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PubMedDate not supplied

Effect of dietary fat saturation and cholesterol on LDL composition and metabolism. In vivo studies of receptor and nonreceptor-mediated catabolism of LDL in cebus monkeys.

The mechanism(s) by which polyunsaturated fats reduce low density lipoprotein (LDL) cholesterol and apolipoprotein (apo) B were investigated in 20 cebus monkeys (Cebus albifrons) fed diets containing corn oil or coconut oil as fat (31% of calories) with or without dietary cholesterol (0.1% by weight) for 3 to 10 years. Coconut-oil feeding compared to corn-oil feeding resulted in significant increases in levels of plasma total cholesterol (176%), very low density lipoprotein (VLDL)-LDL cholesterol (236%), high density lipoprotein (HDL) cholesterol (148%), apo B (78%), and apo A-I (112%). The addition of dietary cholesterol to corn oil compared to corn oil alone resulted in smaller, but significant, increases in levels of total cholesterol (44%), HDL cholesterol (40%), and apo A-I (33%). Although the increases in VLDL-LDL cholesterol were of similar magnitude (52%), they barely failed to reach statistical significance (p less than 0.08), while the changes in apo B levels were negligible. The addition of dietary cholesterol to coconut oil, compared to coconut oil alone, resulted in no significant changes in lipoprotein cholesterol or apoproteins, although levels of VLDL-LDL cholesterol and apo B values increased 22% and 16%, respectively. Although hepatic free cholesterol content was not altered by diet, coconut-oil compared to corn-oil feeding resulted in significant increases in hepatic cholesteryl esters (236%) and triglycerides (325%), the latter increasing still further when dietary cholesterol was added to coconut oil (563%). To further assess the effects of these dietary changes on LDL metabolism, radioiodinated normal and glucosylated LDL kinetics were performed. The production rate of LDL apo B was not altered by diet. With corn-oil feeding, 63% of LDL catabolism was via the receptor-mediated pathway. Coconut-oil compared to corn-oil feeding resulted in a 50% decrease in receptor-mediated LDL apo B fractional catabolic rate (FCR) and a 27% reduction in nonreceptor-mediated LDL apo B FCR. The addition of dietary cholesterol to corn oil, compared to corn oil alone, resulted in no significant effect on LDL apo B catabolism. The addition of dietary cholesterol to coconut oil, compared to coconut oil alone, was associated with no significant change in nonreceptor catabolism of LDL apo B but with a 58% decrease in receptor-mediated catabolism of LDL (p less than 0.059). The diet-induced alterations of LDL catabolism were significantly correlated with hepatic lipids, which were enriched in saturated fatty acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗

PubMed1996

A flow cytometric competition technique for measuring interaction of LDL with cellular LDL-receptors applied to patients with mutant (Arg3500-->Gln) apolipoprotein B.

We report our experience with a method to evaluate binding and uptake in cells of low density lipoprotein (LDL) from heterozygous patients with familial defective apolipoprotein B-100 (FDB-LDL) and LDL from normolipidemic subjects (nonFDB-LDL). The method is based on competition for binding/uptake in Epstein-Barr Virus (EBV)-transformed lymphocytes or COS cells overexpressing an LDL-receptor transgene between fluorescently labeled LDL and the unlabeled LDL of interest, and measurements are by flow cytometry. With EBV-lymphoblasts, the ability of FDB-LDL to displace fluorescent LDL ("Dil"-LDL) from cells at 4 degrees C (binding) was reduced to approximately 1/3 of normal. Displacement of "Dil"-LDL by FDB-LDL from cells at 20 degrees C (binding/uptake) was reduced to less than 1/2 of normal. Similar results were obtained with COS cells. Freezing of serum to -80 degrees C for 24 hours did not affect results, and we could discriminate between binding/uptake of FDB-LDL and nonFDB-LDL prepared from serum that had been stored at -80 degrees C for three months.

Adult↗

PubMed1991

Cholesterol-rich LDL perfused at physiological LDL-cholesterol concentration induces platelet aggregation and PAF-acetylhydrolase activation.

The aim of this research was to perform an in vivo study on the relationships between lipid oxide (LP), platelet aggregation and PAF-acetylhydrolase in a model using perfusion of cholesterol-rich LDL media diluted to physiological LDL-cholesterol concentration. Normal rabbits were infused with LDL (d 1.025-1.063 g/ml) extracted from rabbits previously fed either with standard food (I-LDL group), 1% cholesterol food (II-LDL group) or 1% cholesterol plus probucol (IV-LDL group). CU2+ modified II-LDL was also infused (III-LDL group). After dilution as above, LP increased significantly in III- and II-LDL media. After perfusion, LP significantly increased in III- and II-LDL groups as compared to baseline values and to control. Compared to the I-LDL group, PAF-acetylhydrolase and platelet aggregation significantly increased in III- and II-LDL groups. These data indicate the property of cholesterol- rich LDL to activate PAF-acetylhydrolase and enhance platelet aggregation, even when perfused through a medium containing a physiological LDL-cholesterol concentration.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2005

Colesevelam HCl reduces LDL particle number and increases LDL size in hypercholesterolemia.

BACKGROUND: Although LDL-cholesterol (LDL-C) remains the target of cholesterol-lowering therapy, high levels of LDL particle numbers better differentiate cardiovascular risk than LDL-C concentrations. Colesevelam HCl, a novel bile acid sequestrant, has also been shown to reduce mean LDL-C by 18% with the maximum dose. The effect of colesevelam HCl on LDL particle number and particle size has not been previously published. LDL particle number and particle size were measured by NMR spectroscopy. METHODS: In this multi-center, double-blind, placebo-controlled study, 149 patients with moderate hypercholesterolemia (LDL-C > 160 mg/dL [4.14 mmol/L]; triglycerides < 300 mg/dL [3.39 mmol/L]) were randomized to colesevelam HCl (1.5-3.75 g/d) or placebo for 6 weeks. RESULTS: Colesevelam HCl reduced LDL particle number by 6.8% at a dosage of 3.0 g/d (P = 0.031), and 13.7% (P = 0.0002) at a dosage of 3.75 g/d. Baseline levels of triglycerides or LDL size did not influence changes in LDL particle number. Further, mean LDL particle size increased with colesevelam HCl 3.75 g/d (1.1% increase versus baseline, P < 0.05). CONCLUSION: For patients with moderate hypercholesterolemia, treatment with colesevelam HCl at the recommended starting dose of 3.75 g/d lowered LDL particle number, and increased mean LDL particle size.

Allylamine↗

PubMed2001

Reversible and irreversible non-internalized LDL and methyl LDL accumulation by human fibroblasts.

In previous in vivo animal studies, we showed that low density lipoprotein (LDL) accumulated irreversibly at the edges of healing arterial lesions rather than being internalized and degraded. To see if similar LDL accumulation occurs in vitro, fibroblasts from normal and homozygous familial hypercholesterolemic (FH) subjects were incubated at 37 degrees C with 125I-LDL and 125I-methyl LDL; the latter is not recognized by any known LDL receptor. Normal fibroblast accumulation of LDL and methyl LDL (5 microg/ml) plateaued within 1 h at 200 and 100 ng/mg, respectively. With FH cells, both LDL and methyl LDL accumulation plateaued at 100 ng/mg. Lipoprotein accumulation by both cell types rose steeply at concentrations up to 15-25 microg/ml, and less so at higher concentrations. Except for degradation of LDL by normal cells, degradation was minimal, which indicated that much of the lipoprotein accumulation was unaccompanied by internalization. The accumulation of both lipoproteins by both cell types was greater at 37 degrees C than at 4 degrees C, and was inhibited between 43 and 75% by homologous unlabeled lipoprotein. To see if any accumulation was irreversible, cells were incubated with radiolabeled lipoproteins for 3 h (pulse), then with homologous unlabeled lipoproteins for up to 20 h (chase). About 50% of intact radiolabeled lipoprotein rapidly dissociated from cells into the medium in the first 4 h of the chase period. In contrast, between 4 and 20 h, most of the remaining intact LDL and methyl LDL appeared to be irreversibly bound, because it was released at a rate of only 0-1%/h. Thus, we conclude that, under the conditions studied, both reversible and irreversible non-internalized LDL binding play a major role in LDL accumulation by cultured cells.

Binding, Competitive↗

PubMed1997

LDL from aerobically-trained subjects shows higher resistance to oxidative modification than LDL from sedentary subjects.

We studied the effect of regular intense aerobic exercise on the LDL susceptibility to oxidation and the electronegative LDL-proportion (LDL(-)). A group of 38 well-trained athletes was compared to a group of 38 age-BMI-matched sedentary individuals. Athletes showed higher concentration of total cholesterol (athletes 5.08 +/- 0.70 versus controls 4.65 +/- 0.75 mmol/l, P = 0.0229) and HDL cholesterol (athletes 1.72 +/- 0.47 versus controls 1.46 +/- 0.39 mmol/l, P = 0.0068); total plasma triglyceride, LDL cholesterol and VLDL cholesterol did not differ between trained and untrained subjects. The susceptibility of LDL to oxidation, determined by conjugated dienes formation and expressed as lag phase, was lower in athletes than in sedentaries (trained subjects 47.0 +/- 5.6 versus sedentary subjects 41.9 +/- 5.0 min, P = 0.0002). LDL(-) was similar in both groups (athletes 10.32 +/- 4.70 versus controls 10.26 +/- 3.71%). The antioxidant content in total plasma and isolated LDL (alpha-tocopherol, retinol, lycopene, alpha-carotene and beta-carotene) was quantitated by HPLC in a subgroup of 32 athletes and 32 control subjects. Athletes showed higher amounts of alpha-tocopherol and retinol in plasma, but not in LDL. However, none of these antioxidants correlated with the lag phase time. Trained subjects showed lower prevalence of smoking. However, no differences were observed between smokers and non-smokers concerning lag phase. No significant difference between athletes and sedentaries concerning LDL density, or composition was observed. We conclude that LDL from trained subjects is more resistant to oxidative modification than LDL from sedentary subjects. This observation could not be attributed to conventional antioxidants as alpha-tocopherol and carotene content of LDL was unchanged in trained subjects. Thus, although none of the variables studied appear as a single predictor of the LDL susceptibility to oxidation, an additive effect of the antioxidant content, the presence of some undetermined co-antioxidant, HDL and/or smoking habits cannot be discarded as responsible for the increased resistance to oxidation of LDL in trained subjects.

Adult↗

PubMed1998

Glyc-oxidized LDL impair endothelial function more potently than oxidized LDL: role of enhanced oxidative stress.

Hypercholesterolemia is associated with impairment of endothelial function due to increased levels of LDL. In diabetic patients, however, attenuation of endothelial function occurs even under normocholesterolemic conditions. Here we assessed whether glycation of LDL potentiates their influence on endothelial function, with particular emphasis on the oxidizability of LDL and the role of O2-. Human LDL was glycated by dialyzation for 7 days against buffer containing 200 mmol/l glucose, or sham-treated without glucose, and oxidized by incubation with Cu2+. Glycation significantly enhanced the oxidizability of LDL, as detected by diene formation and by electrophoretic mobility (27.5 mm for oxidized LDL vs. 34 mm for oxidized glycated LDL at 20 h of oxidation). Isolated rings of rabbit aorta were superfused with physiological salt solution, and isometric tension was recorded. Incubation of the aortic rings with sham-treated or with glycated LDL, not oxidized, had no influence on acetylcholine-induced, endothelium-dependent relaxation. Exposure of the aortic rings to oxidized non-glycated LDL caused a significant inhibition (30% at 1 microM acetylcholine) of the endothelium-dependent relaxation only in the presence of diethyl-dithiocarbamate (DDC), an inhibitor of the endogenous superoxide dismutase (SOD). Incubation of aortic rings with oxidized glycated LDL attenuated endothelium-dependent relaxation even in the absence of DDC (by 31% at 1 microM acetylcholine). The presence of DDC potentiated the inhibition of relaxation (65% inhibition at 1 microM acetylcholine), and co-incubation with exogenous SOD and catalase prevented the inhibition of relaxation, indicating a mediator role of O2-. Endothelium-independent relaxation induced by forskolin was unaffected by any of the lipoproteins. Using a chemiluminescence assay, significantly increased O2- production of aortic rings pretreated with oxidized glycated LDL (4101 +/- 360 counts/s) in comparison to control rings (753 +/- 81 counts/s) or arteries pretreated with oxidized non-glycated LDL (2358 +/- 169 counts/s) could be detected, suggesting that enhanced NO-inactivation by O2- could be the underlying mechanism for the stronger impairment of endothelium-dependent dilations by oxidized glycated LDL. Glycation increases the oxidizability of LDL and potentiates its endothelium-damaging influence. The likely mechanism for attenuation of endothelium-dependent dilations is increased formation of O2-, resulting in inactivation of nitric oxide. This mechanism may play an important role in diabetic patients and may contribute to disturbed organ perfusion.

Animals↗

PubMed1998

Drug targeting using low density lipoprotein (LDL): physicochemical factors affecting drug loading into LDL particles.

Low density lipoprotein (LDL) has been found suitable as a targeting carrier for cytotoxic drugs. However, higher drug loading into LDL particles without disrupting their native integrity remains a major obstacle. The purpose of this study is to investigate the different physicochemical factors that may affect drug loading and to characterize LDL-drug conjugates. Doxorubicin (Dox) and 3', 5'-O-dipalmitoyl-5-iodo-2'-deoxyuridine (dpIUdR) were used as reference cytotoxic drugs. Drugs were loaded into LDL particles using the dry film method with or without surfactants, liposomal and the direct addition method. The effects of incubation temperature, time and stoichiometry of LDL-drug conjugates on drug loading were investigated. The LDL-drug conjugates were evaluated for their stability and characterized by differential scanning calorimetry (DSC), denatured gel (SDS-PAGE), and electron microscopy (EM). We have suitably incorporated 45+/-10 Dox and 150+/-25 dpIUdR molecules/LDL particle. A seven-fold increase in Dox incorporation was achieved with the liposomal preparation compared to the dry film method. A 4- to 6-h incubation at 37 degreesC was suitable to restore the native structure of LDL particles. No apo B fragmentation of LDL particles was noted on denatured gel. DSC studies showed no change in the Tm of the LDL and the LDL-drug conjugates. An increase in particle size of LDL-dpIUdR, not LDL-Dox was observed in EM compared to the native LDL which may be related to higher incorporation of dpIUdR. The results indicate that physicochemical factors significantly affect drug loading efficiency and may need to be considered to optimize drug incorporation into LDL particles.

Chromatography, High Pressure Liquid↗

PubMed2001

Low-density lipoprotein (LDL)-induced monocyte-endothelial cell adhesion, soluble cell adhesion molecules, and autoantibodies to oxidized-LDL in chronic renal failure patients on dialysis therapy.

Premature atherosclerosis is a major cause of morbidity and mortality in chronic renal failure patients undergoing dialysis. In this study, we compared autoantibodies to oxidized low-density lipoprotein (OX-LDL), soluble cell adhesion molecules (CAMs), and the effect of both LDL and OX-LDL on monocyte endothelial cell adhesion in chronic renal failure patients on hemodialysis (HD, n = 16) and peritoneal dialysis (PD, n = 17) compared with matched healthy control subjects (C, n = 17). In addition, we studied the effect of supplementation with RRR-alpha-tocopherol (AT) 800 IU/d for 12 weeks on the above measures. LDL and OX-LDL induced adhesion of U937 cells to cultured endothelium, soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intracellular adhesion molecule-1 (sICAM-1), and soluble E-selectin (sE-selectin); autoantibodies to OX-LDL and markers of lipid peroxidation were determined before and after AT supplementation. Native LDL from PD patients induced greater monocyte-endothelial cell adhesion than LDL from C subjects (43.8% +/- 17.0% v25.3% +/- 17.7%, respectively, P = .0028). OX-LDL from chronic renal failure patients on both PD and HD stimulated greater adhesion than OX-LDL from the C subjects (68.0% +/- 18.5% and 57.6% +/- 15.1% v 40.9% +/- 17.3%, respectively, P < .01); OX-LDL from PD patients induced greater adhesion than that from HD patients (P < .01). Plasma methylglyoxal levels were significantly increased in both HD and PD groups, with higher levels in the HD group. Chronic renal failure patients on HD and PD also had higher levels of plasma sVCAM-1 and sE-selectin than C subjects (P < .01), indicating endothelial activation. Titers of autoantibodies to OX-LDL were not elevated in renal failure patients. Supplementation with AT 800 IU/d for 12 weeks, while resulting in significant enrichment with AT in LDL, did not have a significant effect on any of the parameters studied. This study makes the novel observation that the LDL of chronic renal failure patients on HD and PD appears to be potentially more atherogenic, since it induces greater monocyte-endothelial cell adhesion.

Adult↗

PubMed2000

Why is glycated LDL more sensitive to oxidation than native LDL? A comparative study.

It is well established that oxidative modification of low-density lipoprotein (LDL) plays a causal role in human atherogenesis and the risk of atherosclerosis is increased in patients with diabetes mellitus. To examine the influence of different agents which may influence LDL-glycation and oxidation, experiments including glycation with glucose, glucose 6-phosphate, metal chelators (EDTA) and antioxidants (BHT) were performed. The influence of time dependence on the glycation process and the alteration of the electrophoretic mobility of LDL under diverse glycation and/or oxidation conditions was also investigated. The formation of conjugated dienes and levels of lipid peroxides in these different LDL-modifications were estimated. The copper-induced oxidation of LDL in vitro was determined by measurement of thiobarbituric acid reactive substances (TBARS) and expressed as nmol MDA/mg of LDL protein. We found that glycated LDL is more prone to oxidation than native LDL. Using native LDL, the maximal oxidation effect was found to reach a value of 49.72 nmol MDA/mg protein after 8 h. The maximum oxidation of the 31 days, glycated LDL with glucose was 71.76 nmol MDA/mg protein amounting to 144.33% of the value found for native LDL. In the case of glucose 6-phosphate glycation, the maximum oxidation under the same conditions amounted to 173.77% of the value found for native LDL. To measure the extent of glycation, fluorescence of advanced glycation end products (AGEs) was determined (370 nm excitation and 440 nm emission). The most potent glycation agent was glucose 6-phosphate leading to the formation of very high amounts of AGEs. This process was promoted in the absence of EDTA, which prevents the oxidative cleavage of modified Amadori products (ketoamines) to AGEs. We therefore conclude that both processes, glycation and oxidation, result in the modification of LDL. The lower the glycation-rate (+/- EDTA) as measured by relative fluorescence units RFU (generation of AGEs), the lower the additional oxidation rate after glycation as measured by TBARS (generation of MDA equivalents). Glycation and/or oxidation change the electrophoretic mobility of LDL.

Adult↗

PubMed1997

A new function for the LDL receptor: transcytosis of LDL across the blood-brain barrier.

Lipoprotein transport across the blood-brain barrier (BBB) is of critical importance for the delivery of essential lipids to the brain cells. The occurrence of a low density lipoprotein (LDL) receptor on the BBB has recently been demonstrated. To examine further the function of this receptor, we have shown using an in vitro model of the BBB, that in contrast to acetylated LDL, which does not cross the BBB, LDL is specifically transcytosed across the monolayer. The C7 monoclonal antibody, known to interact with the LDL receptor-binding domain, totally blocked the transcytosis of LDL, suggesting that the transcytosis is mediated by the receptor. Furthermore, we have shown that cholesterol-depleted astrocytes upregulate the expression of the LDL receptor at the BBB. Under these conditions, we observed that the LDL transcytosis parallels the increase in the LDL receptor, indicating once more that the LDL is transcytosed by a receptor-mediated mechanism. The nondegradation of the LDL during the transcytosis indicates that the transcytotic pathway in brain capillary endothelial cells is different from the LDL receptor classical pathway. The switch between a recycling receptor to a transcytotic receptor cannot be explained by a modification of the internalization signals of the cytoplasmic domain of the receptor, since we have shown that LDL receptor messengers in growing brain capillary ECs (recycling LDL receptor) or differentiated cells (transcytotic receptor) are 100% identical, but we cannot exclude posttranslational modifications of the cytoplasmic domain, as demonstrated for the polymeric immunoglobulin receptor. Preliminary studies suggest that caveolae are likely to be involved in the potential transport of LDL from the blood to the brain.

Animals↗

PubMed1988

Quantification in vivo of increased LDL content and rate of LDL degradation in normal rabbit aorta occurring at sites susceptible to early atherosclerotic lesions.

While the exact mechanisms that initiate atherosclerotic lesions are unknown, considerable evidence supports a role for low density lipoprotein (LDL). We investigated whether in the normal rabbit, LDL metabolism in areas of aorta that are destined to become lesioned during cholesterol feeding differed from the metabolism in adjacent lesion-resistant aorta. These studies took advantage of the predictable pattern of early atherosclerotic lesions in the cholesterol-fed rabbit. Early lesions occur diffusely in the aortic arch and ascending aorta and distal to branch orifices in the abdominal aorta and the descending thoracic aorta. Arterial rates of irreversible degradation of LDL and concentrations of intact LDL were measured in susceptible and resistant sites with homologous doubly labeled LDL. LDL was labeled directly with 131I and with 125I-tyramine cellobiose. The latter label provides a highly sensitive means to determine the sites and rates of lipoprotein degradation in vivo. The arterial concentration of intact LDL in the lesion-prone aortic arch was 3.12 +/- 0.45 micrograms LDL cholesterol/g (n = 14), 3.6 +/- 0.69 times that in the relatively lesion-resistant descending thoracic aorta (p less than 0.001). The rate of LDL degradation in the aortic arch was 3.14 +/- 0.41 micrograms LDL cholesterol/g/day, 2.14 +/- 0.24 times that in the descending thoracic aorta (p less than 0.001). In the abdominal aorta, the LDL (per gram wet weight) concentration and degradation rate (per square centimeter surface area) at branch sites exceeded that at nonbranch sites by 88 +/- 11% (p less than 0.001) and by 61 +/- 8% (p less than 0.001), respectively. These data provide evidence that in the normal rabbit, which does not develop atherosclerotic lesions, focal elevations of arterial LDL degradation rate and concentrations of intact LDL occur at sites that first develop atherosclerotic lesions in the hypercholesterolemic animal. These differences in LDL metabolism may be linked causally to the propensity to develop atherosclerotic lesions at these sites.

Animals↗

PubMed1996

LDL apheresis reduces the susceptibility of LDL to in vitro oxidation in a diabetic patient with hemodialysis treatment.

OBJECTIVE: We studied whether LDL apheresis would alter the susceptibility of LDL to in vitro oxidative modification induced by copper ion in a diabetic patient undergoing hemodialysis treatment. RESEARCH DESIGN AND METHODS: Isolated LDL was dialyzed against phosphate-buffered saline and resuspended at a concentration of 0.3 mg cholesterol mass/ml buffer LDL oxidation was then initiated by addition of 1.66 mumol/l CuSO4, and the formation of conjugated dienes was continuously monitored spectrophotometrically at 234 nm. RESULTS: The duration of the lag phase of LDL obtained after LDL apheresis was markedly longer than that of LDL obtained before LDL apheresis. The propagation rate and the maximum conjugated diene formation of LDL oxidation were not changed after LDL apheresis. CONCLUSIONS: A LDL subfraction that was susceptible to oxidation was removed by LDL apheresis, resulting in reduction of susceptibility of LDL to in vitro oxidation remaining in plasma. LDL apheresis may be of clinical importance for preventing atherosclerotic disease in diabetic patients undergoing hemodialysis.

Aged↗

PubMed1987

Detection of the low density lipoprotein (LDL) receptor on nitrocellulose paper with colloidal gold-LDL conjugates.

Gold-low density lipoprotein (LDL) conjugates were used to detect the LDL receptor on nitrocellulose paper. Solubilized rat liver membrane proteins were subjected to electrophoresis and electroblotted onto nitrocellulose paper. The receptor was then detected as a red band (within 10 min) by overlaying with the LDL conjugates. The coloration was prevented by unlabeled LDL, EDTA, and suramin but not by unlabeled HDL3. In the dot blot assay, detection with the colloidal gold-LDL conjugates was as sensitive as both the autoradiographic method with 125I-labeled LDL and the biotinylated LDL method; the estimated limit of detection by scanning densitometry was 1.6 femtomoles of receptor protein. When the coloration obtained with the colloidal gold-LDL conjugates was intensified by photochemical silver staining, down to 200 attomoles of the LDL receptor could be detected. In this assay, the EDTA-sensitive binding of colloidal gold-LDL to solubilized hepatic membrane proteins was 12 times higher for rats treated with 17 alpha-EE than for normal rats. The use of colloidal gold-LDL conjugates is therefore a very easy, safe, inexpensive, fast and sensitive method for the detection of the LDL receptor on nitrocellulose paper. Furthermore, with silver staining and scanning densitometry, the colloidal gold-LDL conjugates could be used in a dot blot assay to quantify tissue and cell LDL receptors down to attomolar levels.

Animals↗

PubMed1994

Lipoprotein lipase (LpL) affects low density lipoprotein (LDL) flux through vascular tissue: evidence that LpL increases LDL accumulation in vascular tissue.

A cardinal feature of the atherosclerotic lesion is increased low density lipoprotein (LDL) content of the arterial wall. Such increases in vascular wall LDL could result from either increased flux of circulating LDL across the arterial endothelial barrier or decreased efflux of LDL that has entered the vascular tissue. A number of studies have focused on factors that alter permeability of endothelial cell monolayers and intact blood vessels causing increased LDL influx. In contrast, the current studies were designed to test the hypothesis that lipoprotein lipase (LpL), increases LDL accumulation and decreases LDL efflux from vascular tissue. Frog mesenteric venular microvessels were cannulated and the rates of fluorescently labeled LDL accumulation (N/t) and efflux (T1/2) were measured by quantitative fluorescence microscopy. When the vessels were perfused with a solution containing bovine milk LpL (10(-5) g/ml) and human LDL (protein = 0.68 mg/ml), N/t was > 15x greater than that of control vessels which were perfused with LDL alone. LpL addition did not change albumin permeability, suggesting that increased N/t was not related to changes in vessel permeability. Increased LDL accumulation within the vessel could have resulted from either an increase in LDL influx from the vessel lumen into the vascular tissue or a decrease in efflux of LDL. Therefore, LDL efflux from vascular tissue was determined by measuring the rate of decline in fluorescence intensity of control and LpL-treated vessels after washout of the vessel lumen with a clear, nonfluorescent solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗

PubMed1995

Effect of gemfibrozil treatment in hypercholesterolemia on low density lipoprotein (LDL) subclass distribution and LDL-cell interaction.

Gemfibrozil, a widely used fibric acid derivative, corrects hypercholesterolemia in a non-negligible fraction of patients. To investigate the mechanism of the cholesterol-lowering activity of fibric acids, a study was performed in 12 type IIa hyperlipidemic patients treated with gemfibrozil for 12 weeks. Changes in low density lipoprotein (LDL) structure and composition, agonist capacity of LDL against the LDL-receptor in human skin fibroblasts, LDL-receptor activity in mononuclear cells, lecithin:cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) activity, were evaluated. Plasma total and LDL cholesterol levels decreased by 17% and 20% after 12 weeks of treatment, the reduction being directly correlated with the baseline levels (r = 0.75 and 0.78, respectively). The mean LDL diameter increased significantly, from 25.5 to 26.1 nm, while the relative content of small LDL particles (< 25.1 nm) increased from 23.4% to 32.8% of total LDL. Neither the apolipoprotein (apo) B secondary structure nor the affinity of LDL for the LDL-receptor of fibroblasts were affected. The LDL-receptor activity in patients' mononuclear cells increased 3-fold, the rise being unrelated to the plasma cholesterol reduction. LCAT activity did not change, while CETP activity was reduced by 25% (P = 0.13) after treatment. These findings indicate that gemfibrozil causes significant changes in LDL structure that do not, however, affect the LDL interaction with peripheral cells.

Adult↗

PubMed1994

Flow cytometric evaluation of LDL receptors using DiI-LDL uptake and its application to B and T lymphocytic cell lines.

Low density lipoprotein receptors (LDL-R) on Daudi Burkitt's lymphoma (BL) cells were assessed using fluorescent DiI (3,3'-dioctadecylindocarbocyanine iodide)-LDL and flow cytometric analyses. Receptor-specific binding of DiI-LDL is followed by internalization of the bound complex and lysosomal hydrolysis of the ligand. Increase in the fluorescence intensity per cell is hence used as a measure of DiI-LDL uptake and, implicitly, as an indication of LDL-R presence. Our results show that uptake was observed in > 98% of the Daudi cells, and the level of uptake was significant and clearly distinguishable from autofluorescence, suggesting that: (a) this assay is comparable to the iodinated LDL uptake assay, although the ED50 values for the ligands are different; (b) this assay is comparable to the flow-cytometric detection of LDL-R using a commercial antibody directed against the receptor itself, and superior to a similar assay based on an antibody directed against membrane-bound LDL; (c) LDL uptake could be monitored along with transferrin uptake, suggesting that multiple endocytic receptor activities can be concurrently studied; (d) DiI-LDL uptake can be examined along with fluorescein-conjugated anti-CD10, -CD19, and -CD71, with little cross-interference, offering the added advantage that endocytic uptake and phenotyping can be simultaneously monitored; (e) the expression of LDL-R is intrinsically elevated in diverse cell lines such as Daudi, Raji, Ramos, Jurkat, and WIL2-NS, but not in normal lymphocytes. Our results therefore indicate that flow cytometric analysis of DiI-LDL uptake has potentially useful applications in the detection and study of endocytic receptor LDL-R in B and T lymphocytic cell lines.

B-Lymphocytes↗

PubMed1994

LDL and acetyl-LDL inhibit the NK activity and are taken up by CD56+ lymphocytes.

The effect of LDL and modified LDL (acetyl-LDL) was studied on human natural killer cell-mediated cytotoxicity against K562 cells. Incubation for 24 h of peripheral blood lymphocytes (PBL) with a high concentration (200 micrograms/ml) of LDL decreased the NK activity in some donors. After acetylation of the LDL protein (apoB), the modified-LDL systematically inhibited the NK function of PBL in a time- and dose-dependent manner. Inhibition mediated by acetyl-LDL (AcLDL) was significantly greater than that of LDL, indicating that the apoB modification can mediate the inhibition of the NK function. AcLDL also inhibited the NK activity of peripheral blood mononuclear cells, suggesting that, under our experimental conditions, monocytes are not efficient enough to protect NK cells against the adverse effects of modified-LDL. With a cytofluorimetric analysis, the internalization of acetyl-LDL by PBL was demonstrated and was only 3-4 times lower than LDL internalization in lymphocytes. It appeared to be time, temperature and dose dependent, saturable and different from the internalization mediated by the known scavenger receptors. Finally, CD14- CD3+ lymphocytes and CD14- CD56+ lymphocytes were able to internalize AcLDL in the same way. Our results suggest that in some in vivo circumstances, when the LDL concentration and/or the modified-LDL/LDL ratio increase in tissues, lipoproteins are internalized by NK cells and also can induce adverse effects on the NK function.

Antigens, CD↗