Low-density Lipoprotein Samples Research Articles

The study of structural accessibility of free thiol groups in human low-density lipoproteins

The experimental evidence for the apolipoprotein B100 (apoB) domain structuring in low-density lipoprotein (LDL) was investigated focusing on the accessibility of free thiol groups. Three different spectroscopic methods were combined with the biochemical perturbations of LDL particle. The spectrophotometric method was adapted for LDL and the exposure of free thiols was analyzed in the native LDL and LDL exposed to sequential denaturation. The results indicate that 24-h denaturation does not expose all free thiols in LDL. Using thiol-specific spin labeling and electron paramagnetic resonance spectroscopy (EPR), different populations of labeled thiols were resolved. The comparison of the EPR spectra of native LDL and LDL with selectively blocked thiol groups revealed significant difference in the respective hyperfine splittings. The phenomenon can arise due to different polarity and/or mobility of the nitroxides in the microenvironments of spin label binding sites of these two LDL samples. The results indicate that nine thiol groups in apoB are distributed in different domains of LDL: two are more exposed, two are buried deeply in the lipid matrix of the particle and the rest are located in hydrophobic parts of this extremely complex protein–lipid assembly. These observations provide experimental support for the emerging theoretical models of apoB.

Structural changes of low density lipoproteins with Cu2+ and glucose induced oxidation.

The compositional and structural changes of lipids and apolipoproteins during in vitro oxidation of low density lipoprotein (LDL) are investigated in this study by IR spectroscopy. For comparison, LDL samples containing either copper or glucose at physiological or pathological concentrations are considered in order to know the separate affects of these chemical factors on LDL oxidation. The results show that the initial steps of lipid oxidation proceed through hydrogen atom loss from methylene groups, as well as loss of cholesteryl ester molecules, and later a recovering of carbonyl compounds resulting from aldehyde formation that generally occurs in autooxidation processes. Lipid oxidation is induced by copper ions, and glucose enhances metal ion induced LDL oxidation as determined by conjugated diene formation. As to the protein conformational changes, IR spectroscopy reveals for the first time that LDL oxidation involves formation of beta-sheet structures, these being more abundant in LDL samples with pathological concentrations of glucose or copper. Consequently, the LDL structural changes may contribute to the recognition of oxidized LDL particles by scavenger receptors.

A Suggested Method for the Prediction of the Oxidation Resistance of Low Density Lipoprotein by Determination of the Lag Time

A simple clinical blood test, which measures the total antioxidant status of Low Density Lipoprotein (LDL) and therefore its vulnerability to oxidative stress is suggested, and as a first stage this was tried on a small sample of eight healthy adult males. The body's natural defence and repair systems try to handle all free radicals, but these systems are not hundred percent effective. Thereby, the role of antioxidants (particularly natural antioxidants) becomes evident and vitamins such as Vitamin C, Vitamin E, and compounds like beta-carotene are under especially extensive study. Vitamin C is the most abundant water-soluble antioxidant acting in extracellular fluid, while Vitamin E is the most abundant fat-soluble antioxidant, and it protects the polyunsaturated fatty acids within the LDL from oxidation and helps to prevent the process of atherogenesis. The determination of 'lag-phase' during continuous monitoring of oxidation of LDL in vitro is a convenient and objective procedure for determining the susceptibility of LDL from different donors towards oxidation as well as of pro- and anti-oxidants. The Lag-time for LDL samples, obtained from eight healthy adult males was found to be between 40-50 minutes. The measurement of this Lag-phase could be a highly promising routine method for measuring the total antioxidant status of LDL.

Binding of Low Density Lipoproteins to Lipoprotein Lipase Is Dependent on Lipids but Not on Apolipoprotein B

Lipoprotein lipase (LPL) efficiently mediates the binding of lipoprotein particles to lipoprotein receptors and to proteoglycans at cell surfaces and in the extracellular matrix. It has been proposed that LPL increases the retention of atherogenic lipoproteins in the vessel wall and mediates the uptake of lipoproteins in cells, thereby promoting lipid accumulation and plaque formation. We investigated the interaction between LPL and low density lipoproteins (LDLs) with special reference to the protein-protein interaction between LPL and apolipoprotein B (apoB). Chemical modification of lysines and arginines in apoB or mutation of its main proteoglycan binding site did not abolish the interaction of LDL with LPL as shown by surface plasmon resonance (SPR) and by experiments with THP-I macrophages. Recombinant LDL with either apoB100 or apoB48 bound with similar affinity. In contrast, partial delipidation of LDL markedly decreased binding to LPL. In cell culture experiments, phosphatidylcholine-containing liposomes competed efficiently with LDL for binding to LPL. Each LDL particle bound several (up to 15) LPL dimers as determined by SPR and by experiments with THP-I macrophages. A recombinant NH(2)-terminal fragment of apoB (apoB17) bound with low affinity to LPL as shown by SPR, but this interaction was completely abolished by partial delipidation of apoB17. We conclude that the LPL-apoB interaction is not significant in bridging LDL to cell surfaces and matrix components; the main interaction is between LPL and the LDL lipids.

Characterization of modified low density lipoprotein subfractions by capillary isotachophoresis.

Oxidative modification of low density lipoproteins (LDLs) is an important pathogenetic factor in atherosclerosis. The various steps in oxidative modifications of LDL can be monitored using different methodologies with varying degrees of complexity. In this study, we propose capillary isotachophoresis (CITP) as a suitable tool to detect and measure the degree of oxidation of LDL. LDL was isolated from pooled plasma of healthy volunteers by sequential ultracentrifugation, and oxidation was performed in vitro as well as in cell culture experiments. Native LDL and oxidatively modified LDL were characterized by apo B-100 fluorescence and conjugated diene formation. Samples were separated by CITP combined with sudan black B staining. To underline the inherent advantages of this approach, CITP was compared with classical lipoprotein electrophoresis using agarose gel. We demonstrate the CITP method to be highly sensitive, as changes in peak area of the separated LDL subfractions were detected after only 2 h of oxidation. The leading LDL peaks increased, while the terminating LDL peaks decreased in parallel throughout the duration of oxidation. The LDL samples, oxidized for 4-24 h, also exhibited an increased migration velocity of the fractions. In summary, we present the first study investigating LDL-subfractions separated by CITP and the alterations of these LDL-subfractions after gradual in vitro oxidation and after oxidative modification by monocyte-derived macrophages and vascular smooth muscle cells.

Lipolytic enzyme effect on small low-density lipoprotein particles in women treated with estrogen

Objective: To test whether hydrolysis of low-density lipoprotein (LDL) triglyceride by lipolytic enzymes decreases the size of LDL particles in women treated with estrogen replacement. Methods: Fifteen postmenopausal women received 0.625 mg conjugated equine estrogens daily for 3 months. Plasma concentrations of total cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol were measured before and after therapy. We also assayed levels of total, free, and esterified cholesterol, triglyceride, and protein in LDL. Plasma samples were incubated at 37C for 24 hours and LDL fractions were isolated by ultracentrifugation. After LDL samples were further incubated with or without lipoprotein lipase (500, 700, and 1000 ng/mL) at 37C for 24 hours, LDL triglyceride, LDL protein, and the diameter of LDL particles were measured. Results: Estrogen decreased total cholesterol and increased triglyceride and HDL cholesterol in plasma. Estrogen treatment decreased the ratio of cholesteryl ester/protein, whereas the ratio of triglyceride/protein increased. Estrogen decreased LDL particle diameter. Incubation of plasma increased the ratio of LDL triglyceride/protein from 0.40 ± 0.14 to 0.48 ± 0.15 ( P < .05) and decreased the ratio of LDL cholesteryl ester/protein from 1.17 ± 0.25 to 1.09 ± 0.22 ( P < .05), but LDL particle diameter did not change. Incubation of LDL with lipoprotein lipase reduced the LDL triglyceride/protein ratio, and decreased the diameter of LDL particles from 25.61 ± 0.87 nm to 24.89 ± 0.88 nm (500 ng/mL, P < .05), 24.62 ± 1.20 nm (700 ng/mL, P < .05), and 24.67 ± 1.19 nm (1000 ng/mL, P < .05). Conclusion: In women treated with estrogen, hydrolysis of triglyceride in LDL particles might be accompanied by reduced particle size.

A short-run new analytical ultracentrifugal micromethod for determining low-density lipoprotein sub-fractions using Schlieren refractometry

We have developed a new analytical ultracentrifugal micromethod for the determination of serum low-density lipoprotein (LDL) subclasses directly from ultracentrifugal Schlieren scans. We have used special software for the analysis of this type of single-spin density-gradient ultracentrifugation. The flotation of LDL patterns was obtained by underlayering a physiological salt solution with serum or isolated lipoprotein fractions raised to a density of 1.3 g/mL in the spinning ultracentrifugation capillary band-forming cell. The repeated analysis of Schlieren curves of the same sample from 10 to 100 microL in the 60-100 min full-speed interval time resulted in quite reproducible results. We obtained quantitative results by measuring the Schlieren areas between the sample curves and the reference baseline curve by using computerised numerical and graphic techniques. The decomposition of the integrated curve was carried out using a nonlinear regression program followed by deconvolution algorithm analysis in order to determine the parameters of the composing Gaussian subclasses. The LDL particle concentrations were calculated from the area under the integral of the Gaussian curve using a calibration data constant. The flotation range of the LDL Schlieren curves in the cell was identified with serum from which LDL had been removed by means of precipitation reagents and with centrifugation of isolated LDL aliquots. With this technique, we measured the concentration of LDL and analysed its polydispersity without the need for preceding sequential isolation of the LDL. On the basis of the Schlieren curves, the LDL samples were either physically paucidisperse, having a symmetrical peak within a narrow density range, or were polydisperse, showing an asymmetrical pattern distributed over a broader density region. The described method proved to be useful for a clear and immediate visual presentation of the concentration values of the LDL and for the identification of the heterogeneity of LDL variants without the need for the preparative isolation of that density class.

Direct Evidence for apo B-100-Mediated Copper Reduction: Studies with Purified apo B-100 and Detection of Tryptophanyl Radicals

Copper binding to apolipoprotein B-100 (apo B-100) and its reduction by endogenous components of low-density lipoprotein (LDL) represent critical steps in copper-mediated LDL oxidation, where cuprous ion (Cu(I)) generated from cupric ion (Cu(II)) reduction is the real trigger for lipid peroxidation. Although the copper-reducing capacity of the lipid components of LDL has been studied extensively, we developed a model to specifically analyze the potential copper reducing activity of its protein moiety (apo B-100). Apo B-100 was isolated after solubilization and extraction from size exclusion-HPLC purified LDL. We obtained, for the first time, direct evidence for apo B-100-mediated copper reduction in a process that involves protein-derived radical formation. Kinetics of copper reduction by isolated apo B-100 was different from that of LDL, mainly because apo B-100 showed a single phase-exponential kinetic, instead of the already described biphasic kinetics for LDL (namely α-tocopherol-dependent and independent phases). While at early time points, the LDL copper reducing activity was higher due to the presence of α-tocopherol, at longer time points kinetics of copper reduction was similar in both LDL and apo B-100 samples. Electron paramagnetic resonance studies of either LDL or apo B-100 incubated with Cu(II), in the presence of the spin trap 2-methyl-2-nitroso propane (MNP), indicated the formation of protein-tryptophanyl radicals. Our results supports that apo B-100 plays a critical role in copper-dependent LDL oxidation, due to its lipid-independent-copper reductive ability.

Oxidation of Linoleic Acid in Low-Density Lipoprotein: An Important Event in Atherogenesis.

Contrary to earlier views the main oxidation products of low-density lipoprotein (LDL) are derived from linoleic acid and not arachidonic acid, as determined by GC/MS investigations of the in vitro oxidation of LDL samples. A similar product spectrum, in which epoxyhydroxyoctadecenoic acids such as 1 and 2 have been identified for the first time, is obtained from minimally oxidized (that is, aged) LDL. Since this is still recognized by the LDL receptor, it is concluded that toxic oxidation products are introduced in endothelial cells in vivo and cause damage there.

Is glycation of low density lipoproteins in patients with Type 2 diabetes mellitus a LDL pre-oxidative condition?

The study aimed to evaluate whether low density lipoprotein (LDL) in diabetic patients is more glycated and susceptible to oxidation than in non-diabetic subjects and investigated the hypothesis that LDL glycation is associated with an increased plasma concentration of LDL- (a circulating electronegatively charged LDL), proposed as an index of in vivo oxidation. LDL glycation was measured by a competitive enzyme immunoadsorbent assay, using a monoclonal antibody against glycated apoB in 24 Type 2 diabetic patients and 12 healthy controls. LDL- was separated by ion-exchange HPLC in LDL samples obtained after sequential preparative ultracentrifugation (density range 1.019-1.063). In vitro LDL susceptibility to oxidation was evaluated by following the kinetics of conjugated diene formation and by measuring the lag-phase time in the presence of copper (Cu2+) ions. The percentages of glycated apoB (3.33+/-2.54% vs. 1.24+/-0.71%) and of LDL- (3.88+/-1.49% vs. 2.34+/-1.03%) in total LDL were significantly higher in diabetic patients (P<0.01 for both). LDL- was positively correlated with glycated apoB (r = 0.68, P<0.001). LDL isolated from Type 2 diabetic patients showed a significant decrease (P<0.001) in the resistance to oxidative stress, as indicated by the shorter lag-phase time (91+/-12.6 vs. 120+/-24.5 min). The lag-phase time was inversely correlated with glycated apoB (r = -0.65, P<0.001) and LDL- concentrations (r = -0.69, P<0.001). In this population of Type 2 diabetic patients, LDL were more glycated, more susceptible to in vitro oxidation and had a higher percentage of electronegative LDL. The glycation of apoB is proposed to be associated with a significative increase of in vivo and in vitro LDL oxidation.

Estradiol as an antioxidant: incompatible with its physiological concentrations and function

Estradiol has been documented to inhibit the oxidation of low density lipoprotein (LDL). We show that physiological concentrations of estradiol do not inhibit the oxidation of LDL by copper. LDL samples isolated from a) premenopausal and postmenopausal women and from b) women at different time periods during their menstrual cycle, who differ vastly in plasma estradiol levels, were also oxidized at the same rates by copper. In contrast, LDL samples isolated from c) women who were hyperstimulated during in vitro fertilization (IVF), with estradiol concentrations above 2000 pg/ml, were resistant to oxidation by copper. However, these LDL samples were also oxidized at a higher rate by peroxidases. More importantly, subjects with high estradiol levels also showed an increase in myeloperoxidase (MPO) protein in the plasma. Based on these results, we conclude that at physiologic concentrations, it is unlikely that estradiol could act as an antioxidant. In fact, the ability of estradiol to induce MPO and become a prooxidant might instead suggest that MPO-mediated oxidative clearance of LDL from plasma by liver might favorably influence the outcome of atherosclerosis. —Santanam, N., R. Shern-Brewer, R. McClatchey, P. Z. Castellano, A. A. Murphy, S. Voelkel, and S. Parthasarathy. Estradiol as an antioxidant: incompatible with its physiological concentrations and function. J. Lipid Res. 1998. 39: 2111–2118.

Optimization of the assay for sialic acid determination in low density lipoprotein

Sialic acid level in blood plasma and circulating glycoproteins is considered to be a marker for a number of pathologic conditions, including atherosclerosis, cancer, etc. The precise measurement of sialic acid level is an important laboratory procedure to allow correct interpretation of results. Colorimetric methods commonly used for the measurement of sialic acid are not highly specific, as interfering substances may alter the results. Among these, malondialdehyde and other aldehydes play the decisive role. In the circulation, aldehydes are commonly produced during lipid peroxidation in the lipid core of lipoprotein particles, especially low density lipoprotein (LDL). To establish the impairment to the sialic acid determination in LDL introduced by interfering substances, the optimized assay based on Warren's traditional method was developed and tested in 606 LDL samples. The optimization implies the comparison of color developed using the standard Warren procedure with that due to contaminating agents, mainly thiobarbituric acid-reactive substances (TBARS). In LDL stored at 4°C, the estimates obtained by the modified procedure were 41.5“% or 30.1 nmol/mg lower, on average, compared to the standard procedure (n = 45, P < 0.0001). Even in LDL stored at −70°C, sialic acid estimates obtained by the modified procedure were 6.6"% or 3.6 nmol/mg lower, on average, compared to the standard measurement (n = 561, P < 0.005). Thus, the modified procedure avoids significant distortion of the measurement induced by the presence of interfering agents. —Sobenin, I. A., V. V. Tertov, and A. N. Orekhov. Optimization of the assay for sialic acid determination in low density lipoprotein. J. Lipid Res. 1998. 39: 2293–2299.

Potent Inhibitory Activities of Hydrophobic aci-Reductones (2-Hydroxytetronic Acid Analogs) Against Membrane and Human Low-Density Lipoprotein Oxidation

The effects of selected aci-reductones, which are hydrophobic ascorbate-related analogs including 4-chlorophenyl-2-hydroxytetronic acid (Cpd A), 4-(1,1′-biphenyl)-2-hydroxytetronic acid (Cpd B), and 4-(4′-chloro-1,1′-biphenyl)-2-hydroxytetronic acid (Cpd C), on membrane and low density lipoprotein (LDL) oxidation were assessed. Hepatic microsomal lipid peroxidation was induced by the ascorbate + Fe(II) chemical system. All three agents inhibited membrane lipid peroxidation in a concentration-dependent manner with the order of potency: Trolox (vitamin E) ≤ Cpd A ≪ Cpd B < Cpd C; based on the ec 50 values, Cpd B and Cpd C were 11- and 19-fold, respectively, more potent than Trolox. In contrast to ascorbic acid, all three agents did not display any membrane prooxidative effect in the presence of iron. When human LDL was incubated with 10 μM of Cu(II), LDL oxidation, determined by the formation of thiobarbituric acid reactive substances, followed a typical sigmoidal curve with an initial lag phase. Preincubation of the LDL samples with low micromolar concentrations (1 and 3 μM) of each agents for 30 min before the addition of copper resulted in significant delays of the lag time of LDL oxidation, and the effectiveness of Cpd B and Cpd C was more prominent than that mediated by either Trolox or probucol. Since clinical evidence strongly supports the hypothesis that atherogenesis is initiated by LDL oxidation, the results suggest that these aryl tetronic acid analogs may serve as promising candidates for future therapeutic use as anti-atherogenic agents.

Characterization and quantitation of apolipoprotein B-100 by capillary electrophoresis

The interaction of low density lipoproteins (LDL) with different surfactants was studied by capillary electrophoresis (CE) and sucrose density gradient ultracentrifugation as part of developing a method for quantitation of apoB-100 in serum. A mixture of surfactants consisting of 70% sodium dodecyl sulfate (SDS), 25% sodium myristyl sulfate, and 5% sodium cetyl sulfate was found to delipidate LDL particles more effectively than pure SDS or sodium decyl sulfate. The delipidation products of LDL [apolipoprotein B-100 (apoB-100) and lipids] were resolved as two distinct peaks by CE when using a 3.5 mm 70% SDS mixture, 20% (v/v) acetonitrile, 50 mm sodium borate, pH 9.1 buffer. This CE method was also used to characterize apoB-100 derived from samples of lipoprotein [a] and very low density lipoproteins (VLDL). A CE-based quantitation method for apoB-100 was developed utilizing the observed linear relationship between apoB-100 concentration and its corrected 214 nm absorbance peak area measured on-line by CE. Concentration values of apoB-100 in LDL and VLDL samples were determined by CE and found to be accurate when compared to values obtained by immunoturbidimetric analysis and the Lowry method. Capillary electrophoresis can be used as a precise, accurate, and specific on-line method for the qualitative and quantitative analysis of the apoB-100 component of VLDL and LDL-related lipoproteins.—Cruzado, I. D., S. L. Cockrill, C. J. McNeal, and R. D. Macfarlane. Characterization and quantitation of apolipoprotein B-100 by capillary electrophoresis. J. Lipid Res. 1998. 39: 205–217.

Addition of lutein, lycopene, or β-carotene to LDL or serum in vitro: Effects on carotenoid distribution, LDL composition, and LDL oxidation

Carotenoids are dietary antioxidants transported with plasma lipoproteins, primarily low-density lipoprotein (LDL). In this study in vitro methods were used to increase the amounts of specific, individual carotenoids in LDL. By addition of carotenoid to isolated LDL or to serum, followed by (re)isolation of the lipoproteins, samples of LDL were enriched 4- to 150-fold with lutein, 2- to 15-fold with lycopene, or 3- to 25-fold with β-carotene. Enrichment with specific carotenoids was achieved without affecting the electrophoretic mobility of the lipoprotein, its cholesterol to protein ratio, or the levels of other cartenoids or α-tocopherol. The distributions among lipoproteins of carotenoid added to serum were similar, but not identical, to the distributions of the endogenous carotenoids. In particular, for added lutein, a greater proportion was found in HDL, and for added β-carotene, more was found in very low-density lipoprotein (VLDL). We then studied the effect of enriching LDL with specific carotenoids on its susceptibility to oxidation by copper ions. Lutein, β-cryptoxanthin, lycopene, and β-carotene, the four major plasma carotenoids, and α-tocopherol were destroyed before the formation of lipid peroxidation products. The rates of destruction of the individual carotenoids differed; lycopene was destroyed most rapidly and lutein most slowly. Upon oxidation of β-carotene-enriched LDL, the rates of destruction of β-carotene, lycopene, and lutein were slowed and the lag times before the initiation of lipid peroxidation increased from 19 to 65 min. Neither effect was observed in LDL enriched with lutein or lycopene. Thus, β-carotene was unique among the carotenoids studied in having a small, but significant effect on LDL oxidation in vitro.

The effects of gliclazide and other sulfonylureas on low-density lipoprotein oxidation in vitro

Diabetes is associated with increased oxidant stress. This may contribute to the development of diabetic macrovascular complications through increased oxidation of low-density lipoprotein (LDL), which is thought to be a crucial step in the development of atherosclerosis. The sulfonylurea gliclazide has been shown to have free radical—scavenging activity in vitro, but its effects on LDL oxidation, and these effects of other sulfonylureas, are unknown. To investigate this, we studied the effects of in vitro supplementation with gliclazide 1 μmol/L on copper-induced oxidation of LDL isolated from 20 control subjects and 22 type II diabetic patients. The effects of 1 μmol/L vitamin C, a known water-soluble antioxidant, were studied simultaneously. The resistance to oxidation, expressed as the lag time between the addition of copper and commencement of oxidation, was significantly increased by both gliclazide and vitamin C, and the effect was similar for LDL from diabetic and control subjects. The baseline oxidation lag time was 63.4 ± 2.1 minutes, and increased to 108 ± 4.4 minutes with gliclazide and 88.7 ± 5.6 minutes with vitamin C ( P = .0001, baseline v either treatment). The increase in lag time with gliclazide of 70% ± 3% was greater than the 30% ± 5% increase with vitamin C ( P < .0005). In a separate experiment, LDL isolated from eight control and 10 diabetic subjects was supplemented with 1 μmol/L gliclazide, glibenclamide, glipizide, and tolbutamide. For each LDL sample, all drugs were studied simultaneously and the oxidation lag time was compared against that of untreated LDL. Gliclazide increased the lag time from 53.7 ± 2.4 minutes to 108.4 ± 4.5 minutes ( P = .0001). None of the other sulfonylureas had any effect on lag time. These findings demonstrate that gliclazide is an effective inhibitor of in vitro LDL oxidation, and in this respect, it is more potent on a molar basis than vitamin C. This antioxidant property of gliclazide was not shared by the other sulfonylureas studied.

High fat, high cholesterol diets alter low density lipoprotein size and binding affinity in monkeys

The purpose of this study was to examine the effects of various dietary fats on low density lipoprotein (LDL) binding in an in vitro system where receptor number is not regulated. Cynomolgus monkeys were fed diets containing 37% of energy from fat, with various degrees of saturation, and 0.4 mg/kcal cholesterol or low-fat (13% of energy), low cholesterol (0.03 mg/kcal) chow. Plasma LDL was isolated after 16 weeks. The fatty acid composition of LDL showed enrichment corresponding to the dietary fats consumed, and the high fat, high cholesterol diets produced marked hypercholesterolemia compared to chow feeding. Of those fed the high fat diets, monkeys fed the fish oil diet had the highest LDL cholesterol concentrations, 13.25±0.77 mmol/l, while those fed the safflower oil diet had the lowest, 7.51±3.31. LDL from chow fed monkeys had the lowest binding affinity; the Kd was 26.2±8.7 μg/ml, nearly twice that of the high fat diets (P=0.003). No significant differences in binding were found between the different high fat diets, although there was a trend toward lower affinity in the diets enriched in polyunsaturated fat. LDL size was affected by diet with chow fed monkeys having the smallest average LDL, 259.3±1.7 Å compared to the other groups (P=0.03). Monkeys fed the fish oil diet tended to have smaller LDL, but this was not significantly different from the other high fat diets. Binding affinity was correlated with LDL size, r=0.54, P<0.01. LDL composition, as measured by apo B/cholesterol ratio, was altered by feeding a high fat, high cholesterol diet. The ratio was reduced in the LDL samples from monkeys fed the high fat diets compared to those fed chow, but this ratio was not significantly correlated with binding. Thus, it appears that increasing dietary fat and cholesterol intake increases LDL size and binding affinity, such that LDL metabolism may be altered independently from effects on receptor number; the type of dietary fat does not seem to influence this process when fat and cholesterol content is very high.

Oxidative modification of low density lipoprotein in normal and hyperlipidemic patients: effect of lysophosphatidylcholine composition on vascular relaxation

The elevated level of plasma low density lipoprotein (LDL) in hyperlipidemic patients is an important risk factor for the production of atherosclerosis. Plasma LDL must be modified before it can produce an impairment of endothelium-dependent relaxation in aortic rings or enhancement of uptake by macrophages. The dramatic increase in lysophosphatidylcholine (lysoPC) content in oxidatively modified LDL has been touted as an important biochemical factor for the impairment of endothelium-dependent relaxation. The present study was designed to examine the lysoPC composition of oxidized LDL samples from normal and hyperlipidemic subjects, and their effects on the impairment of endothelium-dependent relaxation. Oxidatively modified LDL from hyperlipidemic patients contained a slightly higher level (17%) of lysoPC, but produced a disproportionately greater impairment of endothelium-dependent relaxation than that from normal subjects. As lysoPC is composed of many molecular species, its composition in oxidized LDL samples was analyzed. In hyperlipidemic patients, lysoPC samples were found to contain a higher proportion of long-chain acyl groups. Subsequent studies revealed that only long-chain lysoPC (C > 16:0) were effective in impairing endothelium-dependent relaxation. Experimental loading of oxidized LDL from normal subjects with long chain lysoPC to mimic levels observed in oxidized LDL from hyperlipidemic patients resulted in further impairment of endothelium-dependent relaxation. We conclude that the greater proportion of long-chain lysoPC found in the oxidized LDL of hyperlipidemic subjects is responsible for the increased impairment of endothelium-dependent vascular relaxation. We propose that the high level of LDL found in the plasma of hyperlipidemic patients, coupled with its enhanced ability to generate long chain species of lysoPC during oxidative modification, are important factors for the development of atherosclerosis in these patients.

A Comparison of the Kinetics of Low-Density Lipoprotein Oxidation Initiated by Copper or by Azobis(2-Amidinopropane)

This article describes the kinetics of low density lipoprotein (LDL) oxidation catalyzed by azobis(2-amidinopropane)·dihydrochloride, ABAP, or by copper. The LDLs were isolated from nonhuman primates fed diets enriched in one of three types of fatty acids: saturated fatty acids, monounsaturated fatty acids, predominantly, oleic acid, or polyunsaturated fatty acids, predominantly linoleic acid. Oxidation was followed by monitoring the formation of conjugated diene hydroperoxides from polyunsaturated fatty acids (PUFA). For both copper and ABAP-initiated oxidation, the rate of LDL oxidation depended on the concentrations of initiator, PUFA, and LDL. Except for the dependence on PUFA concentration the rate of LDL oxidation was not directly influenced by the fatty acid composition of the LDL particle. The two initiators had very different dependence on initiator concentration. Because LDL particles are essentially small, lipid-rich droplets, the kinetic descriptions of LDL oxidation assumed: (1), that there was only one chain per particle, and (2) that the radical chain was terminated when a second radical either entered or was formed in the particle. When two LDL samples having very different lag times were mixed, the oxidation profile was bimodal. This finding demonstrated that the oxidation of native LDL particles was independent of the oxidation state of the other native LDL particles in solution, i.e., LDL particles do not rapidly exchange radicals, for example, hydroperoxyl radicals. Oxidation initiated by ABAP was proportional to [ABAP] 0.5, suggesting that hydroperoxyl radical recombination between the lipid hydroperoxyl radical and the ABAP-hydroperoxyl radical was the chain-terminating step. The reciprocal of the rate of copper oxidation was linearly related to the reciprocal copper concentration, demonstrating that the binding of copper to LDL was necessary to initiate oxidation. This binding constant showed considerable variability among LDL samples. The kinetic descriptions of LDL oxidation reflect the differences in the mechanisms of initiation and termination.

Molecular weight determination of lipoprotein(a) [Lp(a)] in solutions containing either NaBr or D2O: relevance to the number of apolipoprotein(a) subunits in Lp(a).

Molecular weight determination of low-density lipoprotein (LDL) is usually performed in solutions containing high concentrations of salt (up to 13.4 M NaBr) by sedimentation velocity and diffusion experiments, because it does not preferentially bind salt or water. Considering that lipoprotein(a) [Lp(a)] is structurally similar to LDL, differing only by the presence of Apo(a), the molecular weight, M, of Lp(a) has also been measured in solutions containing high concentrations of NaBr. We questioned the suitability of this practice by comparing the apparent molecular weight, Mapp, and partial volume, phi', of Lp(a) determined by sedimentation and flotation equilibrium in a three-component system containing NaBr with the analogous parameters, M and partial specific volume, v, determined in a two-component system containing D2O. LDL served as a control. In agreement with previous findings obtained with different methods, our results indicate no significant differences in M and v of four different LDL samples and apparently no significant preferential binding of solvent components. In contrast, values of Mapp and phi' of Lp(a) evaluated in NaBr are significantly greater than M and v. Preferential binding of solvent components appeared to be a function of Apo(a) mass or the number of kringle IV domains, as expressed by increasing percentage differences between the two sets of parameters, ranging from 4 to 13% in M and 0.2 to 0.5% in v of Lp(a) species having Apo(a) with 15-27 kringle IV domains. Furthermore, our results indicate that the variable Apo(a) kringle IV domains are more involved in this process than the constant domain of Apo(a). These findings indicate that the Lp(a) molecular weight should be determined in D2O and that high concentrations of NaBr should be avoided as their use would lead to overestimated molecular weights and partial specific volumes. Application of this method to the question of how much Apo(a) is released upon the reduction of Lp(a) led to the conclusion that Lp(a) contains only one Apo(a) molecule.