies were 4-7 ,ACi/mg of protein, with no loss of enzymatic activity. Lipoprotein Preparation. LDL (density, 1.025-1.05 g/ml) was isolated from serum of normolipidemic human donors by sequential flotation (17). LDL protein content was quantified (15) with bovine serum albumin as a standard. LDL was iodinated with Na1251 by the method of McFarlane (18), as modified for lipoproteins (19). Labeling efficiencies were 45-60%. The fraction of radioactivity precipitable by 10%to (wt/vol) trichloroacetic acid ranged from 94% to 97%, and lipid-associated radioactivity was