Modified Low Density Lipoprotein Particles Research Articles

Aggregation Susceptibility of Low-Density Lipoproteins-A Novel Modifiable Biomarker of Cardiovascular Risk.

Circulating low-density lipoprotein (LDL) particles enter the arterial intima where they bind to the extracellular matrix and become modified by lipases, proteases, and oxidizing enzymes and agents. The modified LDL particles aggregate and fuse into larger matrix-bound lipid droplets and, upon generation of unesterified cholesterol, cholesterol crystals are also formed. Uptake of the aggregated/fused particles and cholesterol crystals by macrophages and smooth muscle cells induces their inflammatory activation and conversion into foam cells. In this review, we summarize the causes and consequences of LDL aggregation and describe the development and applications of an assay capable of determining the susceptibility of isolated LDL particles to aggregate when exposed to human recombinant sphingomyelinase enzyme ex vivo. Significant person-to-person differences in the aggregation susceptibility of LDL particles were observed, and such individual differences largely depended on particle lipid composition. The presence of aggregation-prone LDL in the circulation predicted future cardiovascular events in patients with atherosclerotic cardiovascular disease. We also discuss means capable of reducing LDL particles’ aggregation susceptibility that could potentially inhibit LDL aggregation in the arterial wall. Whether reductions in LDL aggregation susceptibility are associated with attenuated atherogenesis and a reduced risk of atherosclerotic cardiovascular diseases remains to be studied.

CD36 in Atherosclerosis: Pathophysiological Mechanisms and Therapeutic Implications.

Atherosclerosis is a chronic disease characterized by lipid retention and inflammation in the artery wall. The retention and oxidation of low-density lipoprotein (LDL) in sub-endothelial space play a critical role in atherosclerotic plaque formation and destabilization. Oxidized LDL (ox-LDL) and other modified LDL particles are avidly taken up by endothelial cells, smooth muscle cells, and macrophages mainly through several scavenger receptors, including CD36 which is a class B scavenger receptor and membrane glycoprotein. Animal studies performed on CD36-deficient mice suggest that deficiency of CD36 prevents the development of atherosclerosis, though with some debate. CD36 serves as a signaling hub protein at the crossroad of inflammation, lipid metabolism, and fatty acid metabolism. In addition, the level of soluble CD36 (unattached to cells) in the circulating blood was elevated in patients with atherosclerosis and other metabolic disorders. We performed a state-of-the-art review on the structure, ligands, functions, and regulation of CD36 in the context of atherosclerosis by focusing on the pathological role of CD36 in the dysfunction of endothelial cells, smooth muscle cells, monocytes/macrophages, and platelets. Finally, we highlight therapeutic possibilities to target CD36 expression/activity in atherosclerosis.

Mast Cells as Potential Accelerators of Human Atherosclerosis—From Early to Late Lesions

Mast cells are present in atherosclerotic lesions throughout their progression. The process of atherogenesis itself is characterized by infiltration and retention of cholesterol-containing blood-derived low-density lipoprotein (LDL) particles in the intimal layer of the arterial wall, where the particles become modified and ingested by macrophages, resulting in the formation of cholesterol-filled foam cells. Provided the blood-derived high-density lipoproteins (HDL) particles are able to efficiently carry cholesterol from the foam cells back to the circulation, the early lesions may stay stable or even disappear. However, the modified LDL particles also trigger a permanent local inflammatory reaction characterized by the presence of activated macrophages, T cells, and mast cells, which drive lesion progression. Then, the HDL particles become modified and unable to remove cholesterol from the foam cells. Ultimately, the aging foam cells die and form a necrotic lipid core. In such advanced lesions, the lipid core is separated from the circulating blood by a collagenous cap, which may become thin and fragile and susceptible to rupture, so causing an acute atherothrombotic event. Regarding the potential contribution of mast cells in the initiation and progression of atherosclerotic lesions, immunohistochemical studies in autopsied human subjects and studies in cell culture systems and in atherosclerotic mouse models have collectively provided evidence that the compounds released by activated mast cells may promote atherogenesis at various steps along the path of lesion development. This review focuses on the presence of activated mast cells in human atherosclerotic lesions. Moreover, some of the molecular mechanisms potentially governing activation and effector functions of mast cells in such lesions are presented and discussed.

Electronegative LDL: An Active Player in Atherogenesis or a By- Product of Atherosclerosis?

Low-density lipoproteins (LDLs) are the major plasma carriers of cholesterol. However, LDL particles must undergo various molecular modifications to promote the development of atherosclerotic lesions. Modified LDL can be generated by different mechanisms, but as a common trait, show an increased electronegative charge of the LDL particle. A subfraction of LDL with increased electronegative charge (LDL(-)), which can be isolated from blood, exhibits several pro-atherogenic characteristics. LDL(-) is heterogeneous, due to its multiple origins but is strongly related to the development of atherosclerosis. Nevertheless, the implication of LDL(-) in a broad array of pathologic conditions is complex and in some cases anti-atherogenic LDL(-) properties have been reported. In fact, several molecular modifications generating LDL(-) have been widely studied, but it remains unknown as to whether these different mechanisms are specific or common to different pathological disorders. In this review, we attempt to address these issues examining the most recent findings on the biology of LDL(-) and discussing the relationship between this LDL subfraction and the development of different diseases with increased cardiovascular risk. Finally, the review highlights the importance of minor apolipoproteins associated with LDL(-) which would play a crucial role in the different properties displayed by these modified LDL particles.

Triglycerides and Cardiovascular Disease

A long-standing association exists between elevated triglyceride levels and cardiovascular disease* (CVD).1,2 However, the extent to which triglycerides directly promote CVD or represent a biomarker of risk has been debated for 3 decades.3 To this end, 2 National Institutes of Health consensus conferences evaluated the evidentiary role of triglycerides in cardiovascular risk assessment and provided therapeutic recommendations for hypertriglyceridemic states.4,5 Since 1993, additional insights have been made vis-a-vis the atherogenicity of triglyceride-rich lipoproteins (TRLs; ie, chylomicrons and very low-density lipoproteins), genetic and metabolic regulators of triglyceride metabolism, and classification and treatment of hypertriglyceridemia. It is especially disconcerting that in the United States, mean triglyceride levels have risen since 1976, in concert with the growing epidemic of obesity, insulin resistance (IR), and type 2 diabetes mellitus (T2DM).6,7 In contrast, mean low-density lipoprotein cholesterol (LDL-C) levels have receded.7 Therefore, the purpose of this scientific statement is to update clinicians on the increasingly crucial role of triglycerides in the evaluation and management of CVD risk and highlight approaches aimed at minimizing the adverse public health–related consequences associated with hypertriglyceridemic states. This statement will complement recent American Heart Association scientific statements on childhood and adolescent obesity8 and dietary sugar intake9 by emphasizing effective lifestyle strategies designed to lower triglyceride levels and improve overall cardiometabolic health. It is not intended to serve as a specific guideline but will be of value to the Adult Treatment Panel IV (ATP IV) of the National Cholesterol Education Program, from which evidence-based guidelines will ensue. Topics to be addressed include epidemiology and CVD risk, ethnic and racial differences, metabolic determinants, genetic and family determinants, risk factor correlates, and effects related to nutrition, physical activity, and lipid medications. In the United States, the National Health and …

N(epsilon)-(Carboxymethyl)lysine and Coronary Atherosclerosis-Associated Low Density Lipoprotein Abnormalities in Type 2 Diabetes: Current Status.

In comparison to the general population, individuals with diabetes suffer a 3- to 4-fold increased risk for developing complications of atherosclerosis and vascular insufficiency. This fact should be taken into account to develop a suitable determinant for the early detection of these complications and subsequently reduce the adverse effect of type 2 diabetes. In vitro experiments have shown that the products of glucose auto-oxidation and Amadori adducts are both potential sources of Nε-(carboxymethyl)lysine (CML). Excessive formation of CML on low density lipoprotein (LDL) has been proposed to be an important mechanism for the dyslipidemia and accelerated atherogenesis observed in patients with type 2 diabetes. It has been postulated that the uptake of CML-LDL by LDL receptors is impaired, thereby decreasing its clearance from the blood circulation. Alternatively, the uptake of these modified LDL particles by scavenger receptors on macrophages and vascular smooth muscle cells (SMCs) and by AGE receptors on endothelial cells, SMCs, and monocytes is highly enhanced and this, in turn, is centrally positioned to contribute to the pathogenesis of diabetic vascular complications especially coronary artery disease. The present review summarizes the up-to-date information on effects and mechanism of type 2 diabetes-associated coronary atherosclerosis induced by CML-LDL modification.

In vitro oxidized and glycated human low-density lipoprotein particles characterized by capillary zone electrophoresis

A simple capillary zone electrophoresis (CZE) method was used to determine native, in vitro Cu 2+ and glucose modified low-density lipoprotein (LDL) particles for four healthy subjects. The LDL electropherograms are highly reproducible with good precisions of effective mobility and peak area. The native LDL capillary electrophoresis (CE) profile shows a major peak with lower mobility and two minor peaks with higher mobilities. For three-hour Cu 2+ oxidation, one major peak with mobility close to that of the native major peak, and one minor peak with mobility extending to −47 × 10 −5 cm 2 V −1 s −1 appear. For eighteen-hour Cu 2+ oxidation, one major peak with mobility much higher than that of the native major peak appears. As the reaction time for LDL and Cu 2+ increases from 0 to 24 h, effective mobility of the LDL major peak increases, suggesting that LDL particles become more negatively charged and oxidized as the time increases. The in vitro glycated LDL particles are characterized by a major peak and two minor peaks. Mobility of the major peak is close to that of native major peak, but the second minor peak is much more negatively charged with mobility extending to −53 × 10 −5 cm 2 V −1 s −1. Native, oxidized and glycated LDL particles show distinctive differences in their CZE profiles. Agarose electrophoresis shows that the charge to mass ratios of native, three-hour Cu 2+ and glucose modified LDL particles are similar, but that of eighteen-hour Cu 2+ oxidized LDL particles is higher.

Open tubular capillary electrochromatography: a new technique for in situ enzymatic modification of low density lipoprotein particles and their protein-free derivatives

Electrochromatography with open tubular capillaries coated with human low density lipoprotein (LDL) particles and their protein-free derivatives was studied as a method for their in situ enzymatic modification. LDL particles as monolayers or their protein-free derivatives (lipid microemulsions) were coated on 50 microm i.d. capillaries, which resemble tiny human blood vessels in size, the arterioles. The immobilized LDL particles were exposed to sphingomyelinase, phospholipase A2 or alpha-chymotrypsin at 25 and 37 degrees C. The mobility of the electro-osmotic flow was employed as a surface charge indicator, and the retention factors of steroids were used as hydrophobicity indicators. Moreover, the capillaries were, for the first time, coated with lipid microemulsions containing either LDL-derived or commercial lipids, and the immobilized microemulsions were treated with sphingomyelinase in capillary. The results demonstrate that open tubular capillaries provide a good microreactor for the in situ modification of LDL particles and lipid microemulsions. The technique only requires extremely low quantities of LDL particles, lipid microemulsions, and enzymes. It allows quick and easy alteration of the reaction conditions, and the enzymes can be collected and reused. Asymmetrical flow field flow fractionation provides useful information on the size of the enzymatically modified LDL particles.

Decrease in pH Strongly Enhances Binding of Native, Proteolyzed, Lipolyzed, and Oxidized Low Density Lipoprotein Particles to Human Aortic Proteoglycans

Binding of low density lipoprotein (LDL) to proteoglycans and modification of LDL are key processes in atherogenesis. Recently, it has been demonstrated that during atherogenesis the extracellular pH of atherosclerotic lesions decreases. We have examined the effect of the decreased pH on the binding of LDL to human aortic proteoglycans. The binding of native, oxidized, proteolyzed (alpha-chymotrypsin-treated), or lipolyzed (sphingomyelinase- or phospholipase A(2)-treated) LDL particles to proteoglycans were measured in microtiter well assays at pH 5.5-7.5. We found that the lower the pH, the higher the amount of binding of LDL to proteoglycans. At the lowest pH tested (pH 5.5), the amounts of proteoglycan-bound native, proteolyzed, sphingomyelinase-, and phospholipase A(2)-treated LDL were 20-, 23-, 30-, and 37-fold higher, respectively, than at pH 7.5. Interestingly, although oxidized LDL failed to bind to proteoglycans at neutral pH, there was significant binding at acidic pH. Binding of native and modified LDL to proteoglycans at pH 5.5 was blocked by 1 m NaCl, indicating that at neutral pH LDL binds to proteoglycans via ionic interactions. Inhibition of this binding by acetylation and cyclohexanedione treatment of LDL showed that the positively charged amino acids of apolipoprotein B-100, lysine, and arginine, respectively, mediated the ionic interaction. Taken together, our results suggest that in areas of atherosclerotic arterial intima where the extracellular pH decreases, retention of LDL by proteoglycans is enhanced, leading to extracellular accumulation of LDL and progression of the disease.

Lipolytic enzymes in atherosclerosis as the potential target of inhibitors

It is at present generally accepted that three components—modification of lipoproteins, infection and inflammation play an important role in the evaluation of atherosclerotic lesions. Low density lipoprotein (LDL) can undergo modification and accumulate over time in the artery wall. Modified LDL particles share the ability to induce inflammatory functions of cells associated with atheroma. The present review will focus on the multifunction action of lipases and phospholipases in lipid and lipoprotein metabolism, and atherosclerosis. The development of highly specific lipolytic enzyme inhibitors not only for studying their kinetic effects but also for potential pharmacological application is discussed.

Neopterin and 7,8-Dihydroneopterin Interfere With Low Density Lipoprotein Oxidation Mediated by Peroxynitrite and/or Copper

Low density lipoprotein (LDL) oxidation within the artery wall likely represents a key event in the formation of atherosclerotic lesions. Oxidatively modified LDL particles exert chemotactic properties on macrophages, and the uncontrolled uptake of modified LDL by macrophages leads to the formation of lipid-loaded foam cells, a hallmark of early stage atherosclerosis. Human macrophages stimulated by interferon- n generate reactive oxygen species (ROS), neopterin, and 7,8-dihydroneopterin. Higher concentrations of neopterin were found in atherosclerosis, and earlier studies have provided evidence that these neopterin derivatives are able to interfere with reactive species. We therefore investigated whether they also modulate LDL oxidation mediated by Cu(II) and/or peroxynitrite (ONOO m ). By means of UV-absorption recording the formation of conjugated dienes in the course of lipid oxidation as well as by measuring the relative electrophoretic mobility of oxidized LDL, we found that neopterin is capable of enhancing ONOO m - as well as Cu(II)-mediated LDL oxidation, whereas 7,8-dihydroneopterin mainly protects LDL from oxidation. However, in case of Cu(II)-mediated LDL oxidation, an initial prooxidative effect of 7,8-dihydroneopterin could be observed. We hypothesize that 7,8-dihydroneopterin may chemically reduce Cu(II) to Cu(I) thereby increasing its oxidative capacity. After total reduction of Cu(II), excess 7,8-dihydroneopterin may block the oxidative potential of Cu(I) and thus decrease the oxidation of LDL. These findings confirm the general behavior of pteridines in redox processes and suggest an in vivo contribution to the process of LDL oxidation.

Tetradecylthioacetic acid reduces stenosis development after balloon angioplasty injury of rabbit iliac arteries

Background: tetradecylthioacetic acid (TTA) is a synthetic long-chain fatty acid analogue that inhibits the oxidative modification of low-density lipoprotein particles in vitro. We examined the influence of TTA on the arterial wall response after balloon angioplasty injury in a rabbit iliac model. Methods and results: 14 rabbits were randomized to receiving either TTA fatty acids 800 mg daily perorally (weight 3.6±0.1 kg) or to normal diet (weight 3.5±0.5 kg, P=NS). Angioplasty was performed via right carotidotomy on both iliac arteries using an oversized balloon catheter, the TTA group being pretreated for 3 weeks. After angioplasty, the lumen diameter was 2.37±0.18 versus 2.36±0.13 mm for the TTA and control groups, respectively ( P=NS). At 10 weeks follow-up angiography, minimal luminal diameter was 1.64±0.27 versus 1.13±0.52 mm for the TTA and control groups respectively ( P<0.05). Histomorphometry did not show significant differences in intimal hyperplasia between the two groups (maximal intimal thickness 0.22±0.04 versus 0.19±0.10 mm, P=NS and intimal area 0.32±0.12 versus 0.36±0.23 mm 2, P=NS for the TTA and the control groups, respectively). In the heart, the sum of the n-3 fatty acids was 8.9±2.7 in the TTA group versus 4.3±0.2 mol% in the control group ( P<0.05). The anti-inflammatory fatty acid index, calculated as (22:5 n-3+22:6 n-3+20:3 n-6)/20:4 n-6, was 0.76±0.10 vs. 0.25±0.03 for the TTA and control groups, respectively ( P<0.05). In vitro TTA (100 μM) reduced the proliferation of human smooth muscle cell by more than 50%. Conclusion: treatment with TTA is associated with positive arterial remodeling after angioplasty injury. The significance of the in vitro inhibition of human smooth muscle cell proliferation needs to be further elucidated.

Antibodies Against Oxidized LDL - Theory and Clinical Use

Modification of low density lipoprotein (LDL) particles due to oxidation, glycation and binding of advanced glycation end-products (AGEs) or malondialdehyde (MDA, a final product of lipid peroxidation) is considered most important in the process of atherogenesis. Oxidatively modified LDL are distinguished by another receptor type, which was discovered on the surface of macrophages and was called the scavenger receptor. Uncontrolled intake of LDL converts macrophages to foam cells; their accumulation under the vascular endothelium is considered as the first stage of atherosclerosis. Oxidation of LDL is a complex process taking place in both the extra- and intracellular space. At the end of this oxidative process, modified LDL particles show chemotactic, cytotoxic and immunogenic properties. Oxidized LDL express a large number of epitopes and cause production of polyclonal autoantibodies against these products, especially against apoB100 modified by MDA and 4-hydroxynonenal. IgoxLDL (antibodies against oxidized LDL) can be demonstrated either directly in intimal lesions or as a component of circulating immune complexes. IgoxLDL do not form a homogeneous group but a varied mixture of antibodies-isoantibodies caused by HDL and LDL polymorphism, antibodies against the lipid phase of LDL and antibodies against modified apoB100 of the immunoglobulin class IgA or IgG. Antibodies against oxLDL were found in many diseases other than atherosclerosis such as diabetes mellitus, renovascular syndrome, uremia, rheumatic fever, morbus Bechtjerev or lupus erythematodes. Newborns have practically the same levels of IgoxLDL as their mothers; however, these values did not differ from those in the healthy population of non-pregnant women of the same age. The decrease in IgoxLDL titer was very slow and lasted many months; that is why this parameter cannot be considered suitable for describing the rapid changes during oxidative stress of the organism. Positive correlation of IgoxLDL with antiphospholipids and other antibodies was repeatedly demonstrated; their determination can thus be used as a marker for the description of total production of autoantibodies in various diseases. The changes and correlations of IgoxLDL, anti-b-2-glycoprotein I IgG and antiphospholipid antibodies support the immunological link between thrombotic and atherosclerotic processes in the human body.

Abnormalities in lipoprotein metabolism in hemodialysis patients

Patients on chronic hemodialysis treatment are at elevated atherogenic risk, and dyslipidemia appears to be one of the major risk factors. However, most of these patients exhibit elevated serum triglycerides, whereas serum cholesterol and low-density lipoprotein (LDL) cholesterol levels are in the normal range. This study was therefore designed to examine the influence of hypertriglyceridemia under the condition of hemodialysis and diabetes mellitus on LDL metabolism. LDL was isolated from healthy controls, hypertriglyceridemic diabetic patients, and nondiabetic hemodialysis patients (N = 30, 10 in each group), which were separated into six subfractions by density gradient ultracentrifugation and were characterized concerning lipid/protein composition, degree of glycation, and oxidation. Uptake of 125I-labeled LDL was examined via LDL receptors of HepG2 cells and scavenger receptors of mouse peritoneal macrophages. In hemodialysis patients, serum triglycerides were significantly elevated, whereas cholesterol levels were within the normal range. Triglyceride enrichment occurred in the very low-density lipoprotein (VLDL) class and LDL class, and an accumulation of a highly atherogenic small dense LDL subfraction could be detected predominantly in patients with non-insulin-dependent diabetes mellitus. LDL of hemodialysis patients also contained elevated levels of lipid peroxidation products, which were even higher in diabetic patients. Alterations in composition, size, and configuration of LDL from diabetic and nondiabetic patients on hemodialysis impaired LDL receptor-mediated degradation and enhanced the uptake of these modified LDL particles via nonsaturable scavenger receptors. Diminished LDL receptor-mediated uptake of modified, triglyceride-rich, small dense LDL most likely leads to accumulation of these lipoproteins in vivo, favoring the development of atherosclerotic lesions. Future clinical studies must demonstrate whether patients will benefit from reducing these atherogenic particles by lipid-lowering intervention.

Sphingomyelinase Induces Aggregation and Fusion, but Phospholipase A2 Only Aggregation, of Low Density Lipoprotein (LDL) Particles: TWO DISTINCT MECHANISMS LEADING TO INCREASED BINDING STRENGTH OF LDL TO HUMAN AORTIC PROTEOGLYCANS

During atherogenesis, low density lipoprotein (LDL) particles bind to extracellular matrix proteoglycans in the arterial wall, become modified, and appear as aggregated and fused particles. Sphingomyelinase (SMase) and phospholipase A2 (PLA2) have been found in the arterial wall, and, moreover, lesional LDL shows signs of hydrolysis of both sphingomyelin and phosphatidylcholine. We have now studied the effects of these two lipolytic modifications on the aggregation and fusion of LDL particles by hydrolyzing the particles with Bacillus cereus SMase or bee venom PLA2. In addition, the binding strengths of the modified LDL to human aortic proteoglycans (PG) were analyzed on an affinity column. We found that SMase induced aggregation and fusion of LDL, but PLA2 induced only aggregation of the particles. In addition, the SMase-induced aggregation and fusion of LDL was promoted by pretreatment of LDL with PLA2. Determination of the binding strengths of the hydrolyzed LDL revealed that mere lipolysis of LDL without aggregation or fusion, either by SMase or PLA2, did not affect the binding of the particles to PG. Aggregation and fusion of lipolyzed LDL particles, however, increased their strength of binding to PG. Active lysine residues in apolipoprotein B-100 (apoB-100) appear to be involved in the binding of LDL to PG, and, in fact, quantitative 13C NMR analysis revealed that, in the fused LDL particles, the number of active lysine residues per apoB-100 moiety was increased. Moreover, aggregation and fusion of LDL increased the number of apoB-100 copies and, consequently, the number of active lysine residues per aggregate or fused particle. Our present findings therefore (i) show that treatment of LDL with SMase and PLA2 generates modified LDL particles, which then bind to human aortic PG with increased strength, and (ii) suggest that SMase- and PLA2-induced aggregation and fusion of LDL are potential mechanisms leading to focal retention of extracellular lipid in the arterial wall.

Preparation and characterization of immobilized phospholipase A 2 on chitosan beads for lowering serum cholesterol concentration

Phospholipase A 2 (PLA 2) from cobra venom, which can hydrolyze the S N2 ester bond of 1,2-diacylphosphatides, was immobilized by covalent binding to porous chitosan beads. Immobilization has to be carried out by using the carboxylic groups instead of the amine groups of the enzyme to get reasonable activity retention (higher than 50%). The effects of amount of activating reagent EDC and enzyme loading during the immobilization step were investigated. Since EDC could modify important Asp groups in the enzyme, the EDC/enzyme weight ratio should be less than 10. Although the activity retention of immobilized enzyme increased with enzyme/bead weight ratio, this ratio should be kept to a minimum at 1×10 −3 to optimize coupling yield of enzyme activity and reduce internal diffusion resistance. The kinetic properties and stability of the immobilized enzyme were determined. The immobilized PLA 2 was packed into a column to hydrolyze phospholipid in a circulating packed-bed reactor. The flow rate of the substrate solution should be set at 37.5 cm/min (superficial velocity) to eliminate external diffusion resistance, under which condition the column reactor could be reused up to 10 times with less than 20% loss of activity. Since enzymatic hydrolysis of phospholipid on low density lipoprotein (LDL) particle surface with PLA 2 could result in faster plasma clearance of the modified LDL particles, an in vitro bioreactor containing immobilized PLA 2 should be able to lower serum cholesterol concentration. A significant decrease in total serum cholesterol concentration in hypercholesterolemic rabbits was observed after 90-min treatment.

Effect of soybean phytoestrogen intake on low density lipoprotein oxidation resistance.

The oxidation of low density lipoproteins (LDLs) is thought to take place in the arterial intima when the particles have become isolated from circulating water-soluble antioxidants. We hypothesized that isoflavonoid antioxidants derived from soy could be incorporated into lipoproteins and possibly could protect them against oxidation, which is regarded as atherogenic. Six healthy volunteers received 3 soy bars [containing the isoflavonoid antioxidants genistein (12 mg) and daidzein (7 mg)] daily for 2 weeks. LDLs were isolated from blood drawn at the the end of a 2-week dietary baseline period, after 2 weeks on soy, and after discontinuation of soy. Large increases in plasma isoflavonoid levels occurred during soy feeding, but only minute amounts were stably associated with lipoproteins (less than 1% of plasma isoflavonoids in the LDL fraction). The LDLs were subjected to copper-mediated oxidation in vitro. Compared with off soy values, lag phases of LDL oxidation curves were prolonged by a mean of 20 min (P < 0.02) during soy intake, indicating a reduced susceptibility to oxidation. The results suggest that intake of soy-derived antioxidants, such as genistein and daidzein, may provide protection against oxidative modification of LDL. As only very small amounts of these substances were detected in purified LDL, modified LDL particles may have been produced in vivo by circulating isoflavonoids promoting resistance to oxidation ex vivo.

Proteolysis and Fusion of Low Density Lipoprotein Particles Strengthen Their Binding to Human Aortic Proteoglycans

Lipid droplets resembling those seen in the extracellular space of the arterial intima were generated in vitro when granule proteases of rat serosal mast cells degraded the apolipoprotein B-100 (apoB-100) component of granule-bound low density lipoprotein (LDL), and the particles fused on the granule surface (Paanenen, K., and Kovanen, P. T. (1994) J. Biol. Chem. 269, 2023-2031). Moreover, the binding of the fused particles to the heparin proteoglycan component of the granules was found to be strengthened. We have now treated LDL particles with alpha-chymotrypsin and examined the strength with which the proteolytically modified LDL binds to human aortic proteoglycans on an affinity column. We found that chymotryptic degradation of the LDL particles triggered particle fusion. The higher the degree of proteolytic degradation, the higher were the degree of fusion and the strength of binding to the aortic proteoglycans. Separation of the proteolyzed particles by size exclusion chromatography into two fractions, unfused and fused particles, and analysis of their binding strengths revealed that not only the fused but also the unfused proteolyzed particles bound more tightly to the proteoglycans than did the native LDL particles. To investigate the mechanism underlying this increase in binding strength, we attached [13C]dimethyl groups to the lysines and used NMR spectroscopy to quantify the active lysine residues of apoB-100, which are thought to be located in basic areas of apoB-100 and involved in binding of LDL to proteoglycans. Analysis of the 13C-labeled particles showed that, despite loss of apoB-100 fragments from the particles, the number of active lysine residues in the unfused proteolyzed particles had not decreased. In the fused proteolyzed particles, the number of active lysine residues was markedly increased. Thus, proteolytic fusion appears to increase the number of basic domains of apoB-100, which would explain the observed increase in the strength of binding of the modified LDL particles to arterial proteoglycans. Since the fused particles resemble the small lipid droplets found in the atherosclerotic arterial intima, this LDL modification offers a plausible mechanism for the focal accumulation of lipid droplets in the extracellular proteoglycan matrix during atherogenesis.

Interaction between zinc(II)-phthalocyanine-containing liposomes and human low density lipoprotein

The interaction of human low density lipoprotein (LDL) and small unilamellar liposomes containing the photosensitiser zinc(II)—phthalocyanine (ZnPc) was studied in vitro to determine if ZnPc could be directly incorporated into the lipoprotein in the absence of other serum components. Incubation of LDL with increasing concentrations of liposomes resulted in a progressive increase in the net negative charge of LDL as determined by agarose gel electrophoresis and both ZnPc and liposomal phospholipid were incorporated into the modified LDL particles. Gel chromatography experiments indicated an increase in the molecular mass of modified LDL and immunoaffinity chromatography provided evidence that apoprotein B epitopes on modified LDL were unable to bind to antibody. The study indicated that the liposomal components could be selectively incorporated into LDL by a process that did not appear to involve either aggregation or fusion of particles.

Effect of n−3 fatty acid‐rich fish oil supplementation on the oxidation of low density lipoproteins

This study was aimed at determining the effect of fish oil supplementation on copper-catalyzed oxidation of low density lipoproteins (LDL) from nine hypertriglyceridemic human subjects. A rapid headspace gas chromatographic method was used to measure the volatile oxidation products from LDL. Propanal and hexanal were the major volatile products formed in the oxidation of n-3 and n-6 polyunsaturated fatty acids (PUFA), respectively. Fish oil supplementation resulted in a significant increase in propanal formation from 3.7 to 13.4 nmol/mL LDL (P < 0.01); it also resulted in small decreases in pentanal formation from 14.7 to 11.4 nmol/mL LDL and in hexanal formation from 138 to 108 nmol/mL LDL (P < 0.05). The changes in peroxidation products paralleled the changes in LDL composition, which showed a significant increase in n-3 PUFA from 3.2 to 14.6% (P < 0.01) and a decrease in n-6 PUFA from 43.7 to 35.0% (P < 0.05). Propanal formation was highly and significantly correlated with n-3 PUFA content (r = 0.950, P < 0.001). Since total volatiles remained unchanged, this indicated that the two groups of LDL samples did not differ in overall oxidative susceptibility. Although fish oil intake did not alter the oxidative susceptibility of LDL, the chemically modified LDL particles generated a distinct pattern of volatile oxidation products that reflected changes in their fatty acid composition.