Lipoprotein metabolism in the suckling rat: characterization of plasma and lymphatic lipoproteins

Suckling rat plasma contains (in mg/dl): chylomicrons (85 +/- 12); VLDL (50 +/- 6); LDL (200 +/- 23); HDL1 (125 +/- 20); and HDL2 (220 +/- 10), while lymph contains (in mg/dl): chylomicrons (9650 +/- 850) and VLDL (4570 +/- 435) and smaller amounts of LDL and HDL. The lipid composition of plasma and lymph lipoproteins are similar to those reported for adults, except that LDL and HDL1 have a somewhat higher lipid content. The apoprotein compositions of plasma lipoproteins are similar to those of adult lipoproteins except for the LDL fraction, which contains appreciable quantities of apoproteins other than apoB. Although the LDL fraction was homogeneous by analytical ultracentrifugation and electrophoresis, the apoprotein composition suggests the presence of another class of lipoproteins, perhaps a lipid-rich HDL1. The lipoproteins of lymph showed low levels of apoproteins E and C. The triacylglycerols in chylomicrons and VLDL of both lymph and plasma are rich in medium-chain-length fatty acids, whereas those in LDL and HDL have little or none. Phospholipids in all lipoproteins lack medium-chain-length fatty acids. The cholesteryl esters of the high density lipoproteins are enriched in arachidonic acid, whereas those in chylomicrons, VLDL, and LDL are enriched in linoleic acid, suggesting little or no exchange of cholesteryl esters between these classes of lipoproteins. The fatty acid composition of phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine were relatively constant in all lipoprotein fractions, suggesting ready exchange of these phospholipids. However, the fatty acid composition of phosphatidylethanolamine in plasma chylomicrons and VLDL differed from that in plasma LDL, HDL1, and HDL2. LDL, HDL1, and HDL2 were characterized by analytical ultracentrifugation and shown to have properties similar to that reported for adult lipoproteins. The much higher concentration of triacylglycerol-rich lipoproteins in lymph, compared to plasma, suggests rapid clearance of these lipoproteins from the circulation.     

Influence of lecithin acyl chain composition on the kinetics of exchange between chylomicrons and high density lipoproteins

The kinetics of lecithin exchange between native lipoproteins was characterized for individual molecular species of lecithins of rat mesenteric lymph chylomicrons and rat plasma HDL. Studies were performed in the absence of lipid transfer proteins. Donor (chylomicrons) and acceptor (HDL) particles were present in ratios of 1:1 and 1:10 with respect to total phospholipid. Biphasic exchange kinetics were observed for all major lecithins common to chylomicrons and HDL at both proportions of donor to acceptor particles. During the early rapid phase of exchange, complete in about 30 min, 40-60% of the total lecithin pool was exchanged. Initial exchange rates were most rapid for the more hydrophilic species of the major lecithins normally present in both lipoproteins. Calculated activation energies correspondingly were least for a diunsaturated lecithin (18:1-20:4), intermediate for lecithins were 16:0 in position-1 (16:0-18:2 and 16:0-20:4), and highest for analogous lecithins with 18:0 in position-1. A 10-fold increase in the ratio of acceptor to donor particles affected neither the biphasic nature of the exchange nor the rates of exchange of individual molecular species (consistent with exchange by diffusion rather than by particle collisions). Total equilibration of individual molecular lecithin species was achieved by 24 hr (37 degrees C, donor to acceptor ratio of 1:1) with only a small change in the relative mass of lecithins in chylomicrons and HDL. Novel lecithins containing 18:3, incorporated into chylomicrons, were found to exchange exceedingly rapidly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Composition, removal and metabolic fate of chylomicrons derived from diabetic rats

Tri[14C]acylglycerol-labelled chylomicrons, obtained from cannulated mesenteric lymph of streptozotocin-diabetic donor rats, when intravenously injected into non-diabetic recipient rats, disappeared from the circulation at a significantly slower rate than similarly prepared tri[14C]acylglycerol chylomicrons from non-diabetic donor rats (t1/2, 5.6 +/- 0.7 vs. 3.2 +/- 0.5 min-1, P less than 0.02). The appearance of labelled lipolysis products among plasma lipids (free fatty acid, cholesterol ester and phospholipid fractions) was delayed, indicating decreased availability for lipolysis of the chylomicron-borne triacylglycerol of diabetic origin. Tissue distribution of triacylglycerol, 15 min after the injection of chylomicrons to recipient rats, disclosed a 4-5-fold increase in uptake by muscles (heart and diaphragm) in relation to adipose tissues (epididymal and perirenal sites), in the case of chylomicrons of diabetic derivation. Since a large share of the chylomicron triacylglycerol was taken up by the liver, this tissue was perfused with chylomicron 'remnants' prepared by partial in vitro lipolysis with purified lipoprotein lipase. The 'remnants' of diabetic derivation were taken up by the liver at a 2-3-fold slower rate than those of non-diabetic origin. Chylomicrons derived from diabetic rats were found to be similar in size but markedly depleted of E apolipoproteins as determined by SDS-polyacrylamide gel electrophoresis, isoelectric focussing and a specific immunoassay. Decreases were also seen in A-I apolipoproteins by immunoassay and isoelectric focussing. Chylomicron 'remnants' were also markedly apolipoprotein E-deficient. In vitro incubation of the 'diabetic remnants' with high-density lipoproteins raised their apolipoprotein E content approx. 3-fold and considerably increased their hepatic uptake. Injection of intact chylomicrons preincubated with high-density lipoproteins likewise increased their in vivo removal rate toward the range of that of 'non-diabetic' chylomicrons. We conclude that diabetes-induced changes in the apolipoprotein composition of the chylomicrons and chylomicron remnants play an important role in their removal from the circulation. It appears that their recognition pattern is altered, reducing their ability to interact with receptor sites in the peripheral tissues and the liver, respectively.     

Metabolism of radiolabelled chylomicron lipids in intact and hepatectomized rats

Intact rats removed more radiolabelled triacylglycerol, cholesterol, and cholesterol ester but not phosphatidylcholine (PC) in the first 6 min than hepatectomized rats. There was no difference between intact and hepatectomized rats in the transfer of radiolabelled chylomicron lipids to other lipoproteins. Specific radioactivity measurements demonstrated a net transfer of PC (intact and hepatectomized rats) and unesterified cholesterol (intact rats only) onto both the low density lipoprotein/high density lipoprotein-1 (LDL/HDL1) and HDL2 fractions. [3H]Fatty acids were rapidly incorporated into blood cell phospholipids and into HDL and LDL cholesterol esters of both intact and hepatectomized rats. Substantial rearrangements of [3H]palmitate occurred during lipid uptake by liver.     

Molecular pathways in the transformation of model discoidal lipoprotein complexes induced by lecithin:cholesterol acyltransferase

Incubation (24 h, 37 degrees C) of discoidal complexes of phosphatidylcholine and apolipoprotein A-I (molar ratio 95 +/- 10 egg yolk phosphatidylcholine-apolipoprotein A-I; 10.5 X 4.0 nm, long X short dimension; designated, class 3 complexes) with the ultracentrifugal d greater than 1.21 g/ml fraction transformed the discoidal complexes to a small product with apparent mean hydrated and nonhydrated diameter of 7.8 and 6.6 nm, respectively. Formation of the small product was associated with marked reduction in phosphatidylcholine-apolipoprotein AI molar ratio of the complexes (on average from 95:1 to 45:1). Phospholipase A2 activity of lecithin:cholesterol acyltransferase participated in the depletion process, as evidenced by production of unesterified fatty acids. In the presence of the d greater than 1.21 g/ml fraction or partially purified lecithin:cholesterol acyltransferase and a source of unesterified cholesterol, the small product could be transformed to a core-containing (cholesteryl ester) round product with a hydrated and nonhydrated diameter of 8.6 and 7.5 nm, respectively. By means of cross-linking with dimethylsuberimidate, the protein moiety of the small product was shown to contain primarily two apolipoprotein A-I molecules per particle, while the large product contained three apolipoprotein A-I molecules per particle. The increase in number of apolipoprotein A-I molecules per particle during transformation of the small to the large product appeared to result from fusion of the small particles during core build-up and release of excess apolipoprotein A-I from the fusion product. The results obtained with the model complexes were consistent for the most part with recent observations (Chen, C., Applegate, K., King, W.C., Glomset, J.A., Norum, K.R. and Gjone, E. (1984) J. Lipid Res. 25, 269-282) on the transformation, by lecithin:cholesterol acyltransferase, of the small spherical high-density lipoproteins of patients with familial lecithin:cholesterol acyltransferase deficiency.     

Reverse cholesterol transport in the isolated perfused rat spleen.

1. A method has been developed which enables the rat spleen to be loaded in vivo with [3H]cholesterol to a high specific radioactivity using cholesterol-labelled erythrocytes. The erythrocytes were shown to be rapidly degraded by the spleen and not released intact during subsequent perfusion. 2. When labelled spleens were perfused with whole blood or serum, lipoproteins in the high-density lipoprotein (HDL) range were shown to be the principal lipoprotein vehicles for the removal of cholesterol, the specific radioactivity of cholesterol being much greater in the HDL fractions than in other lipoproteins, particularly in the d 1.175-1.210 fraction. 3. The formation of [3H]cholesteryl ester was restricted to the major HDL fractions. 4. Experiments utilizing individual HDL fractions added to a basal perfusate indicated that HDL1 (d 1.050-1.085) was of less importance in the removal of cholesterol from the spleen than HDL subfractions of higher density. Also, a decrease in density of the lipoproteins was observed during perfusion, concurrent with uptake of cholesterol, especially in the d 1.085-1.125 subfraction. 5. When [3H]cholesterol-labelled spleens were perfused with whole blood, about half of the radioactivity released was detected in erythrocytes, indicating a rapid exchange or transport of cholesterol. Thus erythrocytes could play an important role in the transfer of unesterified cholesterol when the chemical potential gradient is favourable.     

Alterations in erythrocyte membrane lipid composition and fluidity in primary lipoprotein lipase deficiency.

Lipid composition of plasma lipoproteins and erythrocyte ghost membranes has been studied in 16 healthy normolipidaemic subjects and in 16 patients affected by primary lipoprotein lipase deficiency, resulting in severe chylomicronaemia and in cholesterol-depleted low-density lipoproteins and high-density lipoproteins. A significant decrease in membrane cholesterol/phospholipid ratio was observed in lipoprotein lipase deficient patients compared to controls (3.27 +/- 0.33 vs. 3.95 +/- 0.50, mean +/- S.D.; P less than 0.0001). There was also an increase in the erythrocyte membrane phosphatidylcholine/sphingomyelin ratio in lipoprotein lipase deficient patients compared to controls (1.53 +/- 0.10 vs. 1.05 +/- 0.13; P less than 0.0001) due to a concurrent increase in phosphatidylcholine and decrease in sphingomyelin relative concentrations in these patients. Erythrocyte ghost membrane fluidity was determined by fluorescence anisotropy and found to be higher in membranes from lipoprotein lipase deficient patients. This increase in membrane fluidity can be attributed in part to changes in membrane cholesterol and phospholipid concentrations in response to abnormal plasma lipoprotein composition.     

Uptake of endogenous cholesterol by a synthetic lipoprotein

The addition of cholesterol-poor phospholipid liposomes to canine plasma in vivo and in vitro substantially alters the distribution of phospholipids, apoproteins, and, especially, cholesterol. In vivo, intravenously injected phospholipid liposomes remain discrete particles, which are readily distinguished from the normally occurring lipoproteins by their buoyant density and electrophoretic mobility. They acquire unesterified cholesterol from endogenous sources, thereby producing an acute rise in the concentration of this sterol in plasma. The liposomes also accumulate endogenous proteins, one of which is identified as apolipoprotein A-I. In vitro, phospholipid liposomes incubated with plasma acquire unesterified cholesterol and apolipoprotein A-I at the expense of high-density lipoproteins (HDL), the major carrier of cholesterol in normal canine plasma. In exchange, the HDL particles are enriched in phospholipids and become larger. At sufficiently high concentrations, the liposomes nearly completely deplete HDL of its unesterified cholesterol. Thus, there are generated two types of particles, both rich in apolipoprotein A-I and phospholipid, but one (modified HDL) containing mainly esterified cholesterol in its core and the other (modified liposomes) containing mainly unesterified cholesterol at its surface. It is concluded that phospholipid liposomes produce important changes in the distribution of lipids and protein in canine plasma, particularly at the expense of HDL. These changes appear to favor the mobilization of tissue cholesterol into the plasma, and may have application to atherosclerosis.     

Effects of blocking plasma lipid transfer protein activity in the rabbit

Plasma lipid transfer protein activity was completely blocked in rabbits for up to 48 h by infusion with goat antibody to rabbit lipid transfer protein. Lipid transfer protein activity in plasma of control animals, infused with antibody from a non-immune goat, decreased during the experiment but was never less than 50% of pre-infusion levels. During the period that lipid transfer protein activity was completely blocked, there were changes in high-density lipoprotein composition (expressed as % by weight) with a reduction in triacylglycerol from 8.4 +/- 2.4% to 1.0 +/- 0.2% (P less than 0.05) and an increase in esterified cholesterol from 10.7 +/- 1.7% to 14.5 +/- 0.3% (P less than 0.1). In conjunction with the observed changes in high-density lipoprotein composition, there was an increase in high-density lipoprotein particle size from a mean radius of 4.7 to 5.4 nm. The change in composition and particle size was not observed in high-density lipoproteins from control animals. There was a change in the distribution of plasma cholesterol in control animals, with a fall in the proportion of cholesterol in high-density lipoproteins (P less than 0.02) and consequently an increase in the proportion of cholesterol in low-density lipoproteins (P less than 0.02). However, the distribution of plasma cholesterol in animals in which lipid transfer protein activity was inhibited was maintained at original levels during the period of inhibition. Consequently, in these animals, there was a less atherogenic distribution of cholesterol during the period of lipid transfer protein inhibition when compared with control animals. The changes observed in lipoproteins, in the absence of lipid transfer protein activity, demonstrate that lipid transfer protein modifies lipoproteins in vivo and appears to contribute to a more atherogenic lipid profile.     

Cholesterol is necessary for triacylglycerol-phospholipid emulsions to mimic the metabolism of lipoproteins

Emulsions with lipid compositions similar to the triacylglycerol-rich lipoproteins were metabolized similarly to natural chylomicrons or very-low-density lipoproteins when injected intravenously in rats. Radioactive labels tracing the emulsion triacylglycerols and cholesteryl esters were both removed rapidly from the blood stream, but the removal rate of triacylglycerols was faster than that of cholesteryl ester. Most of the removed cholesteryl ester label was found in the liver, but only a small fraction of the triacylglycerol label was found in this organ, consistent with hepatic uptake of the remnants of the injected emulsion. Emulsions otherwise identical but excluding unesterified cholesterol were metabolized differently. The plasma removal of triacylglycerols remained fast, but the cholesteryl esters were removed very slowly. Heparin stimulated lipolysis, but failed to increase the rate of removal of cholesteryl esters from emulsions lacking cholesterol. Evidently, emulsions lacking cholesterol were acted on by the enzyme lipoprotein lipase, but the resultant triacylglycerol-depleted remnant particle remained in the plasma instead of being rapidly taken up by the liver. Therefore, the presence of emulsion cholesterol is a critical determinant of early metabolic events, and the findings point to a similar role for cholesterol in the natural triacylglycerol-rich lipoproteins.     

Structural modification of plasma HDL by phospholipids promotes efficient ABCA1-mediated cholesterol release

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates prebeta(1)-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated prebeta(1)-LpA-I-like particles inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than HDL(3) (IC(50) = 2.20 +/- 0.35 vs. 37.60 +/- 4.78 microg/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased ( approximately 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native prebeta(1)-LpA-I and prebeta(1)-LpA-I-like particles were increased significantly in stimulated cells. Furthermore, glyburide significantly inhibited phospholipid and free cholesterol efflux to DMPC-treated plasma. Removal of apoA-I-containing lipoproteins from normolipidemic plasma drastically reduced free cholesterol efflux mediated by DMPC-treated plasma. Finally, treatment of Tangier disease plasma with DMPC affected the amount of neither prebeta(1)-LpA-I nor free cholesterol efflux. These results indicate that DMPC enrichment of normal plasma resulted in the redistribution of apoA-I from alpha-HDL to prebeta-HDL, allowing for more efficient ABCA1-mediated cellular lipid release. Increasing the plasma prebeta(1)-LpA-I level by either pharmacological agents or direct infusions might prevent foam cell formation and reduce atherosclerotic vascular disease.     

Metabolic heterogeneity of post-lipolysis rat mesenteric lymph small chylomicrons produced in vitro

The study was undertaken to investigate the metabolic rat of post-lipolysis mesenteric lymph small chylomicrons produced in vitro. Small chylomicrons doubly labeled with [3H]cholesterol (more than 70% in cholesteryl esters) and [14C]palmitate-labeled triglycerides were collected from rat mesenteric lymph during periods of fasting. Lipolysis was performed in vitro with lipoprotein lipase purified from bovine milk. More than 98% of the chylomicron-triglycerides could be hydrolyzed to fatty acids. Post-lipolysis chylomicrons were separated by zonal ultracentrifugation, characterized, and tested for biological behavior in intact rats. Following lipolysis the lipoproteins lost nearly all their triglycerides, apoA-I, and apoC, and were relatively enriched with cholesteryl esters, unesterified cholesterol, phospholipids, and apoB. Three preparations were tested for biological behavior: pooled (total) post-lipolysis chylomicrons (diameter approximately 250 A); particles at the ascending part of the zonal effluent (diameter approximately 300 A), and at the descending part (diameter approximately 200 A). After intravenous injection to intact rats, [3H]cholesteryl ester decay was very rapid with pooled lipoproteins and the 300-A preparation (t1/2 = 5-10 min). The 200-A preparation in contrast stayed in circulation much longer (t1/2 = 60-90 min). The study thus demonstrated metabolic heterogeneity of post-lipolysis small chylomicrons and indicated that some may form an LDL-like subpopulation with a plasma lifetime slower than "remnants" but faster than LDL.     

Effect of dietary cholesterol level on the composition of thoracic duct lymph lipoproteins isolated from nonhuman primates

The effect of two different levels of dietary cholesterol (0.16 mg/Kcal and 0.79 mg/cal) on the composition of thoracic lymph duct lipoproteins was studied in two species of nonhuman primates, Ceropithecus aethiops (African green monkey) and Macaca fascicularis (cynomolgus monkey). Diet was infused intraduodenally at a constant rate to facilitate comparisons among animals. The higher level of dietary cholesterol resulted in an increase in the amount of cholesteryl ester in lymph chylomicrons and VLDL. Cholesteryl oleate was the predominant cholesteryl ester present in lymph d less than 1.006 g/ml lipoproteins and it was the predominant cholesteryl ester formed from exogenous radiolabeled cholesterol. The percentage of saturated and monounsaturated cholesteryl esters in lymph chylomicrons and VLDL significantly increased with the higher dietary cholesterol level. The apoprotein distribution of chylomicrons and VLDL was qualitatively similar during infusions of both diets. The apoprotein B of intestinal chylomicrons and VLDL, termed apoprotein B2, was qualitatively similar during low and high cholesterol diet infusion and was significantly smaller than that of plasma LDL apoB, termed apoprotein B1, as indicated by its electrophoretic mobility in SDS-polyacrylamide gels. The major phospholipid present in lymph chylomicrons and VLDL was phosphatidylcholine and the phospholipid composition of the particles was not affected by diet. Lymph d greater than 1.006 g/ml lipoproteins were separated and the cholesterol mass distribution among lipoprotein fractions was found to be similar during both diet infusions. With an increase in the level of dietary cholesterol, the percentage esterification of cholesterol mass and of exogenous cholesterol radioactivity increased in LDL and HDL from lymph. Lymph LDL and HDL contained less free and esterified cholesterol when their composition was compared to that for these lipoproteins in plasma. We conclude that the primary effect of increased dietary cholesterol level was to increase the cholesteryl ester content of all lymph lipoproteins; cholesterol distribution among lymph lipoproteins was unaffected.     

H+ transport by reconstituted gastric (H+ + K+)-ATPase

Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol liposomes by means of a freeze-thaw-sonication technique. Upon addition of MgATP, active H+ transport was observed, with a maximal rate of 2.1 mumol X mg-1 X min-1, requiring the presence of 100 mM K+ at the intravesicular site. However, in the absence of ATP an H+-K+ exchange with a maximal rate of 0.12 mumol X mg-1 X min-1 was measured, which could be inhibited by the well-known ATPase inhibitors vanadate and omeprazole, giving the first evidence of a passive K+-H+ exchange function of gastric (H+ + K+)-ATPase. An Na+-H+ exchange activity was also measured, which was fully inhibited by 1 mM amiloride. Simultaneous reconstitution of Na+/H+ antiport and (H+ + K+)-ATPase could explain why reconstituted ATPase appeared less cation-specific than the native enzyme (Rabon, E.C., Gunther, R.B., Soumarmon, A., Bassilian, B., Lewin, M.J.M. and Sachs, G. (1985) J. Biol. Chem. 260, 10200-10212).     

Identification and partial characterization of discrete apolipoprotein B containing lipoprotein particles produced by human hepatoma cell line HepG2

The purpose of this study was to test the use of human hepatocarcinoma HepG2 cells as a model for studying the formation and secretion of human hepatic lipoproteins. To this end, we determined the rate of accumulation and percent composition of neutral lipids and apolipoproteins in the culture medium of HepG2 cells and isolated and partially characterized the apolipoprotein B (ApoB) containing lipoprotein particles. The rates of accumulation in the medium of HepG2 cells, grown in minimum essential medium during a 24-h incubation, of triglycerides, cholesterol, and cholesterol esters expressed as microgram/(g of cell protein X h) were 373 +/- 55, 167 +/- 14, and 79 +/- 10, respectively; the secretion rates for apolipoproteins B, A-I, E, A-II, and C-III were 372 +/- 36, 149 +/- 14, 104 +/- 13, 48 +/- 4, and 13 +/- 1 microgram/(g of cell protein X h), respectively. The major portion of ApoB was present in very low density lipoproteins (VLDL) and low-density lipoproteins (LDL) (84%), with the remainder occurring in high-density lipoproteins (HDL) (16%). Approximately 10-13% of ApoA-I and ApoA-II were present in VLDL and LDL, while 60% of ApoE occurred in HDL and 40% in VLDL and LDL. To separate ApoB-containing lipoproteins, secreted lipoproteins were fractionated by either sequential immunoprecipitation or immunoaffinity chromatography with antibodies to ApoB and ApoE. Results showed that 60-70% of ApoB occurred in the culture medium as lipoprotein B (LP-B) and 30-40% as lipoprotein B:E (LP-B:E). Both ApoB-containing lipoproteins represent polydisperse systems of spherical particles ranging in size from 100 to 350 A for LP-B and from 200 to 500 A for LP-B:E. LP-B particles were identified in VLDL, LDL, and HDL, while LP-B:E particles were only present in VLDL and LDL. The major neutral lipid of both ApoB-containing lipoproteins was triglyceride (50-70% of the total neutral lipid content); cholesterol and cholesterol esters were present in equal amounts. The LP-B:E particles contained 70-90% ApoB and 10-30% ApoE. The ApoB was identified in both types of particles as B-100. A time study on the accumulation of ApoB-containing lipoproteins showed that LP-B particles were secreted independently of LP-B:E particles.     

Plasma clearance of chylomicrons labeled with retinyl palmitate in healthy human subjects

To estimate hepatic uptake of chylomicron remnants in humans, chylomicrons and intestinal very low density lipoproteins (VLDL) were endogenously labeled with retinyl esters, harvested by plasmapheresis, and pulse-injected into the donor 44 hr after plasmapheresis. Plasma decay of retinyl palmitate was measured in eight healthy volunteers. Retinyl palmitate plasma disappearance obeyed an apparent first order function in seven studies and, in one study, a biexponential function with the second, slow exponential accounting for only 13% of the retinyl palmitate plasma decay. The mean fractional removal of rate was 0.037 +/- 0.037 min-1 (mean +/- SD) in a one-compartment model. The apparent volume of distribution, Vd, was 109 +/- 25% of the estimated plasma volume. Plasma clearance of retinyl palmitate was 130 +/- 97 ml/min calculated as Vd x Ke. Mean T 1/2 was 29 +/- 16 min. Both in vitro and in vivo the retinyl palmitate remained largely within chylomicrons and intestinal VLDL. Only 4.3% was transferred from chylomicrons to other lipoprotein classes during in vitro incubation for 5 hr. After plasma was stored for 42 hr, 5% was transferred to higher density lipoproteins. During 12 hr after a test meal containing retinyl palmitate, only 6.4 +/- 1.5% of the retinyl palmitate absorbed was found in the LDL fraction and 3.1 +/- 3.8% in the d 1.063 g/ml lipoproteins. We conclude that retinyl palmitate is a useful marker for chylomicrons and their remnants in humans and that the plasma clearance of retinyl palmitate-labeled chylomicrons is probably an estimate of chylomicron remnant plasma clearance in man.     

Experimental nephrotic syndrome: removal and tissue distribution of chylomicrons and very-low-density lipoproteins of normal and nephrotic origin

Lymph chylomicrons and plasma VLDL, 14C-labelled in vivo, were isolated from normal and nephrotic rats and injected into normal or nephrotic recipients. In normal recipients, the half-life of chylomicrons of nephrotic vs. normal origin was significantly longer (5.2 +/- 0.5 vs. 3.5 +/- 0.4 min-1). The nephrotic chylomicrons were larger in size, deficient in apo-E and apo A-I, rich in triacylglycerol and cholesterol, but poor in phospholipids, indicating that factors related to composition affected their removal. The half-life of nephrotic vs. normal VLDL, given to normal recipients, was unexpectedly shorter, (4.5 +/- 0.2 vs. 5.8 +/- 0.2 min-1). The nephrotic VLDL were also triacylglycerol- and cholesterol-rich and phospholipid-poor, but had a large diameter spread and contained a dense fraction according to the zonal ultracentrifugation pattern, suggesting the presence of faster removable IDL-like particles. When nephrotic rats received normal particles, a pronounced removal delay was seen, paralleling the extent of plasma triacylglycerol elevation. The half-life of chylomicrons was 8.3 +/- 1.4 and 15.2 +/- 2.5 min-1 in moderately and severely nephrotic rats, respectively, that of VLDL was 11.72 +/- 2.1 and 37.8 +/- 7.1 min-1 correspondingly. The chylomicron-triacylglycerol uptake was reduced both by adipose tissues and muscles of normal or nephrotic recipients, with some increase in entry into lungs, kidneys and spleen. Tissue distribution patterns of VLDL-triacylglycerol was similar to that of chylomicrons, except that the liver took up approx. 90% of the label. The low share of triacylglycerol uptake by tissues rich in lipoprotein lipase indicates that the activity of this enzyme was unlikely to limit the rate of removal. Lipoprotein lipase activity in adipose tissue and heart was slightly decreased in moderately nephrotic rats and declined only by approx. 35% in severely nephrotic ones. These results indicate that the removal defect in nephrosis seems to be due, in part, to changes in the composition of triacylglycerol-rich particles, compromising their accessibility to lipolysis and, in part, to their abundance, saturating the lipolytic capacity.     

Dual pathways for the secretion of lysosomal cholesterol into a medium from cultured macrophages

The removal of cholesterol from macrophages is important for reversing foam cell formation. In a previous study, we demonstrated that mouse peritoneal macrophages in culture secrete significant amounts of unesterified cholesterol from the lysosomes into the medium during endocytosis and subsequent metabolism of cholesterol-containing liposomes [Furuchi, T., Aikawa, K., Arai, H., and Inoue, K. (1993) J. Biol. Chem. 268, 27345-27348]. In this study, we found that at least two distinct mechanisms are involved in this process. The efflux of unesterified cholesterol into the medium was greatly suppressed by pregnenolone, an inhibitor of lysosomal cholesterol transport, but an appreciable proportion of the unesterified cholesterol was still released into the medium. Analysis of the medium containing the secreted cholesterol by NaBr density gradient ultracentrifugation revealed that the unesterified cholesterol was distributed in two different density peaks (bottom and d =/ approximately 1.1). The d =/ approximately 1.1 peak material formed high-density lipoprotein (HDL)-like particles that were produced and secreted by the macrophages. The lipid components of these particles were phosphatidylcholine and sphingomyelin, while the sole protein component was apolipoprotein E (apo E). Treatment with pregnenolone completely abolished the production of these HDL-like particles but had little effect on the bottom fractions. These data indicate that macrophages release lysosomal cholesterol via both pregnenolone-sensitive and -insensitive pathways, and that only the cholesterol secreted through the pregnenolone-sensitive pathway is associated with endogenously synthesized apo E-containing HDL-like particles. Moreover, we found that the pregnenolone-sensitive pathway operated independently of the presence or absence of exogenous HDL, whereas secretion via the pregnenolone-insensitive pathway was greatly stimulated by exogenously added HDL.     

Characteristics of chylomicron binding and lipid uptake by endothelial cells in culture.

Bovine vascular endothelial cells bind chylomicrons via a high affinity membrane receptor site. Subsequent to binding, the chylomicron apoprotein was neither internalized nor degraded by either sparse or confluent (contact-inhibited) cells. However, the adsorption of chylomicrons was associated with interiorization of chylomicron cholesteryl ester and triglyceride and the hydrolysis of these lipids to free cholesterol and unesterified fatty acids by a lysosome-dependent pathway. This pathway was active in both subconfluent and contact-inhibited cells. The chylomicron free cholesterol so produced inhibited endogeneous cholesterol synthesis measured in terms of the incorporation of [1-14C]-acetate into sterol. An excess of high density lipoprotein was 2- to 3-fold more effective in reducing both binding of chylomicrons and interiorization of chylomicron lipid than was low density lipoprotein. Chylomicron binding was not "down-regulated" by preincubation of the cells with low density lipoprotein or chylomicrons. The results are discussed in the context of cholesterol sources for contact-inhibited endothelial cells which do not interiorize low density lipoprotein cholesterol.     

Cholesterol distribution and movement in the Mycoplasma gallisepticum cell membrane.

The time course and extent of transfer of [14C]-cholesterol from resting Mycoplasma gallisepticum cells or membrane preparations to high-density lipoproteins were studied. More than 90% of the total cholesterol in isolated, unsealed membrane preparations was exchanged in a single kinetic process. In intact cells, however, cholesterol exists in two different environments. Cholesterol in one environment, representing approximately 50% of the total unesterified cholesterol, is readily exchanged with the cholesterol of high-density lipoproteins, with a half-time of about 4 h at 37 degrees C. The rate of exchange of [14C]cholesterol from the other environment was exceedingly slow, with a half-time of about 18 days. The fraction of the total cholesterol in the readily exchangeable cholesterol pool in intact cells increased somewhat upon aging of the culture. Electron spin resonance spectra of nitroxide-labeled stearic acids incorporated into membranes of M. gallisepticum cells indicated increased rigidity at the late exponential phase of growth. These results suggest that cholesterol is present in approximately equal concentrations on both surfaces of the M. gallisepticum membrane and that in resting cells the rate of movement of cholesterol molecules from the inner to outer halves of the lipid bilayer is exceedingly slow or nonexistent.