Transposition and distribution of cholesterol in rat erythrocytes.
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Animals↗
Biomedical subjects
D B Zilversmit
Publications and source records attributed to D B Zilversmit.
Chylomicron remnant cholesteryl esters as the major constituent of very low density lipoproteins in plasma of cholesterol-fed rabbits.
Feeding rabbits 500 mg of cholesterol daily for 4 to 15 days greatly increased the concentration of esterified cholesterol in lipoproteins of d less than 1.006 g/ml. The origin of hypercholesterolemic very low density lipoproteins was investigated by monitoring the degradation of labeled lymph chyomicrons administered to normal and cholesterol-fed rabbits. Chylomicrons were labeled in vivo by feeding either 1) [3H]cholesterol and [14C]oleic acid or 2) [14C]cholesterol and [3H]retinyl acetate. After intravenous injection of labeled chylomicrons to recipient rabbits, [14C]triglyceride hydrolysis was equally rapid in normal and cholesterol-fed animals. Normal rabbits rapidly removed from plasma both labeled cholesteryl and retinyl esters, whereas cholesterol-fed rabbits retained nearly 50% of doubly labeled remnants in plasma 25 min after chylomicron injection. Ultracentrifugal separation of plasma into subfractions of very low density lipoproteins showed that chylomicron remnants in cholesterol-fed animals are found among all subclasses of very low density lipoproteins. Analysis of cholesteryl ester specific activity-time curves for the very low density lipoproteins subfraction from hypercholesterolemic plasma showed that nearly all esterified cholesterol in large very low density lipoproteins and approximately 30% of esterified cholesterol in small very low density lipoproteins was derived from chylomicron degradation. Apparently, nearly two-thirds of the esterified cholesterol in total very low density lipoproteins from moderately hypercholesterolemic rabbits is of dietary origin.
Animals↗
Validation of a stable emulsion for the assay of lipoprotein lipase activity.
The preparation of a stable, stock, radioactive, trioleoyl glycerol emulsion in glycerol and its application to the assay of lipoprotein lipase are described. The data presented indicate that this stock emulsion combines the convenience and reproducibility of commercially available, nonradioactive emulsions with the sensitivity of sonicated, radioactive, aqueous emulsions. Furthermore, in practice, this glycerol-based emulsion appears to be specific for the protamine-sensitive extrahepatic lipase, having little or no reactivity toward the hepatic lipase measured in postheparin plasma.
Emulsions↗
A new rat liver phospholipid exchange protein.
The soluble fraction from several mammalian tissue homogenates is known to stimulate phospholipid exchange between cell membrane fractions or artificial vesicles. All phospholipid exchange proteins purified to data exhibit an acidic isoelectric point. Using an assay that measures the transfer of [32P] phosphatidylcholine from liposomes to beef heart mitochondria, we report the presence of a new phospholipid exchange protein with a basic isoelectric point (8.4) in rat liver cytosol. A purification procedure, consisting of pH adjustment to 5.1, gel filtrations on Sephadex G 75 and DE 52 cellulose, isoelectric focusing between a pH of 5 and 10, and gel filtration on Sephadex G-50, yielded a fraction with high phosphatidylcholine exchange activity per mg of protein. This fraction exhibits a major band and two minor bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the major band (18 700) is close to that for basic exchange protein fraction obtained by gel filtration (17 000). The distribution of basic and acidic exchange proteins differs markedly in various tissues and animal species. About 50 and 35% of phosphatidylcholine exchange activity from rat liver and rat intestine respectively are due to basic phospholipid exchange proteins. In contrast, no basic exchange protein was found in beef heart and only a small amount in beef liver. In the latter organ, less than 10% of phosphatidylcholine exchange activity was due to a basic phospholipid exchange protein fraction.
Animals↗
Asymmetry and transposition rates of phosphatidylcholine in rat erythrocyte ghosts.
Purified phospholipid exchange protein from beef heart cytosol is used to accelerate the exchange of phospholipids between labeled sealed ghosts and phosphatidylcholine/cholesterol liposomes. The purified protein accelerates the transfer of phosphatidylcholine and, to a lesser degree, that of sphingomyelin, phosphatidylinositol, and lysophosphatidylcholine. The presence of exchange protein does not accelerate the exchange of phospholipids between intact red blood cells and liposomes, but 75% of the phosphatidylcholine of sealed ghosts is readily available for exchange. The remaining 25% is also exchangeable but at a slower rate. When the exchange is assayed between inside-out vesicles and liposomes, 37% of the phosphatidylcholine is readily available, and 63% is exchanged at a slower rate. These results are consistent with an asymmetric distribution of phosphatidylcholine in isolated erythrocyte membrane fractions. The sum of the forward and backward transposition of phosphatidylcholine between the inside and outside layers of sealed ghost membranes amounts to 11% per hour, and the half-time for equilibration is 2.3 h. Significatnly lower values are obtained for the inside-out vesicles (half-time for equilibration: 5.3 h). These results suggest that, during the formation of the vesicles, the asymmetry of phosphatidylcholine is partially preserved, but structural changes occur in the membrane that affect the rate of membrane transposition of phosphatidylcholine.
Animals↗
Phospholipid exchange proteins in rat intestine.
The 105,000 g supernatant and pH 5.1 supernatant fractions from rat intestinal homogenates stimulate phosphatidylcholine exchange between [32P] phosphatidylcholine liposomes and beef heart mitochondria. This active fraction shows the characteristics of a protein. Isoelectric focusing of the intestinal pH 5.1 fraction shows two peaks of phosphatidylcholine exchange activity: one at an acidic pH (4.5-5.3), the other in a basic pH range (8-9). The second peak of activity appears to be a new phospholipid exchange protein. The anatomic distribution of phosphatidylcholine exchange activity in intestine has been investigated. Expressed per mg of protein, phosphatidylcholine exchange activity is higher in mucosa than in the intestinal wall. No significant differences have been found between villi and crypts cells or between jejunal and ileal villi. Futhermore, exchange activity per mg of protein in mucosa is unaffected by fasting or by feeding a high fat or high cholesterol diet. This suggests that phospholipid exchange activity in the absorptive cells is not a rate limiting step in the process of fat absorption.
Animals↗
Role of triglyceride-rich lipoproteins in atherogenesis.
The hypothesis that chylomicron cholesterol is a causal factor in the accumulation of cholesterol in atheromatous lesions is compatible with the finding reported here that hypercholesterokmia in the cholesterol-fed rabbit is due, at least, in part, to the accumulation of chylomicron remnants. The presence of lipoprotein lipase in aorta suggests the possibility that chylomicron remnants are formed on the arterial surface and that they may be deposited in the deeper layers of the arterial wall without prior release into the bloodstream. Consequently, the cholesterol-rich lipo-proteins in the plasma may be the product rather than the precursor of the atherogenic process.
Animals↗
Disposition of plasma cholesterol in the pregnant hypocholesterolemic rabbit.
Processes that might explain the hypocholesterolemia observed in pregnant rabbits were studied in animals fed a cholesterol-free chow diet. Excretion of endogenous steroids, measured after administration of a single intravenous dose of radioactive cholesterol, averaged 75 mg/day in controls and 96 mg/day in pregnant hypocholesterolemic rabbits. The mass of maternal plasma cholesterol transferred to the fetuses was measured in animals in which a constant plasma cholesterol specific activity was maintained. Placental transfer during the first 28 days of gestation ranged from 2 to 12 mg/day for mothers carrying litters of one to eight fetuses. It is concluded that loss of maternal plasma cholesterol due to placental transfer is small compared to loss by fecal steroid excretion. Because large decreases in plasma cholesterol concentration during pregnancy and increases at parturition were not accompanied by changes in the slopes of the specific activity-time curves, it appears most likely that cholesterol in isotopic equilibrium with plasma cholesterol is temporarily and reversibly sequestered in maternal tissues of the pregnant rabbit.
Animals↗
Stimulation of cholesterol ester exchange by lipoprotein-free rabbit plasma.
A fraction in normal and hypercholesterolemic rabbit plasma of density greater than 1.25 stimulates the exchange of cholesterol esters between very low density and low density lipoproteins from hypercholesterolemic rabbit plasma. The exchange does not result from lecithin:cholesterol acyltransferase activity. The active factor appears to be a high molecular weight globulin with an isolelectric point of 5.2.
Animals↗
Catalytic properties of phospholipid exchange protein from bovine heart.
A protein which catalyzes the exchange of phosphatidylcholine between membranes has been purified from heart tissue homogenates up to 300-fold by acidic pH precipitation, (NH4)2SO4 precipitation, gel filtration, and ion-exchange chromatography. Binding of the protein to phosphatidylcholine liposomes as measured by Sepharose chromatography was nondetectable. However, isoelectric focusing experiments showed that individual molecules of phosphatidylcholine were transferred from liposomes to the soluble, partially purified protein. Exchange of phospholipid between liposomes and mitochondria was not affected by the presence of moderate amounts of cholesterol in liposomes. A search for competitive inhibitors among moieties similar to phosphatidylcholine failed to show strong binding sites in the hydrophilic part of the substrate. High concentrations of Na+, Ca2+ and Mg2+ impaired the exchange activity.
Animals↗
Use of phospholipid exchange protein to measure inside-outside transposition in phosphatidylcholine liposomes.
The exchange of phosphatidylcholine between (32P)phosphatidylcholine lipososomes and unlabeled mitochondria was catalyzed by a purified phospholipid exchange protein from bovine heart cytosol. The loss of (23P)phosphatidylcholine from the liposomes appeared to proceed in two stages: with 100 units of phospholipid exchange protein per ml the half-time of the initial stage was about 10 min and that of the final stage 4 days or greater. Agarose-gel chromatography of the liposomes showed an elution compatible with a homofwnwoua pool od amLL single walled vesicles. Treatment of phosphatidyl (14C) choline liposomes with phospholipase D (phosphatidylcholine phosphantidohydrolase) showed that labeled phospholipid removable during the rapid exchange phase was subject to hydrolysis by the phospholipase, but that the labeled phospholipid left after the rapid exchange was completed cound not by hydrolyzed by phospholipase D. It is proposed that the rapidly exchanging phosphatidylcholine constitutes the outer layer of the liposome bilayer. The long half-lives of 4 days or more probably represent the transposition of phosphatidylcholine from the inner to the outer layer of the liposome bilayer.
Animals↗
Mechanisms of cholesterol accumulation in the arterial wall.
The rate of cholesterol accumulation is a function of three separate processes: the transfer of lipid or lipoprotein from blood plasma to the artery, the binding and sequestering of lipids in the arterial wall and the solubilization and removal of lipid from the artery. These processes have been studied with lipids or lipoproteins labeled with radioisotopes by autoradiographic and quantitative chemical procedures. More recently immunochemical procedures have been applied to this problem. Studies have been performed with intact animals, isolated organs and cell cultures. In addition, homogenates have been used successfully to study intraarterial transformations of lipids, (for example, cholesterol esterification). Although epidemiologic and clinical studies, as well as animal experiments, have provided evidence that the concentration of plasma low or very low density lipoproteins parallels the rate of atherogenesis, the nature of the causal chain linking plasma lipoproteins to atherosclerosis is as yet unclear. A possible link between plasma lipoproteins, arterial liproprotein lipase and atherogenesis has been postulated.
Animals↗
Purification of phospholipid exchange proteins from beef heart.
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Animals↗
Lipoprotein lipase activity in bovine aorta.
A lipoprotein lipase in the bovine arterial wall has been identified and partially characterized. The enzyme has a Km apparent of 1 mM for triolein in a phosphatidylcholine stabilized emulsion. The lipase was stimulated 20- to 30-fold by the addition of heated rat plasma to the assay medium. The activity exhibited a pH optimum at 8.6. Protamine sulfate (1.0 mg/ml) inhibited the activity by 50%, whereas 1.4 M sodium chloride inhibited by 85%. Sodium fluoride, an inhibitor of the hormone-sensitive lipase, had no effect on the activity. Additions of low concentrations of heparin or Ca-2+ to the enzyme caused a slight stimulation of the lipolytic activity. A crude sectioning of the aorta revealed specific activity of lipoprotein lipase to be highest at the endothelial side of the artery.
Animals↗
Cholesterol catabolism in the rabbit in fasted and fed states.
Urinary and fecal endogenous steroid excretion of fed or fasted New Zealand white rabbits was determined by the isotopic steady state method after subcutaneous implantation of radioactive cholesterol. While plasma cholesterol was increasing during a 9-day fast, fecal steroid excretion decreased to 10% of the excretion rates in the fed state. Refeeding the fasted rabbits led to a decrease in plasma cholesterol and an increase in fecal endogenous steroid excretion. Urinary steroid excretion, which represented 18% of total endogenous steroid excretion for fed animals, decreased during fasting and increased during refeeding, but these changes were relatively small. The small intestine, cecum, and colon of fed or fasted rabbits had similar endogenous steroid was acidic steroid. During attempts to alter the circulating bile acid concentration by supplying deoxycholate (200 mg/day) to fed rabbits or cholestyramine (2 g/day) to fasted rabbits, plasma cholesterol concentration did not change to the same extent as during fasting or refeeding, respectively. The decreased cholesterol catabolism and the hypercholesterolemia that are seen in the fasting rabbit may result from decreased clearance of plasma cholesterol.
Animals↗
Effects of dietary roughage on cholesterol absorption, cholesterol turnover and steroid excretion in the rat.
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Absorption↗
Influx of cholesterol into plasma in rabbits with fasting hyperbetalipoproteinemia.
New Zealand white rabbits exhibited as much as a threefold increase in plasma cholesterol but no change in hepatic cholesterol when fasted for 7-9 days. Agarose electrophoresis and ultracentrifugation of plasma samples showed that only low density lipoprotein increased during fasting. Fasting changed the composition of the low density lipoprotein by increasing the percentage of cholesterol and decreasing the percentage of triglyceride while protein and phospholipid remained the same. Rates of cholesterol secretion into plasma, measured by Triton WR 1339 injection, and rates of plasma cholesteryl ester synthesis, determined by [2-(14)C]mevalonate injection, were similar for fed and fasted rabbits. These findings suggest that fasting hypercholesterolemia in rabbits did not result from increased production of low density lipoproteins. Triton WR 1339 was shown to inhibit plasma cholesterol esterification in vitro.
Animals↗
Validation of a dual-isotope plasma ratio method for measurement of cholesterol absorption in rats.
Several methods for measuring cholesterol absorption in the rat have been compared. After administration of an oral dose of labeled cholesterol ((14)C or (3)H) and an intravenous dose of colloidal labeled cholesterol ((3)H or (14)C) the ratio of the two labels in plasma or whole blood 48 hr or more after dosing compared closely to the ratio of areas under the respective specific activity-time curves. The area ratio method is independent of a time lag between the appearance of oral and intravenous label in the bloodstream. Both measures of cholesterol absorption agree fairly well with a method based on measuring the unabsorbed dietary cholesterol in a pooled fecal sample. The plasma isotope ratio method gave more reproducible results than the fecal collection method when the measurement was repeated in the same animals 5 days after the first measurement. Cholesterol absorption was overestimated by the use of Tween 20-solubilized labeled cholesterol for the intravenous dose. The plasma disappearance curves of injected labeled colloidal cholesterol and cholesterol-labeled chylomicrons infused intravenously over a 3.5-hr period in the same animal coincided within experimental error from the first day until 75 days after injection. The plasma isotope ratio method for cholesterol absorption gave the same results in rats practicing coprophagy as in those in which this practice was prevented. The addition of sulfaguanidine to the diet lowered cholesterol absorption as measured by the plasma isotope ratio to the same degree as that measured by the fecal collection method.
Animals↗