A B S T R A C T High density lipoprotein was isolated from pooled rat serum and mesenteric lymph of lymph fistula rats. In most experiments, 5,5'-dithionitrobenzoic acid, an inhibitor of the enzyme lecithin:cholesterol acyltransferase, was added during the collection of lymph to prevent modification of the lipid composition of newly secreted lipoproteins. Negative staining electron microscopy of lymph high density lipoprotein revealed discoidal particles (190+3 x 55+1 A) which tended to form rouleaux, smaller spherical particles were also present. Serum high density lipoprotein contained only spherical particles (diameter 93+4 A). Lipid analysis showed that lymph high density lipoprotein was enriched in phospholipid and deficient in cholesterol esters when compared to serum high density lipoprotein. The phospholipid to cholesterol esters ratio was greatest in basal lymph high density lipoprotein when compared to fatty lymph and serum high density lipoprotein. From analysis of the lipid compositional data and direct particle measurement by electron microscopy, it could be determined that -50% of basal lymph high density lipoprotein and 30% of fatty lymph high density lipoprotein was discoid. Basal lymph high density lipoprotein was enriched in apoA-I and deficient in the arginine-rich peptide, and the apoprotein composition of fatty lymph high density lipoprotein more closely resembled serum. These observations demonstrate that intestinal lymph contains two types of high density

The major apoprotein of rat mesenteric lymph chylomicrons has been isolated and characterized and shown to be identical to apoprotein Al ( There is ample evidence that the intestine is an important source of chylomicrons and very low density lipoproteins (1, 2), but less is known concerning the role of the intestine in metabolism of high density lipoprotein (HDL). The available evidence has shown that the liver actively synthesizes nascent HDL which is secreted as disc-shaped particles (3). In addition, studies with isolated perfused rat livers indicate that nascent hepatic HDL is deficient in the major apoprotein of circulating HDL, apoprotein A1 (apo Al) (3,4), apparently acquiring this apoprotein after secretion from the liver. Some question, therefore, exists as to whether there are significant extrahepatic sites of apo A1 synthesis. The sites of synthesis of apo A1 assume increasing importance in view of the recent recognition that this apoprotein is an activator of lecithin:cholesterol acyltransferase (LCAT; EC 2.3.1.43) (5) and may be important in the regulation of cellular cholesterol content (6).Data from several laboratories (7,8) (150-200 g) of the CD strain (Charles River Laboratories, Boston, MA) were used for all studies. Cannulation of the main mesenteric lymphatic duct and duodenum and maintenance of the animals have been described (12). All animals were studied 16-24 hr after surgery. They were maintained in restraining cages and allowed free access to 0.9% (wt/vol) saline and 5% (wt/vol) dextrose. When indicated, Intralipid (Cutter Medical Laboratories, Berkeley, CA) was infused intraduodenally by a constant-infusion pump at a rate of 1 ml/hr. Bile diversion was performed by ligating the common bile duct near the duodenum and cannulating the duct, to permit complete external diversion of bile. Sham-operated rats underwent an abdominal incision and were then allowed access to water and food.Isolation of Lipoprotein Subfractions and apo A1 Purification. Lymph was defibrinated, and chylomicrons were isolated by ultracentrifugation at 3 X 106 g min. Chylomicrons were separated from the subnatant fraction by means of a tube slicer and purified by passage through agarose columns (12). In some cases, lipoproteins of density <1.006 g/ml were isolated from lymph by ultracentrifugation at 108 g min. HDL (1.063-1.210 g/ml) was isolated from mesenteric lymph of lymph-fistula rats and from pooled fresh serum obtained from 12 rats by ultracentrifugation (3.6 X 106 g hr) (13). The HDL fraction was recentrifuged at density 1.21 g/ml and then dialyzed exhaustively (48-72 hr) against 0.15 M NaCl.Purified chylomicrons were delipidated overnight at 40 with 20 volumes of ethanol/ether, 3:2 (vol/vol), and the protein precipitate was washed three times with ether. Delipidated chylomicron apoproteins were solubilized in 0.01 M Tris buffer/8 M urea/1 mM EDTA, pH 8.2, and dialyzed against the same buffer for 24 hr. apo A1 was isolated from the chylomicron apoproteins by chromatography in 8 M urea on a Sep...

The synthesis of apolipoprotein B (apoB) was examined in human fetal and adult intestine and liver. Intestine and liver were minced and then incubated with [3H]leucine, homogenized, and subjected to immunoprecipitation with antiserum that recognized both apoB-100 and apoB-48 (forms of apoB found in low density lipoproteins and in chylomicrons, respectively

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A B S T R A C T To study the metabolic fate of chylomicron phospholipid and apoproteins, 15 mg of doubly labeled ([3H]leu, [32P]phospholipid) rat mesenteric lymph chylomicrons were injected as an intravenous bolus into conscious rats. The specific radioactivity, composition, pool size, and morphology of the plasma lipoproteins were determined after 2-60 min. After injection of chylomicrons, there was a rapid transfer of radioactivity into high density lipoproteins (HDL). At peak specific activity in HDL (2-5 min), 35% of injected apoprotein and 25% of phospholipid radioactivity were recovered in HDL (d 1.063-1.21 g/ml), with smaller recoveries in other lipoproteins and liver. There was an initial rapid rise of 32P specific activity in HDL and d 1.02-1.063 lipoproteins (low density lipoproteins [LDL]), but whereas LDL specific activity subsequently converged with that of d < 1.02 lipoproteins, HDL specific activity decayed more rapidly than LDL or d < 1.02 lipoproteins.Lipolysis of chylomicrons was associated with a transfer of phospholipid mass into LDL and HDL. At 5 min, 80% of injected triglyceride had been lipolyzed and there was a significant increase in phospholipid mass in LDL and a smaller increase in HDL. At 10 min,