Amount Of Cholesteryl Ester Research Articles

Abstract 392: Nanoparticle-based Biosensors for Measuring Apolipoprotein A-I and HDL Function

Background: High-density lipoproteins (HDL) are dynamic circulating nanoparticles that carry cholesterol and have numerous functions. Rapid measurement of HDL, the HDL-associated protein apolipoprotein A-I (apoAI), or HDL function(s) is not currently possible. Such technology may provide new opportunities for HDL monitoring. We utilized nanoparticles to develop a method of sequestering apoAI from solution, including human serum, and for measuring the activity of the HDL-maturing and apoAI-dependent enzyme, lecithin:cholesterol acyl transferase (LCAT). Methods: Gold nanoparticles were surface-functionalized with phospholipids. The synthesized particles were incubated with various amounts of apoAI or with purchased, LDL-depleted, human serum samples where the apoAI amount was measured using an ELISA. After incubation, the particles were easily isolated and apoAI sequestration by the nanoparticles was measured using ELISA. As apoAI is a cofactor that activates LCAT, nanoparticles were incubated with various amounts of apoA1, LCAT, and free cholesterol or with the LDL-depleted human serum samples (diluted) for 1 hour at 37°C to enable binding of apoAI, cholesterol, and LCAT activity. The nanoparticles were easily isolated and the particle-associated cholesteryl ester was measured using an Amplex Red Cholesterol Assay kit. Results: The nanoparticles sequester apoAI according to the concentration of apoAI in solution (R = 0.668, p = 0.035, N = 10). Also, data reveal that the LCAT binds the nanoparticle-apoAI structures and catalyzes the conversion of cholesterol to cholesteryl ester. The amount of cholesteryl ester bound to the nanoparticles is directly dependent on the amount of apoAI (R = 0.729, p = 0.016, N=10). Conclusion: These results demonstrate that specific, functionalized nanoparticles can be added to serum to sequester apoAI in dose-response. Also, the formed conjugate structures activate LCAT whereby the amount of cholesteryl ester formed on the nanoparticle directly correlates with apoAI. Overall, this technology provides a framework and new approach for developing next generation HDL- and HDL-function specific biosensors.

Lipid Deposition on Contact Lenses when Using Contemporary Care Solutions.

There remains only a small amount of data from human studies demonstrating the effect of contact lens/lens care solution combinations on the deposition of lipids. Therefore, information on the degree to which modern materials deposit lipids when used with contemporary care solutions would be valuable. The present study aims to determine the effect of lens care system combinations on levels of total lipid, cholesterol, and cholesteryl esters extracted from three different contact lenses (CLs) when used with four contemporary care systems. Experienced CL wearers were recruited to participate in this study. Combinations of three CLs (etafilcon A [ETA], galyfilcon A [GALY], and senofilcon A [SENO]) and four CL care solutions (Biotrue, ClearCare, OPTI-FREE PureMoist, and RevitaLens Ocutec) were investigated. A total of 791 CLs were analyzed. Subjects were randomized to one lens type and then used all four lens care solutions in random sequence for 10-14 days before the CLs were collected and analyzed for the amount of cholesterol, cholesteryl esters, and total lipids. The mean range of cholesterol recovered across the different care solutions was 0.34-2.77 μg/lens, 3.48-4.29 μg/lens, and 4.75-6.20 μg/lens for ETA, SENO, and GALY lenses, respectively. Use of OPTI-FREE PureMoist with ETA lenses led to a significantly greater amount of cholesterol being recovered when compared to the use of the other solutions with ETA lenses (P < .05). The mean range of cholesteryl esters recovered across different care solutions was 1.31-2.02 μg/lens, 6.43-7.19 μg/lens, and 7.96-10.13 μg/lens for ETA, SENO, and GALY lenses, respectively. There were no differences in the amount of cholesteryl esters and total lipids extracted for a given lens type when used with any of the four care solutions (P > .05). This study did not demonstrate conclusively that any of the solution/CL combinations were superior to any of the other combinations when the amounts of lipid deposition were compared among the tested lenses.

Hormone-sensitive lipase in Japanese flounder Paralichthys olivaceus: the potential function of the inclinator muscle of fin as a lipid storage site

Hormone-sensitive lipase (HSL) is an important regulator of lipolysis in mammals, but little is known about its function in other animals. For a better understanding of fish HSL and potential application of the HSL-mediated lipolysis in aquaculture, we cloned HSL cDNAs from Japanese flounder Paralichthys olivaceus. The full-length cDNAs of two HSL genes, designated HSL1 and HSL2, consisted of 2,922 and 2,832 bp, respectively. RT-PCR showed that their transcripts were abundant in the inclinator muscle of fin, liver, and skeletal muscle. Quantitative real-time PCR further revealed that the transcripts of HSL1 and HSL2 genes were broadly expressed in all tissues tested, with the highest abundance in the inclinator muscle of fin. The inclinator muscle of fin also contained many adipocytes. In addition to triacylglycerols, considerable amounts of cholesteryl esters and non-esterified fatty acids were detected in the lipids extracted from the inclinator muscle of fin. Finally, in situ hybridization localized the HSL1 transcripts to the adipocytes observed in the inclinator muscle of fin. Together, these results suggest that the inclinator muscle of fin is a lipid storage site that releases non-esterified fatty acids for aerobic fin movement, possibly through HSL-mediated lipolysis.

Beyond LDL Cholesterol in Assessing Cardiovascular Risk: apo B or LDL-P?

Plasma concentrations of LDL cholesterol (LDL-C)4 are positively associated with increased risk of atherosclerotic cardiovascular disease. There is a variety of robust evidence indicating that this association is causal in nature. First, rare and common genetic variants that specifically influence LDL-C concentrations are also strongly associated with cardiovascular risk (1). Second, interventions that reduce LDL-C, especially but not exclusively statin therapy, reproducibly reduce cardiovascular events (2). In fact, the data with statins are so strong that they are often used in patients whose LDL-C concentrations are not particularly increased, a setting in which statins have still been shown to reduce cardiovascular risk. Thus there is substantial interest in lipoprotein-related biomarkers that provide information about future cardiovascular risk above and beyond LDL-C itself. LDL contains a core of hydrophobic lipid, mostly cholesteryl ester, a shell of phospholipids, and single molecule of the large protein apolipoprotein B-100 (apo B). It has long been appreciated that LDL particles vary in size and density, which is largely due to variation in the amount of cholesteryl ester in the particle (3). Because the LDL-C measurement is a measure of the amount of cholesterol being carried in LDL, it is not a reliable measure of LDL particle concentration: small, dense LDL particles have much less cholesterol than large, buoyant LDL particles. Interestingly, however, there is evidence that small, dense LDL may be more atherogenic, which has led to the concept that the overall number of LDL particles may be more predictive of cardiovascular risk than the LDL-C concentration itself (3). Several methods have emerged that allow a more direct quantification of the number of LDL particles. Because an LDL particle contains a single molecule of apo B, it is possible to directly estimate the number of particles through a simple measurement of apo B …

Identification of Early Biomarkers during Acetaminophen-Induced Hepatotoxicity by Fourier Transform Infrared Microspectroscopy

Acetaminophen is a widely prescribed drug used to relieve pain and fever; however, it is a leading cause of drug-induced liver injury and a burden on public healthcare. In this study, hepatotoxicity in mice post oral dosing of acetaminophen was investigated using liver and sera samples with Fourier Transform Infrared microspectroscopy. The infrared spectra of acetaminophen treated livers in BALB/c mice show decrease in glycogen, increase in amounts of cholesteryl esters and DNA respectively. Rescue experiments using L-methionine demonstrate that depletion in glycogen and increase in DNA are abrogated with pre-treatment, but not post-treatment, with L-methionine. This indicates that changes in glycogen and DNA are more sensitive to the rapid depletion of glutathione. Importantly, analysis of sera identified lowering of glycogen and increase in DNA and chlolesteryl esters earlier than increase in alanine aminotransferase, which is routinely used to diagnose liver damage. In addition, these changes are also observed in C57BL/6 and Nos2 −/− mice. There is no difference in the kinetics of expression of these three molecules in both strains of mice, the extent of damage is similar and corroborated with ALT and histological analysis. Quantification of cytokines in sera showed increase upon APAP treatment. Although the levels of Tnfα and Ifnγ in sera are not significantly affected, Nos2 −/− mice display lower Il6 but higher Il10 levels during this acute model of hepatotoxicity. Overall, this study reinforces the growing potential of Fourier Transform Infrared microspectroscopy as a fast, highly sensitive and label-free technique for non-invasive diagnosis of liver damage. The combination of Fourier Transform Infrared microspectroscopy and cytokine analysis is a powerful tool to identify multiple biomarkers, understand differential host responses and evaluate therapeutic regimens during liver damage and, possibly, other diseases.

Mild Testicular Hyperthermia Transiently Increases Lipid Droplet Accumulation and Modifies Sphingolipid and Glycerophospholipid Acyl Chains in the Rat Testis

Spermatogenesis is known to be vulnerable to temperature. The aim of this study was to investigate the effects on testicular lipids of the transient germ cell loss that is induced by mild testicular hyperthermia. Adult rat testes were exposed once a day to 43 °C for 15 min for 5 days and the effects were followed for several weeks. Two week after the last heat exposure, spermatocytes and early spermatids had virtually disappeared and the seminiferous tubules were populated mostly by mature spermatids and spermatozoa. One week later, the latter were also absent and mostly Sertoli cells populated the tubules. During these 3 weeks, glycerophospholipids (Gpl) and triacylglycerols with long-chain polyunsaturated fatty acids (PUFA) (e.g., 22:5n-6) and species of sphingomyelin and ceramide with nonhydroxy and 2-hydroxy very long-chain (VLC) PUFA (e.g., 28:4n-6, 2-OH 30:5n-6) decreased alongside the germ cells. Concomitantly, the amounts of cholesteryl esters and ether-linked triglycerides increased, both lipids accumulating long-chain and very-long-chain polyenes. This concurred with a considerable buildup of lipid droplets in Sertoli cells, evidently containing these neutral lipids, apparently formed during germ cell-derived membrane lipid catabolism. Between week 4 and week 6, new cohorts of spermatocytes appeared, and by week 12 most cell changes were reversed. Accordingly, as germ cell differentiation proceeded, 22:5n-6-rich Gpl augmented and spermatocyte-associated sphingolipids with nonhydroxy VLCPUFA appeared before their 2-hydroxy counterparts. The unique fatty acids of rat testicular lipids after mild hyperthermia reveal lipid catabolic and biosynthetic reactions that occur in normal spermatogenesis.

Dietary cholesterol reverses resistance to diet-induced weight gain in mice lacking Niemann-Pick C1-Like 1

Niemann-Pick C1-Like 1 (NPC1L1) mediates intestinal cholesterol absorption. NPC1L1 knockout (L1-KO) mice were recently shown to be resistant to high-fat diet (HFD)-induced obesity in one study, which was contrary to several other studies. Careful comparison of dietary compositions in these studies implies a potential role of dietary cholesterol in regulating weight gain. To examine this potential, wild-type (WT) and L1-KO mice were fed one of three sets of diets for various durations: (1) a HFD without added cholesterol for 5 weeks; (2) a high-carbohydrate diet with or without added cholesterol for 5 weeks; or (3) a synthetic HFD with or without added cholesterol for 18 weeks. We found that L1-KO mice were protected against diet-induced weight gain only on a diet without added cholesterol but not on a diet containing 0.16% or 0.2% (w/w) cholesterol, an amount similar to a typical Western diet, regardless of the major energy source of the diet. Food intake and intestinal fat absorption were similar between the two genotypes. Intestinal cholesterol absorption was blocked, and fecal cholesterol excretion increased in L1-KO mice. Under all diets, L1-KO mice were protected from hepatosteatosis. In conclusion, increasing dietary cholesterol restores diet-induced weight gain in mice deficient in NPC1L1-dependent cholesterol absorption.

Causes and prevention of tamoxifen-induced accumulation of triacylglycerol in rat liver

Tamoxifen can induce hepatic steatosis in women. In this study, we wanted to elucidate the mechanism behind the tamoxifen-induced accumulation of triacylglycerol in liver in female rats, and we hoped to prevent this development by combination treatment with the modified fatty acid tetradecylthioacetic acid (TTA). The increased hepatic triacylglycerol level after tamoxifen treatment was accompanied by decreased acetyl-coenzyme A carboxylase (ACC) and FAS activities, increased glycerol-3-phosphate acyltransferase (GPAT) activity, and a tendency to increased diacylglycerol acyltransferase (DGAT) activity. The activities and mRNA levels of enzymes involved in beta-oxidation, ketogenesis, and uptake of lipids from liver were unaffected by tamoxifen, whereas the uptake of lipoproteins was unchanged and the uptake of fatty acids was decreased. Combination treatment with tamoxifen and TTA (Tam+TTA) normalized the hepatic triacylglycerol level and increased the activities of ACC, FAS, GPAT, and DGAT compared with tamoxifen-treated rats. The activities and mRNA levels of enzymes involved in beta-oxidation, ketogenesis, and uptake of lipids were increased after Tam+TTA treatment. In conclusion, tamoxifen increased the hepatic triacylglycerol level, probably as a result of increased triacylglycerol biosynthesis combined with unchanged beta-oxidation. The tamoxifen-induced accumulation of triacylglycerol was prevented by cotreatment with TTA, through mechanisms of increased mitochondrial and peroxisomal beta-oxidation.

Functional evidence for anti-oxidant action of fluvastatin on low-density lipoprotein using isolated macrophages and aorta.

1. Fluvastatin has been reported to have not only a hypocholesterolaemic effect, but also a protective effect on low-density lipoprotein (LDL) from oxidation. We functionally evaluated the anti-oxidant effect of fluvastatin on oxidation of LDL by copper ions in vitro using mouse macrophages and rabbit aorta preparations. 2. After native LDL (N-LDL) from rabbit plasma had been pre-incubated in the presence or absence of fluvastatin (10 micromol/L) for 4 h, the N-LDL was mildly oxidized by incubation with 5 micromol/L CuCl2 for 5 h and two oxidized LDL, fluvastatin-pretreated (Flu-OxLDL) and -non-treated (OxLDL), were prepared. The level of thiobarbituric acid-reactive substances (TBARS) in Flu-OxLDL and OxLDL markedly increased compared with N-LDL. The degree of increment was significantly less in Flu-OxLDL than OxLDL. 3. When macrophages were incubated with Flu-OxLDL or OxLDL, the amount of cholesteryl ester that accumulated in the macrophages markedly increased compared with N-LDL. The degree of increment was significantly less in Flu-OxLDL than OxLDL. 4. Acetylcholine-induced endothelium-dependent relaxations in rabbit aortic rings were impaired in the presence of either Flu-OxLDL or OxLDL. The degree of impairment was significantly less in Flu-OxLDL. 5. The increased TBARS level, facilitated cholesteryl ester accumulation in macrophages and impaired endothelium-dependent relaxation elicited by OxLDL were not affected by simultaneous treatment with fluvastatin (10 micromol/L). 6. These findings indicate that fluvastatin can protect plasma LDL from oxidative modification and, thereby, prevent cholesterol accumulation in macrophages and endothelial dysfunction in blood vessels. This additional anti-oxidative effect of fluvastatin may be beneficial for preventing the progression of atherosclerosis.

HIGH PERFORMANCE THIN-LAYER CHROMATOGRAPHIC ANALYSIS OF NEUTRAL LIPIDS IN THE MARINE SNAILS ILYANASSA OBSOLETUS AND LITTORINA LITTOREA INFECTED WITH LARVAL TREMATODES

High performance thin-layer chromatography analysis was used to analyze neutral lipids in the digestive gland-gonad (DGG) complex of the marine snails Ilyanassa obsoletus and Littorina littorea infected with larval trematodes. The results were compared with the DGGs of snails not infected with larval trematodes. The most abundant neutral lipid in both infected and non-infected snails was triacylglycerols, with percentages ranging from 2.8% in I. obsoletus snails infected with unidentified armatae cercariae to 24.7% in I. obsoletus snails infected with Zoogonius rubellus. L. littorea snails infected with an unidentified armatae cercaria contained 6 to 7 times the amount of cholesteryl ester than uninfected snails or those infected with Cryptocotyle lingua cercariae. I. obsoletus snails infected with Z. rubellus or an unidentified armatae cercaria contained 2 to 3 times more free sterol than the uninfected controls. Larval trematode parasitism was found to alter the neutral lipid profiles of the marine snails I. obsoletus and L. littorea.

The effect of cyclic AMP analogues on cholesteryl ester synthesis and hydrolysis in cultured rat peritoneal macrophages.

The accumulation of large amounts of cholesteryl ester in macrophages which have invaded the artery wall leads to the formation of foam cells, and is one of the early events in the development of atherosclerosis. The activities of both acyl Coenzyme A: cholesterol acyltransferase (ACAT), which is responsible for cholesterol esterification, and cholesteryl ester hydrolase (CEH) have been shown to be increased in foam cells ( 1 ), so that the cholesteryl ester undergoes a continual cycle of formation and hydrolysis ( l,2). Cyclic AMP (CAMP) has been reported to both stimulate (3) and inhibit (4) ACAT activity in the murine macrophage cell line 5774, while in a recent study we were unable to demonstrate any effect (5). CEH activity, on the other hand, has been shown to be increased by dibutyryl cAMP (Bt2cAMP) in two other murine cell lines, P388DI and WEHl (6.7). In the present work, we have investigated the role of cAMP in the regulation of cholesteryl ester metabolism and foam cell formation in cultured rat peritoneal macrophages using cellpermeable cAMP analogues. Macrophages were collected from male Wistar rats (250300g) by peritoneal washing (8) 2 days after intraperitoneal injection of 3ml thioglycollate (3%). Cells from 3-4 rats were pooled for each preparation. After washing, the cells were cultured as monolayers in supplemented Dulbecco's modified Eagle's medium at 37OC in 95% air l 5% CO?. Rat lipoprotein of density < 1.050glml was isolated from rat serum by ultracentrifugation and acetylated by incubation with acetic anhydride (9). Cholesterol esterification was assayed by the incorporation of radioactivity from potassium ('HI =!ca:c in:o cholesteryl esters during a 5h incubation period. Lipids were extracted with chloroform: methanol (2: 1, v:v) and separated by thin layer chromatography. For the determination of CEH activity, the cells were removed from the plates with a plastic scraper, washed with phosphate-buffered saline and disrupted by sonication. The release of I I-'%]oleate from cholesterol I I '%)oleate was then measured in the resulting homogenate (10). The cAMP analogues used were N6 benzoyl cAMP (BEAMP), B-(4-chlorophenylthio) cAMP (CPTcAMP), 8 chloro cAMP (ClcAMP), N6 monobutyryl cAMP (MBcAMP), 8-(6-amino hexyl) amino cAMP (AHA CAMP), 8-piperidino cAMP (PIP CAMP) and dibutyryl cAMP (BtZcAMP). Significance limits were calculated using Student's t test. Cholesterol esterification in the cultured macrophages was unaffected by a range of cAMP analogues at a concentration of

Formation of pregnenolone- and dehydroepiandrosterone-fatty acid esters by lecithin-cholesterol acyltransferase in human plasma high density lipoproteins

Pregnenolone- (PREG-), and dehydroepiandrosterone- (DHEA-) fatty acid esters (FA) are present in human plasma, where they are associated with lipoproteins. Because plasma has the ability to form PREG-FA and DHEA-FA in vitro from their unconjugated steroid counterparts, we postulated that the LCAT enzyme might be responsible for their formation. Here we show that lecithin-cholesterol acyltransferase (LCAT) has PREG and DHEA esterifying activities. First, VLDL, IDL, LDL, and HDL were isolated by the sequential ultracentrifugation micromethod from the plasma of fasting men and women and tested for their ability to form PREG-FA, DHEA-FA, and cholesteryl esters in vitro from their respective unconjugated counterparts. The results showed that the three steroids were esterified only in HDL subfractions. The rate of tritiated PREG esterification was clearly higher than that of tritiated cholesterol and DHEA, both in total plasma and isolated HDL, and no gender difference was observed. Second, human and guinea pig LCAT were purified and used in phosphatidylcholine-reconstituted vesicles containing human apoAI to show their ability to esterify tritiated cholesterol, PREG, and DHEA in the absence of unlabeled steroid. The amount of cholesteryl ester, PREG-FA, and DHEA-FA increased after incubation as a function of time and amount of purified LCAT, showing that PREG is preferentially acylated by LCAT compared to cholesterol and DHEA. The PREG and DHEA esterifying activities of LCAT were cofactor-dependent, as shown by the absence of acylation without apoAI. Finally, we determined by HPLC the fatty acid moiety of PREG-GA and DHEA-FA formed in human plasma and guinea pig and rat sera in vitro after incubation with unconjugated tritiated PREG and DHEA. We showed that the fatty acid moieties of newly formed tritiated PREG-FA and DHEA-FA were similar to that reported for cholesteryl esters in the plasma of the three species. We conclude that LCAT has a lecithin-steroid acyltransferase activity and that PREG is probably the preferential substrate of this enzyme. In addition, the fact that the differences in the fatty acid moieties of cholesteryl esters of human, guinea pig, and rat plasmas are also observed for PREG-FA and DHEA-FA suggests that the LCAT is the sole circulating enzyme that has PREG and DHEA esterifying activities.

MRS EVALUATION OF BRAIN-METABOLITES IN EXTRACTS FROM CELL-CULTURES, HUMAN TUMORS AND NORMAL TISSUE FROM BRAIN - CHOLESTERYL ESTER, CHOLINE CONTAINING COMPOUNDS AND CREATINE AS MARKERS FOR DEVELOPMENT, DIFFERENTIATION AND PATHOLOGY

A multi stage extraction procedure which gives the possibility to analyze both water soluble and lipid components stemming from the same specimen has been developed in our laboratory. Metabolites from brain cancer biopsies have been compared to metabolites in normal human brain, developing mouse brain and primary mouse cell cultures of neurons and astrocytes from cerebral cortex and cerebellum. Extraction with perchloric acid (PCA) dissolves water.soluble components such as choline containing compounds, creatine, amino acids, carbohydrates and high energy phosphates. The water insoluble fraction left after the PCA treatment was extracted with chloroform/methanol, 2/1 (vol/vol). C-13 and H-1 NMR spectra showed characteristic lipid resonances identified as those from long chain saturated and unsaturated fatty acids and cholesterol. Only glioblastomas contained detectable amounts of cholesteryl ester suggesting this compound as marker for brain pathology. It was shown that cholesterol but not cholesteryl ester is present in cultures of neurons either alone or together with astrocytes. H-1 NMR spectra of PCA extracts from biopsies showed that the creatine/choline containing compounds (Cr/Cho) ratios decreased and the N-acetylaspartate/Cho ratios decreased in glioblastomas as compared to normal brain. Cell cultures retain the Cr/Cho ratio characteristic of the developmental stage of the tissue they were prepared from.

High density lipoprotein loses its effect to stimulate efflux of cholesterol from foam cells after oxidative modification.

In this study, we performed oxidative modification of high density lipoprotein (HDL) in vitro. The amount of lipid peroxide increased when either HDL2 or HDL3 was incubated with phosphate-buffered saline containing 5 microM CuSO4 for 24 h at 37 degrees C, indicating that both fractions of HDL were oxidatively modified. This modification resulted in denaturation of apolipoprotein AI on SDS/PAGE and increased the negative charge on agarose gel electrophoresis. When incubated with macrophage-derived foam cells, native HDL caused a marked efflux of cholesterol from them, leading to a decrease in the amount of cholesteryl ester in the cells. However, oxidized HDL showed a lessened effect on the decrease of cholesteryl ester in foam cells. These data suggest that oxidative modification of HDL may stimulate development of atherosclerosis by limiting efflux of cholesterol from foam cells.

Short- and long-term effects of biliary drainage on hepatic cholesterol metabolism in the rat

The present study concerns short- and long-term effects of interruption of the enterohepatic circulation (EHC) on hepatic cholesterol metabolism and biliary secretion in rats. For this purpose, we employed a technique that allows reversible interruption of the EHC, during normal feeding conditions, and excludes effects of anaesthesia and surgical trauma. [3H]Cholesteryl oleate-labelled human low-density lipoprotein (LDL) was injected intravenously in rats with (1) chronically (8 days) interrupted EHC, (2) interrupted EHC at the time of LDL injection and (3) intact EHC. During the first 3 h after interruption of the EHC, bile flow decreased to 50% and biliary bile acid, phospholipid and cholesterol secretion to 5%, 11% and 19% of their initial values respectively. After 8 days of bile diversion, biliary cholesterol output and bile flow were at that same level, but bile acid output was increased 2-3-fold and phospholipid output was about 2 times lower. The total amount of cholesterol in the liver decreased after interruption of the EHC, which was mainly due to a decrease in the amount of cholesteryl ester. Plasma disappearance of LDL was not affected by interruption of the EHC. Biliary secretion of LDL-derived radioactivity occurred 2-4 times faster in chronically interrupted rats as compared with the excretion immediately after interruption of the EHC. Radioactivity was mainly in the form of bile acids under both conditions. This study demonstrates the very rapid changes that occur in cholesterol metabolism and biliary lipid composition after interruption of the EHC. These changes must be taken into account in studies concerning hepatic metabolism of lipoprotein cholesterol and subsequent secretion into bile.

Abnormalities of composition and of in vitro lipolysis products of human small very low density lipoproteins in hypertriglyceridemia

Small (S f 20–100) very low density lipoprotein (VLDL) particles were prepared by density gradient ultracentrifugation of plasma from normolipidemic and type IV hypertriglyceridemic post-infarction patients and healthy controls. The small VLDL separated from the plasma of severely hypertriglyceridemic post-infarction patients were found to contain twice the amount of cholesteryl esters per particle, compared with small VLDL from normolipidemic patients and healthy controls. There was a linear increase in the percentage of cholesterol that was esterified in the small VLDL with the serum VLDL triglyceride concentration (r = 0.66). When incubated for two hours with bovine lipoprotein lipase in excess and bovine albumin as a free fatty acid acceptor at one and the same triglyceride concentration in the medium, the end-product isolated by ultracentrifugation varied as a function of the serum VLDL triglyceride level. The amount of glyceride-glycerol recovered after two hours of incubation with lipoprotein lipase was 13.3 ± 1.3% (mean ± SEM) of the initial values and did not correlate with the VLDL triglyceride level. With rising serum VLDL triglyceride concentration, the product isolated in the low density lipoprotein (LDL) density region (1.006 < d < 1.063 kg/1) contained more total cholesterol and phospholipids. The linear correlation coefficients for these relations were 0.65 and 0.58 for cholesterol and phospholipids respectively. The ratio of total cholesterol to insoluble protein in the LDL density range after lipolysis rose with increasing serum VLDL triglyceride level ( r = 0.68). The end-product was further characterized by density gradient ultracentrifugation of the incubate. In vitro LDL derived by lipolysis of normolipidemic small VLDL was denser than in vitro LDL of hypertriglyceridemic small VLDL. A significant relation was found between the percentage of cholesteryl esters of total cholesterol in the substrate and the relative amount of total cholesterol recovered in the LDL density fraction after lipolysis ( r = 0.69). We suggest that the enrichment with cholesteryl esters of small VLDL from type IV hypertriglyceridemic patients is caused by lipid transfer from LDL and high density lipoprotein (HDL) and that the change in VLDL particle composition influences the precursor-product relationship to LDL.

Characterization of lipoprotein receptors on rat Fu5AH hepatoma cells.

The rat hepatoma cell line Fu5AH has the unusual property of accumulating massive amounts of cholesteryl ester upon incubation with hypercholesterolemic serum, and especially when incubated with beta-very low density lipoproteins (beta-VLDL) from cholesterol-fed dogs. The present study was designed to identify and characterize the lipoprotein receptors that mediate the cholesteryl ester accumulation. The beta-VLDL and cholesterol-induced apolipoprotein (apo) E-containing high density lipoproteins (apoE HDLc) bound to Fu5AH cells with very high affinity (Kd approximately equal to 10(-10) M), whereas low density lipoproteins (LDL) bound with unusually low affinity (Kd approximately equal to 10(-8) M). Receptor binding activity of 125I-labeled beta-VLDL, 125I-labeled apoE HDLc, and 125I-labeled LDL was abolished by incubation in the presence of an excess of unlabeled LDL or of a polyclonal antibody to the bovine adrenal apoB,E(LDL) receptor. The receptors were completely down-regulated by preincubating Fu5AH cells with beta-VLDL, but much higher levels of beta-VLDL were required than for down-regulation of fibroblast apoB,E(LDL) receptors. Receptor binding was abolished by reductive methylation of the lysyl residues of the apolipoprotein of the beta-VLDL and by an apoE monoclonal antibody (1D7) that blocks receptor binding. The Fu5AH receptor was further characterized by using the bovine adrenal apoB,E(LDL) receptor antibody. A single protein (Mr approximately equal to 130,000) was identified in Triton extracts of whole cells, and two proteins (Mr approximately equal to 130,000 and 115,000) were found in Fu5AH cell membranes disrupted by homogenization. The Mr approximately equal to 115,000 protein was released from the membranes and did not react with an antibody to the carboxyl-terminal (cytoplasmic) domain of the apoB,E(LDL) receptors. These studies indicate that Fu5AH cells express apoB,E(LDL) receptors that have unusually low affinity for apoB-continuing lipoproteins, require large amounts of cholesterol to induce down-regulation, and are susceptible to specific proteolysis in cell homogenates. These apoB,E(LDL) receptors are responsible for the receptor-mediated uptake of beta-VLDL and chylomicron remnants by Fu5AH cells.

Lipoprotein lipase hydrolysis of trioleoylglycerol in a phospholipid interface. Effect of cholesteryl oleate on catalysis.

The effect of cholesteryl oleate on the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol was determined in monolayers of egg phosphatidylcholine at a constant surface pressure of 24 mN m-1. The phospholipid monolayers contained 1.0 to 7.5 mol % trioleoylglycerol and various amounts (0 to 20 mol %) of cholesteryl oleate. The initial rates of trioleoylglycerol hydrolysis were determined with lipoprotein lipase purified from bovine milk. In phospholipid monolayers containing 5.0 or 7.5 mol % trioleoylglycerol, the further addition of cholesteryl oleate caused a decrease in lipoprotein lipase activity. In contrast, addition of cholesteryl oleate to phospholipid monolayers containing 1.0 or 2.5 mol % trioleoylglycerol enhanced enzyme activity; a 3-fold enhancement was observed with 5.0-7.5 mol % cholesteryl oleate. Based on force-area measurements, the cholesteryl ester-mediated decrease in lipoprotein lipase activity observed at high substrate concentrations may be explained by displacement of trioleoylglycerol from the interface, thereby reducing the interfacial trioleoylglycerol concentration available for enzyme catalysis. One explanation for the cholesteryl oleate-mediated enhancement of lipoprotein lipase activity at low trioleoylglycerol concentrations is that the additional spreading of cholesteryl oleate disrupts microemulsions of trioleoylglycerol, thereby increasing the effective monomer substrate concentration available for enzyme catalysis. Based on these monolayer studies with model systems, we suggest that the relative amount of cholesteryl esters in plasma triacylglycerol-rich lipoproteins plays a regulatory role in determining the rate at which triacylglycerols are cleared from the circulation.

Platelet secretory products inhibit lipoprotein metabolism in macrophages.

Macrophages possess a receptor that binds low-density lipoproteins (LDL) containing lysine residues modified by acetylation (Ac LDL), acetoacetylation (AcAc LDL) or malondialdehyde treatment. This receptor (referred to as the Ac LDL receptor or scavenger receptor) internalizes the bound lipoprotein. As a consequence, massive amounts of cholesteryl esters accumulate so that macrophages in culture resemble foam cells found in atherosclerotic lesions. In an effort to identify an unmodified mammalian macromolecule that binds to the Ac LDL receptor, we investigated whether platelet secretory products affect the receptor-mediated endocytosis of chemically modified lipoproteins. Platelets are a potential source of such activity because they exist in close association with foam cells in developing atherosclerotic lesions. Our study demonstrates that human blood platelets secrete a product that inhibits the binding and uptake of AcAc LDL by mouse peritoneal macrophages and the subsequent accumulation of cholesteryl esters. This is the first indication that an endogenous macromolecule interacts with Ac LDL receptor on macrophages.

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.