[PDF][PDF] Rat intestine secretes spherical high density lipoproteins

GP Forester, AR Tall, CL Bisgaier, RM Glickman - J Biol Chem, 1983 - researchgate.net
GP Forester, AR Tall, CL Bisgaier, RM Glickman
J Biol Chem, 1983researchgate.net
MATERIALS AND METHODS Operative Procedures and Collection of Samples-Sprague-
Dawley rats (250-300 g) were subjected to cannulation of the main mesenteric lymphatic
and duodenum after anesthesia with sodium pentobarbital, 30-50 mg/kg, as previously
described (1). The animals were allowed to recover from anesthesia and were infused with a
solution of 5% dextrose and 0.9% NaCl at 10 ml/kg/h and were given ad libitum access to
the same solution. Lymph was collected on ice for 24-48 h with the 1ecithin: cholesterol …
MATERIALS AND METHODS
Operative Procedures and Collection of Samples-Sprague-Dawley rats (250-300 g) were subjected to cannulation of the main mesenteric lymphatic and duodenum after anesthesia with sodium pentobarbital, 30-50 mg/kg, as previously described (1). The animals were allowed to recover from anesthesia and were infused with a solution of 5% dextrose and 0.9% NaCl at 10 ml/kg/h and were given ad libitum access to the same solution. Lymph was collected on ice for 24-48 h with the 1ecithin: cholesterol acyltransferase inhibitor 5, 5 ‘-dithiobis (nitrobenzoic acid), 1 mM, at pH 7.4. At the end of the experimental period, the animals were again anesthesized with sodium pentobarbital and exsanguinated into a solution of 1 mM 5, 5” dithiobis (nitrobenzoic acid) and sodium heparin, 100 units/ml. Preparation of HDL and Their Subfractions-HDL were prepared by sequential preparative ultracentrifugation between the densities of 1.07 and 1.21 g/ml, then were fractionated by isopyknic density gradient ultracentrifugation as previously described (6). HDL were concentrated to 1 ml in a stirred, high pressure ultrafiltration cell (Amicon Corp., Lexington, MA) using a PMlO membrane, then dialyzed against NaBr solution of density of 1.11 g/ml. The dialyzed HDL were placed at the center of a gradient of solutions of NaBr of densities 1.18, 1.15, 1.11 (sample), 1.09, and 1.07 g/ml, 1 ml each. The material was centrifuged for 72 h at 50,000 rpm in an SW50. 1 rotor (Beckman). Subfractions of 325 p1 were removed by pipette and densities were determined by refractometry (6). HDL subclasses were prepared by pooling individual fractions according to measured density.
Morphologic Studies-Holo-HDL or subclasses of HDL separated by isopyknic density gradient ultracentrifugation were analyzed by polyacrylamide gradient gel electrophoresis according to the method described by Anderson et al.(7). Fractions were dialyzed against a buffer of 0.09 M Tris base, 0.15 M boric acid, 1 g/liter of EDTA, and 0.2 g/liter of sodium azide, pH 8.4. They were concentrated to 2-6 mg/ml by va, cuum dialysis in a collodion bag apparatus (Schleicher & Schuell). After addition of bromphenol blue and sucrose, fractions were applied to a 4-30% polyacrylamide slab gradient gel (Pharmacia Corp., Piscataway, NJ) with 60-80 pg of protein per lane. Protein standards (thyroglobulin, apoferritin, catalase, lactic dehydrogenase, bovine serum albumin) of diameter 7.4-13.3 nm (Pharmacia COT.) were utilized for calibration of sizes of lipoproteins identified. Gels were stained with Coomassie blue (8) or oil red 0 (0.04% in 60% ethanol) and scanned by a transmission densitometer (Transidyne Corp., Ann Arbor, MI). Negative-stain electron microscopy was performed as previously described (1, 9). The microscope was calibrated with latex particles of known size.
researchgate.net