Exchange Of Free Cholesterol Research Articles

ANGPTL3 Is Involved in the Post-prandial Response in Triglyceride-Rich Lipoproteins and HDL Components in Patients With Coronary Artery Disease.

It is well-established that there exists an inverse relationship between high-density lipoprotein (HDL) cholesterol and triglyceride (TG) levels in the plasma. However, information is lacking on the impact of post-prandial triglyceride-rich lipoproteins (TRLs) on the structure of HDL subclasses in patients with coronary artery disease (CAD). In this study, the data of 49 patients with CAD were analyzed to evaluate dynamic alterations in post-prandial lipid profiles using nuclear magnetic resonance-based methods. An enzyme-linked immunosorbent assay was used to quantify the serum angiopoietin-like protein 3 (ANGPTL3). After glucose supplementation, the expression of hepatic ANGPTL3 was evaluated both in vitro and in vivo. Compared to fasting levels, the post-prandial serum TG level of all participants was considerably increased. Although post-prandial total cholesterol in HDL (HDL-C) remained unchanged, free cholesterol in HDL particles (HDL-FC) was significantly reduced after a meal. Furthermore, the post-prandial decrease in the HDL-FC level corresponded to the increase in remnant cholesterol (RC), indicating the possible exchange of free cholesterol between HDL and TRLs after a meal. Moreover, CAD patients with exaggerated TG response to diet, defined as TG increase >30%, tend to have a greater post-prandial increase of RC and decrease of HDL-FC compared to those with TG increase ≤30%. Mechanistically, the fasting and post-prandial serum ANGPTL3 levels were significantly lower in those with TG increase ≤30% than those with TG increase >30%, suggesting that ANGPTL3, the key lipolysis regulator, may be responsible for the different post-prandial responses of TG, RC, and HDL-FC.

Comparison of the capacity of β-cyclodextrin derivatives and cyclophanes to shuttle cholesterol between cells and serum lipoproteins

Previous studies from this laboratory have demonstrated that low concentrations of cyclodextrins (<1.0 mm), when added to serum, act catalytically as cholesterol shuttles to accelerate the exchange of free cholesterol between cells and serum lipoproteins. As cholesterol shuttles, cyclodextrins have the potential to serve as pharmacological agents for modifying cholesterol metabolism. In the present study, we have quantitated the cholesterol-shuttling capacity of a series of newly synthesized β-cyclodextrin derivatives (βCDs), with varying structure, and two double-decker cyclophanes. The general protocol is as follows. [3H]cholesterol-labeled CHOK1 cells are incubated for 2 h with the test compounds alone or together with 5% human serum, and efflux of the cellular [3H]cholesterol is measured. As methyl β-cyclodextrin (MβCD) served as the basis for comparison, initial experiments were conducted that demonstrated there was a dose-dependent stimulation of cell cholesterol efflux as the concentration of MβCD increased, with an EC50 that was calculated to be 0.05 mm. To determine the cholesterol-shuttling capacity of the newly synthesized compounds, cell cholesterol efflux is measured when the compounds are present alone, at a concentration of 0.05 mm, or together with 5% human serum.▪Our results demonstrate that the double-decker cyclophanes are the most efficient cholesterol shuttles. Under our experimental conditions, methyl β-cyclodextrin (MβCD) approximately doubles the efflux of cell cholesterol to serum, whereas one of the double-decker cyclophanes produces a 4-fold stimulation in efflux. Four of the β-cyclodextrin derivatives (βCDs) display shuttling ability similar to that of MβCD. Furthermore, there does not appear to be a structural pattern among the other βCDs which could explain their shuttling capacity.—Christian, A. E., H-S. Byun, N. Zhong, M. Wanunu, T. Marti, A. Fürer, F. Diederich, R. Bittman, and G. H. Rothblat. Comparison of the capacity of β-cyclodextrin derivatives and cyclophanes to shuttle cholesterol between cells and serum lipoproteins. J. Lipid Res. 1999. 40: 1475–1482.

Cholesterol kinetics in subjects with bile fistula. Positive relationship between size of the bile acid precursor pool and bile acid synthetic rate.

Our aim was to identify and quantitate cholesterol pools and transport pathways in blood and liver. By studying bile fistula subjects, using several types of isotopic preparations, simultaneous labeling of separate cholesterol pools and sampling all components of blood and bile at frequent intervals, we developed a comprehensive multicompartmental model for cholesterol within the rapidly miscible pool. Data in six components (bile acids, esterified cholesterol in whole plasma, and free cholesterol in blood cells, bile, alpha lipoproteins, and beta lipoproteins) were modeled simultaneously with the SAAM program. The analysis revealed extensive exchange of free cholesterol between HDL and liver, blood cells, and other tissues. There was net free cholesterol transport from HDL to the liver in most subjects. The major organ that removed esterified cholesterol from blood was the liver. A large portion (4,211 mumol) of total hepatic cholesterol comprised a pool that turned over rapidly (t1/2 of 72 min) by exchanging mainly with plasma HDL and was the major source of bile acids and biliary cholesterol. Only 6% of hepatic newly synthesized cholesterol was used directly for bile acid synthesis: the analysis showed that 94% of newly synthesized cholesterol was partitioned into the large hepatic pool (putative plasma membrane free cholesterol) which exchanged rapidly with plasma lipoproteins. Bile acid synthetic rate correlated directly with the size of the large hepatic pool. In conclusion, hepatic and blood cholesterol pools and transports have been quantitated. HDL plays a central role in free cholesterol exchange/transport between all tissues and plasma. In humans, the metabolically active pool comprises a large portion of total hepatic cholesterol that, in part, regulates bile acid synthesis.

Exchange of free cholesterol between plasma and erythrocytes from hyperthyroid and hypothyroid ratsin Vitro

In our previous studies, we found that circulating thyroid hormone levels alter cholesterol partition between plasma and erythrocytes by changing the phospholipid content of erythrocytes (Ruggiero, F. M., et al. (1984) Horm. Metabol. Res. 16, 37-40; Ruggiero, F. M., et al. (1987) Lipids 22, 148-151). As an extension of this work, we now followed the exchange of free cholesterol between plasma and erythrocytes in control, hyperthyroid and hypothyroid rats under various experimental conditions in vitro. In control rats, erythrocytes incubated with plasma at 37 degrees C for 4 hr lose 10% of cholesterol which was esterified by lecithin:cholesterol acyltransferase (LCAT) present in the plasma. In hyperthyroid rats, erythrocytes incubated with plasma lose 30% of cholesterol within the same time. By contrast, in the case of hypothyroid rats incubation for 4 hr was necessary to transfer 24% of free cholesterol from plasma to erythrocytes. Inhibition of cholesterol esterification did not affect the loss of erythrocyte cholesterol in control and in hyperthyroid rats. Ca2+ increased the LCAT activity in the plasma of these rats. The findings shed light on the role of thyroid hormones in regulating cholesterol levels in plasma through active cholesterol transfer between plasma and erythrocytes.

The Dynamics of Free Cholesterol Exchange May Be Critical for Endothelial Cell Membranes in the Brain

The Dynamics of Free Cholesterol Exchange May Be Critical for Endothelial Cell Membranes in the Brain

Effects of Dietary Beef and Soy Protein on Tissue Composition and Low Density Lipoprotein Uptake in Young Pigs

To study effects of dietary protein source on plasma cholesterol homeostasis, six littermate pairs of 8-wk-old, castrated male pigs were assigned randomly to high fat diets containing either lean beef or soy protein isolate, with beef tallow as the major fat source in both diets. Diets were fed for 6 wk, with blood samples taken weekly and analyzed for lipid composition. At 6 wk, each pig was injected with [3H]cholesterol- and [14C]sucrose-labeled low density lipoproteins, and serial blood samples were taken. Pigs were then killed, and several tissues were sampled. Results show that dietary beef or soy protein, when fed with beef tallow as a fat source, has differential effects on rates of uptake of intact LDL by heart, M. sartorius muscle and subcutaneous adipose tissue, and on rates of uptake of LDL cholesterol by net free cholesterol exchange in subcutaneous and perirenal adipose tissues. Dietary protein source affected the composition of only two tissues analyzed. Pigs fed the beef-based diet had greater lipid concentrations in M. sartorius muscle, whereas pigs fed the soy protein-based diet had greater lipid concentrations in heart. Source of dietary protein did not influence in vivo kinetics of LDL cholesterol removal from blood plasma.

Phospholipase A 2-treated human high-density lipoprotein and cholesterol movements: exchange processes and lecithin: cholesterol acyltransferae reactivity

Human HDL 3 ( d 1.125−1.21 g/ml) were treated by an exogenous phospholipase A 2 from Crotalus adamenteus in the presence of albumin. Phosphatidylcholine hydrolysis ranged between 30 and 90% and the reisolated particle was essentially devoid of lipolysis products. 1. (1) An exchange of free cholesterol was recorded between radiolabelled erythrocytes at 5–10% haematocrit and HDL 3 (0.6 mM total cholesterol) from 0 to 12–15 h. Isotopic equilibration was reached. Kinetic analysis of the data indicated a constant rate of free cholesterol exchange of 13.0 μM/h with a half-time of equilibration around 3 h. Very similar values of cholesterol exchange, specific radioactivities and kinetic parameters were measured when phospholipase-treated HDL replaced control HDL. 2. (2) The lecithin: cholesterol acyltransferase reactivity of HDL 3, containing different amounts of phosphatidylcholine, as achieved by various degrees of phospholipase A 2 treatment, was measured using a crude preparation of lecithin: cholesterol acyltransferase (the d 1.21–1.25 g /ml plasma fraction). The rate of esterification was determined between 0 and 12 h. Following a 15–30% lipolysis, the lecithin:cholesterol acyltransferase reactivity of HDL 3 was reduced about 30–40%, and then continued to decrease, though more slowly, as the phospholipid content was further lowered in the particle. 3. (3) The addition of the lecithin:cholesterol acyltransferase preparation into an incubation medium made of labelled erythrocytes and HDL 3 promoted a movement of radioactive cholesterol out of cells, above the values of exchange, and an accumulation of cholesteryl esters in HDL. This reflected a mass consumption of free cholesterol, from both the cellular and the lipoprotein compartments upon the lecithin:cholesterol acyltransferase action. As a consequence of a decreased reactivity, phospholipase-treated HDL (with 2/3 of phosphatidylcholine hydrolyzed) proved much less effective in the lecithin:cholesterol acyltransferase-induced removal of cellular cholesterol.

Cholesterol transport and uptake in miniature swine fed vegetable and animal fats and proteins. 2. LDL uptake and cholesterol distribution in tissues.

In a 2 X 2 factorial arrangement, miniature pigs were fed four diets containing vegetable protein/fat (soybean) and animal protein (egg white)/fat (beef tallow) to demonstrate the effects of protein and fat source on tissue cholesterol concentrations, uptake of intact low density lipoproteins (LDL) and free cholesterol exchange from LDL to tissues. Soybean oil feeding, compared with beef tallow feeding, resulted in greater concentrations of cholesterol in aorta, heart, and large and small intestines. Similar trends were seen in liver, adipose tissue and skeletal muscle. Dietary protein source had little or no effect on tissue cholesterol concentrations. Uptake of intact LDL, as measured by using [14C]sucrose-LDL, tended to be greater in pigs fed soybean oil or soy protein. Net exchange of free cholesterol from LDL, as measured with [3H]cholesterol, tended to be greater when vegetable products were fed. Relative contributions of whole tissues to total uptake by either mechanism were not influenced by diet. Mechanisms in addition to uptake of cholesterol from LDL seem to be involved in the greater accumulation of tissue cholesterol resulting from polyunsaturated fat feeding.

Cholesterol exchange in platelets, erythrocytes and megakaryocytes

Cholesterol exchange between plasma and human platelets and erythrocytes and guinea pig platelets, erythrocytes and megakaryocytes was studied. The characteristics of exchange of cholesterol between [ 3H]cholesterol-labeled plasma and human platelets and erythrocytes were similar: exchange per cell was independent of cell concentration in whole plasma, decreased only 2-fold over a wide range of cell concentrations in low concentrations of plasma and approached a plateau at 1/3 normal plasma cholesterol concentration, and there was no net change in the cholesterol content of either cell. The activation energy for exchange for both cells was 47 kJ/mol. In all experiments, erythrocyte cholesterol was labeled to approximately twice the specific activity of platelet cholesterol. Guinea pig megakaryocyte cholesterol exchanged at 25–33% of the rate of guinea pig platelet cholesterol in vitro. Similarly, when guinea pigs were fed [ 3H]cholesterol], erythrocyte cholesterol specific activity after 24 h was 90%, platelet 50–65%, and megakaryocyte 20–26% that of plasma. Guinea pig platelets incubated with plasma radiolabeled in free and esterified cholesterol incorporated radioactivity from free but not esterified cholesterol. The similarity of free cholesterol exchange in platelets and erythrocytes in vitro and in vivo and the apparent inability of platelets to take up cholesterol esters from lipoproteins suggest that the interaction between normal platelets and normocholesterolemic plasma is limited to cholesterol exchange.

In vivo influx of free and esterified plasma cholesterol into human aortic tissue without atherosclerotic lesions.

In order to determine the in vivo influx of plasma cholesterol into human aortic intimamedia tissue, specimens of the ascending aortic wall without visible atherosclerosis were obtained from patients undergoing aortic valve replacement. Before the operation the patients were intravenously injected with autologous plasma in which the lipoproteins were labeled with radioactive cholesterol. The influence of the duration of the exposure time (0.3-114 h) and of the distribution of radioactivity between free and esterified cholesterol in plasma on the amount of radioactivity found in the arterial wall was studied by the simultaneous use of 3H- and 14C-cholesterol. It was shown that the influx of free and esterified cholesterol into the intima-media layer of the tissue could be calculated from a set of linear equations that relate the labeled sterols in the tissue to the average specific activities in plasma. In nine patients between 50 and 70 yr of age with 4.2-5.9 mM total cholesterol in plasma, the influx of free cholesterol and of esterified cholesterol was 1.2-8.8 and 1.0-12.5 nmol X cm-2 X d-1, respectively. Both hydrolysis and esterification of the sterol fractions in the aortic tissue and exchange of free cholesterol between the plasma lipoproteins and the tissue were demonstrated. The cholesterol content of the intima-media layer was 0.6-2.3 mumol X cm-2. This corresponds to the influx of esterified cholesterol during a period of only 0.1-3.5 yr, which is short compared with the lifespan of the patient. Our data thus suggest that removal of esterified cholesterol from aortic tissue without visible atherosclerosis represents a major importance for the cholesterol concentration in the tissue.

Free cholesterol distribution during in vitro lipolysis of rat plasma very low density lipoprotein: lack of a role for blood and heart cells.

In the present study, an attempt was made to quantify free cholesterol transfer from lipolyzed VLDL to HDL, blood cells and heart cells. The experiments were carried out in vitro or in the isolated perfused rat heart with rat plasma VLDL labelled biosynthetically with [14C]-palmitic acid and [3H]cholesterol, and with bovine milk lipoprotein lipase, human blood cells (erythrocytes, leucocytes and platelets) or rat plasma HDL. Exchange and transfer of free cholesterol was followed by radioactivity and specific activity determinations. The study demonstrated an exchange of free cholesterol between VLDL and blood cells (6-10 h) and VLDL and HDL (120 min). However, none of the blood cells tested served as acceptor for lipolysis-generated free cholesterol, whereas HDL did. In the isolated perfused rat heart, a maximum of 25% of the free cholesterol radioactivity lost from VLDL was found in the tissue. Since exchange must have contributed to this process, the transfer of free cholesterol molecules to the heart is necessarily lower. The study thus demonstrated minimal or possibly no net transport of free cholesterol from VLDL to cells and cell membranes.

Central role of high density lipoprotein in plasma free cholesterol metabolism.

This study was designed to provide direct information on the in vivo metabolism in man of free (unesterified) cholesterol in the major lipoprotein classes. Five human subjects were administered one or two (simultaneous) of the following; [2-(14)C] mevalonic acid, high density lipoprotein (HDL)-free [(14)C] cholesterol, low density lipoprotein (LDL)-free [(14)C] cholesterol, and very low density lipoprotein (VLDL)-free [(3)H]cholesterol. Blood was then obtained at frequent intervals for at least 9 h, and the alpha(HDL) and beta(LDL + VLDL) lipoproteins were quickly separated by heparin-manganese precipitation to prevent ex vivo exchange of free cholesterol. After the administration of [(14)C]mevalonic acid the specific activity (disintegrations per minute/micromole) of free cholesterol in the alpha- and beta-lipoproteins increased for 3 h. During this period the alpha-free cholesterol specific activity was higher than the beta specific activity. After administration of VLDL and LDL labeled with free cholesterol, the alpha-free cholesterol specific activity reached a peak value within 20 min, at which time it was considerably lower than the beta-free cholesterol specific activity. When HDL labeled with free cholesterol was administered, a precursor product relationship was observed between the alpha-free cholesterol (precursor) and beta-free cholesterol (product) specific activities.A multicompartmental model was developed that contained the simplest structure necessary to fit all of the data obtained. The kinetic analysis revealed the presence of extensive exchange (20-85 mumol/min) of free cholesterol between HDL and a tissue pool(s) enriched with newly synthesized free cholesterol. It was found that virtually all (>95%) of the free cholesterol in the beta-lipoproteins (LDL+VLDL) cycles directly through HDL. The free cholesterol in LDL appears to behave in the same fashion as the free cholesterol in VLDL. The results show that there are marked differences in the kinetic behavior of the free cholesterol fractions of alpha- and beta-lipoproteins. There is extensive recycling of free cholesterol between HDL and tissue pools, and between HDL and the beta-lipoproteins; this recycling has been quantitated. The findings support the view that in vivo, the free cholesterol in HDL plays a central role in exchange reactions and in the vascular-tissue cholesterol transport system.

In vivo study of cholesterol turnover in tissues of adult sows

The in vivo study of free and esterified cholesterol turnover was carried out in 15 tissues of adult Large White sows maintained at a constant weight for 10–12 weeks. They received a single intravenous injection either of [1- 14C] acetate, or of an autologous red cell suspension or of plasma, previously labelled in vitro (for red cells) or in vivo (for plasma) with tritiated cholesterol. The tissues can be separated into four groups according to their relative rate of free cholesterol exchange between plasma and tissues. The liver and the lungs have a very fast exchange rate whereas the brain and the spinal cord have a very slow one. The whole lipoprotein particle transfer — an exclusive model for the esterified cholesterol transport from plasma to tissues — has been found in all sow tissues. When [1- 14C] acetate is used as a substrate for cholesterol synthesis, lungs, adrenal glands and heart do not seem — or at an extremely low rate — to convert acetate into cholesterol whereas an intense cholesterol synthesis takes place in the small intestine. Its contribution to cholesterol synthesis in sows — taking into account the cholesterol transfer processes — reaches 70 per cent.

The in vivo transfer of free and esterified cholesterol from plasma into the arterial wall of hypercholesterolemic rabbits

With a new technique based on the simultaneous use of 3H and 14C-cholesterol, the fluxes of free and esterified cholesterol into the thoracic aorta of the hypercholesterolemic rabbits have been measured. By additional use of 3H-leucine as tracer for apoproteins, it was shown that esterified cholesterol in plasma enters the arterial tissue mainly as part of a lipoprotein influx. In contrast, only between 20 and 90% of the influx of free cholesterol could be accounted for by an influx of plasma lipoproteins. The excess probably represents an exchange of free cholesterol between the plasma lipoproteins and the intimal surface of the artery. Some 5-40% of the esterified cholesterol which had entered the artery during a 3-6 hours period was hydrolyzed in the artery during that period, whereas only negligible amounts of newly-entered free cholesterol was esterified. The arterial influx of very low, low and high density lipoproteins or albumin was determined in the same animal. The influx of each fraction was expressed as an intimal clearance, i.e., the influx divided by its plasma concentration. The intimal clearances of these macromolecules decreased linearly with the logarithm of their diameters. This suggests that the arterial influx of plasma lipoproteins and of albumin proceed by the same mechanism, which involves a steric hindrance for these macromolecules in the influx pathway.

Exchange of free cholesterol between low density lipoproteins and human adipocytes.

Free cholesterol uptake from low density lipoproteins (LDL) by fat cells was investigated using exchange-labelled [1,2-3H]cholesterol-LDL and isolated human adipocytes, prepared by collagenase digestion of subcutaneous fat tissue biopsies. Radioactivity appeared in the cellular lipids within 2 min and accumulated at a slower rate approaching linearity for 3 h. At 5 and 120 min, 90% of the recovered cellular radioactivity was found in the bulk lipid fraction and the remainder was distributed among organelle fractions. Uptake of [3H]cholesterol was linearly related to substrate concentrations up to 5 micrograms unesterified cholesterol - LDL . mL-1. At higher concentrations up to 22 micrograms unesterified cholesterol - LDL . mL-1, [1,2-3H]cholesterol accumulated more gradually but did not reach a plateau. Metabolic inhibitors, sodium azide plus sodium fluoride, had no effect on radiocholesterol uptake. Native LDL and high density lipoprotein at equivalent unesterified cholesterol concentrations decreased [1,2-3H]cholesterol uptake, to a similar extent suggesting isotope dilution. The results demonstrate a rapid, receptor-independent uptake of cholesterol from LDL by adipocytes, consistent with an exchange mechanism. This process could explain the rapid labelling of adipose tissue cholesterol in vivo following intravenous administration of radiocholesterol and efflux of free cholesterol from adipose tissue during starvation.

Cholesterol exchange between fat cells, chylomicrons and plasma lipoproteins

Isolated rat adipocytes were incubated with serum lipoproteins or lymphchylomicrons which contained 14C-labeled cholesterol. The specific activity of lipoprotein free cholesterol decreased and that of cellular free cholesterol increased linearly up to 7 h. At this time the cell cholesterol specific activity was only 11% of that of medium cholesterol indicating that the rate of exchange was slow. The specific activity of lipoprotein esterified cholesterol remained unchanged while that of cells showed a slight increase suggesting esterification of incorporated free cholesterol. No detectable change of the lipoprotein or cellular cholesterol concentration occurred indicating that the uptake of radioactive free cholesterol was due to exchange without net movement of sterol. The radioactive cholesterol was incorporated into both membrane fraction and the fat droplet of the adipocytes. The rate of cholesterol exchange was temperature-dependent but it was not influenced by the metabolic state of the cells and not by addition of metabolic inhibitors. Trypsin or pronase treatment of the cells were without influence on the rate of the exchange and denaturation of the plasma lipoproteins with formalin increased the rate of exchange. These results indicate that the exchange of cholesterol is a physical chemical process, which is not linked to energy metabolism of the cells, and which is not mediated by either specific lipoprotein receptors on fat cell membranes or pinocytic uptake of lipoproteins. The rate of free cholesterol exchange showed a linear correlation with the concentration of lipoprotein particles in the medium. The relative transfer rate was highest for chylomicrons and decreased in order chylomicron remnants > very low density lipoprotein > low density lipoprotein > high density lipoprotein. A saturation of the system could be obtained only with high density lipoprotein.

Cholesterol exchange between the red blood cells and plasma of young and old rats

The exchange of unesterified cholesterol between plasma and red blood cells of young (1.5 month) and old (1–2 years) rats was studied using [ 3H]-cholesterol as label. Using labeled plasma and unlabeled red blood cells, no equilibrium of specific activity of unesterified cholesterol with the specific activity of cholesterol of red blood cells occurred in either young or old rats. Using labeled red blood cells and unlabeled plasma, equilibration of radioactivity of cholesterol was observed in both young and old rats. However, a more rapid exchange of free cholesterol was observed when labeled old red blood cells were incubated with unlabeled plasma. This apparent paradox may be explained by the existence of a larger, more readily exchangeable pool of free cholesterol in red blood cells of old rats than of young rats. Red blood cells and plasma of old rats contained significantly more free cholesterol than those of young rats but phospholipid levels were comparable.

Movement of cholesterol in vitro in rat blood and quantitation of the exchange of free cholesterol between plasma and erythrocytes

After administration of [4-(14)C]cholesterol to rats, blood was obtained and incubated for 6 hr or less. Incubation resulted in a net loss of erythrocyte cholesterol and, simultaneously, in an increase of esterified cholesterol in plasma and alpha-lipoproteins. Erythrocyte labile cholesterol was shown to be the sole precursor of esterified cholesterol. However, the relation between loss and esterification was not absolute. Loss of erythrocyte cholesterol could be inhibited without affecting esterification and vice versa. A catenary turnover model is proposed, which links in vivo erythrocyte labile cholesterol and plasma esterified cholesterol. Free cholesterol also exchanged between erythrocytes and lipoproteins. The topological model, as tested by analog computer, appears to be a bicompartmental system governed by nonconstant exchange fluxes. They are exponential functions of time and vary from 0.065 to 0.020 mg/hr/g of blood. The fitting of the curves obtained by analog computer analysis to the experimental curves requires esterification as described above. Variation of the exchange fluxes would be the consequence of lipoprotein structural alterations. If this is true, the initial value of the measured flux in vitro is identical with the in vivo value, and the turnover time of erythrocyte cholesterol is 9.2 hr. Initial exchange flux is not dependent on plasma cholesterol level or on the age of the rats, but it is temperature dependent. Addition of amphotericin B to the plasma does not modify exchange fluxes, but erythrocyte cholesterol loss is increased.

Mouvements de cholesterol in vitro entre les α- et les β- lipoproteines plasmatiques du rat et entre chacune d'elles et les globules rouges

After administration of [4- 14C]cholesterol to rats, blood was obtained and eventually the labeled plasma subdivided into α- and β-lipoprotein classes ( d > 1.063 and d < 1.063). Incubation of plasma, or of α- or β-lipoproteins with unlabeled erythrocytes, was performed at 37° for 7h or less. The changes in free and esterified cholesterol content and specific activity were studied. On the basis of these data, models of exchange were proposed, and checked by analogue computer. 1. 1. Incubation of α- lipoprotein solutions in the presence of erythrocytes, results, first, in exchange of free cholesterol and, second, in a net loss of erythrocyte cholesterol (42%) which is related to an increase of free and esterified cholesterol in the α- lipoproteins. From the isotopic results, it is calculated that 80% of cholesterol which become esterified derives from cholesterol lost by erythrocytes, and only 20% from α- lipoprotein- free cholesterol. Similar observations can be made about β- lipoproteins during their incubation with erythrocytes, but the changes are smaller. 2. 2. The fitting of the experimental curves, by analogue computer, is performed varying the value of the exchange rate of free cholesterol, and that of the partition coefficient in relation with the esterification pathway. The rate of exchange, determined by this method, is not constant during the course of incubation. Considering 1 g of red cells, the rate decreases from 0.029 to 0.007 mg/h in the case of α-lipoproteins and from 0.024 to 0.019 mg/h in that of β- lipoproteins. The repartition of the amount of cholesterol esterified by the way of erythrocytes or lipoprotein cholesterol, calculated from isotopic results, is close to that obtained by analogue computer. 3. 3. During short-time incubation of plasma (2 h), the newly formed cholesterol esters are associated only with the α-lipoproteins. The rate of exchange of free cholesterol between α- and β- lipoproteins is about 0.06 mg/h in 1 g of plasma. Furthermore, the results suggest the occurrence of exchange of ester cholesterol. 4. 4. Finally, dialysis carried out at 5° before incubation is followed by a net esterification of only α- lipoproteins (37 %). Moreover, addition of erythrocytes to a solution of α- lipoproteins results in a picking up of free cholesterol by the erythrocytes. This phenomenon is not so obvious in the case of β- lipoproteins. All these results are discussed keeping in mind the heterogeneity of isolated lipoprotein fractions.

Quantitation of the in vitro free cholesterol exchange of human red cells and lipoproteins

The exchange of free cholesterol in vitro between human red blood cells and low density lipoproteins (LDL) was quantified. The flux of sterol between LDL and red cells was relatively constant over a wide range of concentrations of free cholesterol in lipoproteins. In a system containing a suspension of red blood cells in a mixed solution of high density lipoproteins (HDL) and LDL, the fractional rate of exchange of HDL cholesterol was most rapid followed by LDL and lastly, by red cells. Increasing the ionic strength of the incubation media had no effect on the exchange of cholesterol between LDL and red cells. However, when both HDL and LDL were incubated with red cells in a buffer of increased ionic strength, total red cell cholesterol exchange was unaltered, but proportionately more exchange occurred with HDL and less with LDL. Addition of acetone to the buffer increased the exchange of cholesterol between LDL and red cells but produced no increment in red cell-HDL exchange.