Human Plasma Low Density Lipoproteins Research Articles

The anti-ischemic and antioxidant activity of the pharmacological agonist of galanin receptor GalR2 and carnosine in in vitro and in vivo model systems

Antioxidant and anti-ischemic properties of the pharmacological agonist of galanin receptor GalR2 WTLNSAGYLLGPβAH (Gal) and its C-terminal fragment, dipeptide carnosine (βAH), were studied in the model of regional ischemia and reperfusion of the rat heart in vivo in the dose range of 0.5-5.0 mg/kg and Cu²⁺-induced free radical oxidation of low density lipoproteins (LDL) of human plasma in vitro for peptide concentrations of 0.01 mM and 0.1 mM. Gal was obtained by automatic solid phase synthesis using the Fmoc methodology; its structure was characterized by 1H-NMR spectroscopy and MALDI-TOF mass spectrometry. Intravenous administration of the optimal dose of Gal (1 mg/kg) to rats after ischemia was more effective than carnosine in reducing of the myocardial infarct size and the activity of creatine kinase-MB and lactate dehydrogenase in blood plasma at the end of reperfusion. It also improved the metabolic state of the reperfused myocardium and reduced the formation of peroxidation products during reperfusion. Gal reduced more effectively the formation of adducts of hydroxyl radicals in the interstitium of the area at risk (AAR) of the rat heart than carnosine. Carnosine at a dose of 1 mg/kg more effectively increased the activity of catalase and glutathione peroxidase in the AAR by the end of reperfusion compared to Gal. In a model of Cu²⁺-initiated oxidation of human plasma LDL 0.1 mM carnosine demonstrated a significantly more pronounced reduction in the formation of lipid radicals compared to Gal. The results show that Gal can be considered as a promising agent that reduces myocardial injury during reperfusion and oxidative stress.

Application of enzymatic fluorometric assays to quantify phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in human plasma lipoproteins

Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM) are important surface components of plasma lipoproteins, including very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). However, the pathophysiological roles of PC, PE and SM in lipoproteins have not been well characterized owing to the difficulties in quantifying phospholipid classes in lipoproteins. In this study, we assessed the precision and accuracy of the enzymatic fluorometric assays for measuring PC, PE and SM in VLDL, LDL and HDL, which were isolated from human plasma by ultracentrifugation. The within-run coefficients of variation (CV) for the measurements of PC, PE and SM in lipoproteins were 1.5–2.8 %, 1.1–2.4 % and 0.9–2.3 %, respectively, whereas the between-run CVs for the PC, PE and SM assays were 2.7–4.7 %, 2.1–4.5 % and 1.6–3.3 %, respectively. Excellent linearity and almost complete recovery were achieved for all assays measuring PC, PE and SM in VLDL, LDL and HDL. Our preliminary results using these enzymatic fluorometric assays suggested that the phospholipid compositions were different among VLDL, LDL and HDL. In conclusion, we established high-throughput enzymatic fluorometric assays to quantify PC, PE and SM in human plasma VLDL, LDL and HDL, which will be useful for further investigation of pathophysiological roles of phospholipids in lipoproteins.

Features and quantitative characteristics of the interaction of benzo[a]pyrene with fractions of the human blood plasma lipoproteins

The paper considers the features and quantitative characteristics of the interaction of human blood plasma low (LDL), very low (VLDL) and high density lipoproteins (HDL) with a polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P). Material and methods. The studies were performed using preparative ultracentrifugation of human plasma lipoprotein fractions and fluorescence spectroscopy. Results. The spectral characteristics of tryptophan fluorescence of VLDL, LDL and HDL fractions during titration with B[a]P solution are presented. The interaction of LP fractions with B[a]P was accompanied by quenching of tryptophanil fluorescence: the greatest decrease in fluorescence at saturating concentrations of B[a]P was noted for the HDL fraction — over 80 %; the effect of B[a]P was weaker for LDL (65-74 %) and for VLDL (50-60 %) of the initial level. Based on the results of the tryptophan fluorescence quenching curves and taking into account the molecular weights, quantitative characteristics of the interaction of the LP fractions with B[a]P were obtained: the association constants were of the order of (1.3 - 2.5) × 10-6 M, and the number of binding sites for B[a] P on LP particles ranged from 19 for HDL and up to 43 for VLDL. Conclusion. The quantitative characteristics of the interaction obtained for the complexes of LP with B[a]P allow us to consider the LP of blood plasma as real transport forms for B[a]P and other hydrophobic compounds into the cells of organs and tissues of the body.

Clearance of Carbonyl-Modified Low-Density Lipoproteins in Rabbits

The elimination kinetics of carbonyl-modified low density lipoproteins (LDL) from rabbit bloodstream was studied using isolated LDL of rabbits and humans after preliminary biotinylation or labeling with FITC. Rabbit or human blood plasma LDL were isolated using differential ultracentrifugation in a density gradient; after labeling by biotinylation or by FITC, LDL were modified with various low molecular weight natural dicarbonyls: malondialdehyde (MDA), glyoxal or methylglyoxal. Native (control) and dicarbonyl-modified biotinylated or FITC-labeled LDL were injected into the ear vein of rabbits, and blood samples were taken at certain time intervals. The content of biotinylated LDL in blood plasma was determined by an enzyme immunoassay method; FITC-labeled LDL was determined from the fluorescence spectra. It has been found that glyoxal- and methylglyoxal-modified rabbit and human LDL circulate in the bloodstream of rabbits for almost the same period as native (unmodified) LDL. In contrast to this, MDA-modified rabbit and human LDL were very quickly eliminated from the rabbit bloodstream. Dicarbonyl-modified LDL from human blood plasma is not associated with red blood cells or endothelial cells. We found that using the Oxidized LDL ELISA kits (Mercodia, Sweden) it was possible to identify mainly MDA-modified LDL. The level of MDA-modified LDL in the blood plasma of CHD patients sharply decreased during therapy with evolocumab, the hypocholesterolemic inhibitor of PCSK9 (proprotein convertase of subtilisin/kexin type 9), which activates LDL reutilization in the liver cells. These results explain the extremely rapid clearance of MDA-modified LDL in our experiments by their increased utilization in hepatocytes. The results obtained indicate a high atherogenicity of glyoxal- and methylglyoxal-modified LDL, long-term circulating in the bloodstream.

Antioxidant Properties of Galanin and Its N-Terminal Fragments in in vitro and in vivo Oxidative Stress Modeling.

Antioxidant properties of rat galanin GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH2 (Gal), N-terminal fragment of galanin (2-15 aa) WTLNSAGYLLGPHA (G1), and its modified analogue WTLNSAGYLLGPβAH (G2) were studied in vivo in the rat model of regional myocardial ischemia and reperfusion and in vitro in the process of Cu2+-induced free radical oxidation of human blood plasma low-density lipoproteins. Intravenous administration of G1, G2, and Gal to rats after ischemia induction reduced the infarction size and activities of the necrosis markers, creatine kinase-MB and lactate dehydrogenase, in blood plasma at the end of reperfusion. G1, G2, and Gal reduced formation of the spin adducts of hydroxyl radicals in the interstitium of the area at risk during reperfusion, moreover, G2 and Gal also reduced formation of the secondary products of lipid peroxidation in the reperfused myocardium. It was shown in the in vivo experiments and in the in vitro model system that the ability of galanin peptides to reduce formation of ROS and attenuate lipid peroxidation during myocardial reperfusion injury was not associated directly with their effects on activities of the antioxidant enzymes of the heart: Cu,Zn-superoxide dismutase, catalase, and glutathione peroxidase. The peptides G1, G2, and Gal at concentrations of 0.01 and 0.1 mM inhibited Cu2+-induced free radical oxidation of human low-density lipoproteins in vitro. The results of oxidative stress modeling demonstrated that the natural and synthetic agonists of galanin receptors reduced formation of the short-lived ROS in the reperfused myocardium, as well as of lipid radicals in blood plasma. Thus, galanin receptors could be a promising therapeutic target for cardiovascular diseases.

Rapid affinity chromatographic isolation method for LDL in human plasma by immobilized chondroitin-6-sulfate and anti-apoB-100 antibody monolithic disks in tandem

Low-density lipoprotein (LDL) is considered the major risk factor for the development of atherosclerotic cardiovascular diseases (ASCVDs). A novel and rapid method for the isolation of LDL from human plasma was developed utilising affinity chromatography with monolithic stationary supports. The isolation method consisted of two polymeric monolithic disk columns, one immobilized with chondroitin-6-sulfate (C6S) and the other with apolipoprotein B-100 monoclonal antibody (anti-apoB-100 mAb). The first disk with C6S was targeted to remove chylomicrons, very-low-density lipoprotein (VLDL) particles, and their remnants including intermediate-density lipoprotein (IDL) particles, thus allowing the remaining major lipoprotein species, i.e. LDL, lipoprotein(a) (Lp(a)), and high-density lipoprotein (HDL) to flow to the anti-apoB-100 disk. The second disk captured LDL particles via the anti-apoB-100 mAb attached on the disk surface in a highly specific manner, permitting the selective LDL isolation. The success of LDL isolation was confirmed by different techniques including quartz crystal microbalance. In addition, the method developed gave comparable results with ultracentrifugation, conventionally used as a standard method. The reliable results achieved together with a short isolation time (less than 30 min) suggest the method to be suitable for clinically relevant LDL functional assays.

Electronegative Low-Density Lipoprotein L5 Impairs Viability and NGF-Induced Neuronal Differentiation of PC12 Cells via LOX-1

There have been striking associations of cardiovascular diseases (e.g., atherosclerosis) and hypercholesterolemia with increased risk of neurodegeneration including Alzheimer’s disease (AD). Low-density lipoprotein (LDL), a cardiovascular risk factor, plays a crucial role in AD pathogenesis; further, L5, a human plasma LDL fraction with high electronegativity, may be a factor contributing to AD-type dementia. Although L5 contributing to atherosclerosis progression has been studied, its role in inducing neurodegeneration remains unclear. Here, PC12 cell culture was used for treatments with human LDLs (L1, L5, or oxLDL), and subsequently cell viability and nerve growth factor (NGF)-induced neuronal differentiation were assessed. We identified L5 as a neurotoxic LDL, as demonstrated by decreased cell viability in a time- and concentration-dependent manner. Contrarily, L1 had no such effect. L5 caused cell damage by inducing ATM/H2AX-associated DNA breakage as well as by activating apoptosis via lectin-like oxidized LDL receptor-1 (LOX-1) signaling to p53 and ensuring cleavage of caspase-3. Additionally, sublethal L5 long-termly inhibited neurite outgrowth in NGF-treated PC12 cells, as evidenced by downregulation of early growth response factor-1 and neurofilament-M. This inhibitory effect was mediated via an interaction between L5 and LOX-1 to suppress NGF-induced activation of PI3k/Akt cascade, but not NGF receptor TrkA and downstream MAPK pathways. Together, our data suggest that L5 creates a neurotoxic stress via LOX-1 in PC12 cells, thereby leading to impairment of viability and NGF-induced differentiation. Atherogenic L5 likely contributes to neurodegenerative disorders.

Analysis of low-density lipoprotein-associated proteins using the method of digitized native protein mapping.

The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel-cutting, and quantitative LC-MS/MS (in data-independent acquisition mode, or MS(E) ), was improved by using a new MS/MS mode, ion mobility separation enhanced-MS(E) (HDMS(E) ), and applied to analyze the area of human plasma low-density lipoprotein (LDL). An 18 mm × 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid-cut into 72 square gel pieces and subjected to quantitative LC-MS/MS. Compared with MS(E) , HDMS(E) showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. A total of 253 proteins were assigned with LC-HDMS(E) and the quantity distribution of each was reconstructed as a native protein map. The maps showed that Apo B-100 was the most abundant protein in the grid-cut area, concentrated at pI ca. 5.4-6.1 and apparent mass ca. 1000 kDa, which corresponded to four gel pieces, squares 39-42. An Excel macro was prepared to search protein maps which showed protein quantity peaks localized within this concentrated region of Apo B-100. Twenty-two proteins out of the 252 matched this criterion, in which 19 proteins have been reported to be associated with LDL. This method only requires several microliters of a plasma sample and the principle of the protein separation is totally different from the commonly used ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL.

Isoforms of Hsp70-binding human LDL in adult Schistosoma mansoni worms.

Schistosoma mansoni is one of the most common parasites infecting humans. They are well adapted to the host, and this parasite's longevity is a consequence of effective escape from the host immune system. In the blood circulation, lipoproteins not only help to conceal the worm from attack by host antibodies but also act as a source of lipids for S. mansoni. Previous SEM studies showed that the low-density lipoprotein (LDL) particles present on the surface of adult S. mansoni worms decreased in size when the incubation time increased. In this study, immunocytochemical and proteomic analyses were used to locate and identify S. mansoni binding proteins to human plasma LDL. Ultrathin sections of adult worms were cut transversely from the anterior, medial and posterior regions of the parasite. Immunocytochemical experiments revealed particles of gold in the tegument, muscle region and spine in male worms and around vitelline cells in females. Immunoblotting and 2D-electrophoresis using incubations with human serum, anti-LDL antibodies and anti-chicken IgG peroxidase conjugate were performed to identify LDL-binding proteins in S. mansoni. Analysis of the binding proteins using LC-MS identified two isoforms of the Hsp70 chaperone in S. mansoni. Hsp70 is involved in the interaction with apoB in the cytoplasm and its transport to the endoplasmic reticulum. However, further studies are needed to clarify the functional role of Hsp70 in S. mansoni, mainly related to the interaction with human LDL.

Pathophysiological role and clinical significance of lipoprotein-associated phospholipase A₂ (Lp-PLA₂) bound to LDL and HDL.

Lipoprotein-associated phospholipase A2 (Lp-PLA2), also named as platelet-activating factor (PAF)-acetylhydrolase, exhibits a Ca2+-independent phospholipase A2 activity and catalyzes the hydrolysis of the ester bond at the sn-2 position of PAF and oxidized phospholipids (oxPL). These phospholipids are formed under oxidative and inflammatory conditions, and may play important roles in atherogenesis. The vast majority of plasma Lp-PLA2 mass binds to low-density lipoprotein (LDL) while a smaller amount is associated with high-density lipoprotein (HDL). Lp-PLA2 is also bound to lipoprotein (a) [Lp(a)], very low-density lipoprotein (VLDL) and remnant lipoproteins. Several lines of evidence suggest that the role of plasma Lp-PLA2 in atherosclerosis may depend on the type of lipoprotein particle with which this enzyme is associated. Data from large Caucasian population studies have supported plasma Lp-PLA2 (primarily LDL-associated Lp-PLA2) as a cardiovascular risk marker independent of, and additive to, traditional risk factors. On the contrary, the HDL-associated Lp-PLA2 may express antiatherogenic activities and is also independently associated with lower risk for cardiac death. The present review presents data on the biochemical and enzymatic properties of Lp-PLA2 as well as its structural characteristics that determine the association with LDL and HDL. We also critically discuss the possible pathophysiological and clinical significance of the Lp- PLA2 distribution between LDL and HDL in human plasma, in view of the results of prospective epidemiologic studies on the association of Lp-PLA2 with future cardiovascular events as well the recent studies that evaluate the possible effectiveness of specific Lp-PLA2 inhibitors in reducing residual cardiovascular risk.

Abstract 140: Electronegative LDL and Beta-Amyloid Synergistically Induce Platelet Activation that Can Be Inhibited by Novel MicroRNA-145 and NF-κB Decoys

Background: In addition to its role in Alzheimer’s disease, β-amyloid (Aβ) stimulates platelet aggregation by activating NF-κB. L5, a highly electronegative atherogenic subfraction of low-density lipoprotein (LDL), also induces platelet activation. Our preliminary experiments showed that Aβ and L5 function synergistically; thus, we examined the underlying mechanisms and tested a novel therapeutic approach using oligodeoxynucleotide (ODN) decoys. Methods and Results: Human plasma LDL was separated into 5 subfractions, L1-L5, with increasing electronegativity. L5, but not L1-L4, induced human platelets to release Aβ from α-granules. By phosphorylating IκB kinase β (IKKβ), both L5 and Aβ induced degradation of kappa B inhibitor (IκBα) to activate NF-κB. This led to, by way of c-Jun N-terminal kinase (JNK), the activation of platelet receptor GPIIbIIIa and platelet aggregation. L5- and Aβ-induced IκBα degradation was inhibited by ubiquitin-specific peptidase 31 through deubiquitination, which was in turn inhibited by microRNA (miR)-145. However, a specific miR-145 decoy ODN prevented IκBα degradation by inhibiting miR-145 (Figure), whereas scramble ODN had no effect. Furthermore, a specific NF-κB decoy prevented NF-κB-mediated GPIIbIIIa activation (Figure). Compared to L1, L5 injected into C57/BL6 mice (5 mg/kg of each twice a week for 6 weeks) shortened tail-bleeding time by 38% (n=6; P <0.05), which was prevented by NF-κB decoy but not scramble ODN. Conclusions: Atherogenic L5 LDL potentiates Aβ-mediated platelet activation and aggregation. Novel miR-145 and NF-κB decoys effectively blocked the synergistic effect of L5 and Aβ and may reduce the risk for thrombotic stroke.

Electronegative low-density lipoprotein induces cardiomyocyte apoptosis indirectly through endothelial cell-released chemokines

Cardiomyocyte apoptosis has a critical role in the pathogenesis of heart failure. L5, the most negatively charged subfraction of human plasma low-density lipoprotein (LDL), induces several atherogenic responses in endothelial cells (ECs), including apoptosis. We hypothesized that L5 also contributes to cardiomyocyte apoptosis and studied whether it does so indirectly by inducing the secretion of factors from ECs. We examined apoptosis of rat cardiomyocytes treated with culture-conditioned medium (CCM) of rat ECs that were exposed to L5 or L1 (the least negatively charged LDL subfraction). Apoptosis at early and late time points was twofold greater in cardiomyocytes treated with L5 CCM than in those treated with L1 CCM. The indirect effect of L5 on cardiomyocyte apoptosis was significantly reduced by pretreating ECs with inhibitors of phosphatidylinositol 3-kinase (PI3K) or CXC receptor 2 (CXCR2). Studies with cytokine protein arrays revealed that L5 CCM, but not L1 CCM, contained high levels of ELR(+) CXC chemokines, including lipopolysaccharide-induced chemokine (LIX) and interleukin (IL)-8. The L5-induced release of these chemokines from ECs was abolished by inhibiting the lectin-like oxidized LDL receptor-1 (LOX-1). Addition of recombinant LIX or IL-8 to CCM-free cardiomyocyte cultures increased apoptosis and enhanced production of tumor necrosis factor (TNF)-α and IL-1β by increasing the translocation of NF-κB into the nucleus; these effects were attenuated by inhibiting PI3K and CXCR2. In conclusion, L5 may indirectly induce cardiomyocyte apoptosis by enhancing secretion of ELR(+) CXC chemokines from ECs, which in turn activate CXCR2/PI3K/NF-κB signaling to increase the release of TNF-α and IL-1β.

ELECTRONEGATIVE LOW-DENSITY LIPOPROTEIN INDUCES CARDIOMYOCYTE APOPTOSIS THROUGH A CHEMOKINE-CYTOKINE CROSSTALK MECHANISM

Cardiomyocyte apoptosis plays a critical role in the pathogenesis of heart failure. Human plasma low-density lipoprotein (LDL) can be resolved by charge into five subfractions, L1-L5. L5, the most negatively charged subfraction, induces several atherogenic responses in endothelial cells (ECs),

Peroxidation of lipoproteins in multiple sclerosis

Human plasma low density lipoproteins (LDL) and high density lipoproteins (HDL) are involved in the transport of lipids, modulate membrane lipid composition and regulate signal transduction. HDL-like lipoproteins have been shown also in human cerebrospinal fluid and it has been hypothesized that they could have a role in lipid transport in central nervous system. After synthesis, lipoproteins are susceptible to lipid peroxidation triggered by reactive oxygen species (ROS and RNS) produced by peripheral and brain cells. Aim of the paper has been to review the scientific literature on the role of lipid peroxidation of LDL and HDL in the molecular mechanisms of multiple sclerosis (MS). Several studies have demonstrated a significant increase in lipid peroxidation products in brain, plasma and cerebrospinal fluid of MS patients. The increase of antibodies against ox-LDL in plasma and the presence of ox-LDL in demyelinating plaques in MS brain suggests that the disease is associated with oxidative damage of lipoproteins. The impairment of antioxidant systems or an increase in the production of ROS and RNS could contribute to lipoprotein peroxidation in MS. Oxidized lipoproteins show several alterations of their functions, they are neurotoxic and have pro-inflammatory properties. Therefore lipoprotein lipid peroxidation products could be involved in demyelination and axonal injury in MS.

Chemical composition-oriented receptor selectivity of L5, a naturally occurring atherogenic low-density lipoprotein.

Anion-exchange chromatography resolves human plasma low-density lipoprotein (LDL) into 5 subfractions, with increasing negative surface charge in the direction of L1 to L5. Unlike the harmless L1 to L4, the exclusively atherogenic L5 is rejected by the normal LDL receptor (LDLR) but endocytosed into vascular endothelial cells through the lectin-like oxidized LDL receptor-1 (LOX-1). Analysis with SDS-PAGE and 2-dimensional electrophoresis showed that the protein framework of L1 was composed mainly of apolipoprotein (apo) B100, with an isoelectric point (pI) of 6.620. There was a progressively increased association of additional proteins, including apoE (pI 5.5), apoAI (pI 5.4), apoCIII (pI 5.1), and apo(a) (pI 5.5), from L1 to L5. LC/MSE was used to quantify protein distribution in all subfractions. On the basis of weight percentages, L1 contained 99% apoB-100 and trace amounts of other proteins. In contrast, L5 contained 60% apoB100 and substantially increased amounts of apo(a), apoE, apoAI, and apoCIII. The compositional characteristics contribute to L5's electronegativity, rendering it unrecognizable by LDLR. LOX-1, which has a high affinity for negatively charged ligands, is known to mediate the signaling of proinflammatory cytokines. Thus, the chemical composition-oriented receptor selectivity hinders normal metabolism of L5, enhancing its atherogenicity through abnormal receptors, such as LOX-1.

Effects of phospholipase A2 and its products on structural stability of human LDL: relevance to formation of LDL-derived lipid droplets

Hydrolysis and oxidation of LDL stimulate LDL entrapment in the arterial wall and promote inflammation and atherosclerosis via various mechanisms including lipoprotein fusion and lipid droplet formation. To determine the effects of FFA on these transitions, we hydrolyzed LDL by phospholipase A(2) (PLA(2)), removed FFA by albumin, and analyzed structural stability of the modified lipoproteins. Earlier, we showed that heating induces LDL remodeling, rupture, and coalescence into lipid droplets resembling those found in atherosclerotic lesions. Here, we report how FFA affect these transitions. Circular dichroism showed that mild LDL lipolysis induces partial β-sheet unfolding in apolipoprotein B. Electron microscopy, turbidity, and differential scanning calorimetry showed that mild lipolysis promotes LDL coalescence into lipid droplets. FFA removal by albumin restores LDL stability but not the protein conformation. Consequently, FFA enhance LDL coalescence into lipid droplets. Similar effects of FFA were observed in minimally oxidized LDL, in LDL enriched with exogenous FFA, and in HDL and VLDL. Our results imply that FFA promote lipoprotein coalescence into lipid droplets and explain why LDL oxidation enhances such coalescence in vivo but hampers it in vitro. Such lipid droplet formation potentially contributes to the pro-atherogenic effects of FFA.

Human LDL core cholesterol ester packing: three-dimensional image reconstruction and SAXS simulation studies

Human LDL undergoes a reversible thermal order-disorder phase transition associated with the cholesterol ester packing in the lipid core. Structural changes associated with this phase transition have been shown to affect the resistance of LDL to oxidation in vitro studies. Previous electron cryo-microscopy studies have provided image evidence that the cholesterol ester is packed in three flat layers in the core at temperatures below the phase transition. To study changes in lipid packing, overall structure and particle morphology in three dimensions (3D) subsequent to the phase transition, we cryo-preserved human LDL at a temperature above phase transition (53°C) and examined the sample by electron microscopy and image reconstruction. The LDL frozen from 53°C adopted a different morphology. The central density layer was disrupted and the outer two layers formed a "disrupted shell"-shaped density, located concentrically underneath the surface density of the LDL particle. Simulation of the small angle X-ray scattering curves and comparison with published data suggested that this disrupted shell organization represents an intermediate state in the transition from isotropic to layered packing of the lipid. Thus, the results revealed, with 3D images, the lipid packing in the dynamic process of the LDL lipid-core phase transition.

Langmuir–Blodgett films of polyaniline for low density lipoprotein detection

Langmuir–Blodgett (LB) films of polyaniline (PANI) were utilized for the fabrication of impedimetric immunosensor for detection of human plasma low density lipoprotein (LDL) by immobilizing anti-apolipoprotein B (AAB) via EDC–NHS coupling. The modified electrodes were characterized by electrochemical impedance spectroscopy (EIS) and scanning electron microscopy. AAB/PANI–SA LB immunoelectrodes studied by EIS spectroscopy revealed detection of LDL in the wide range of 0.018 μM (6 mg/dl) to 0.39 μM (130 mg/dl), covering the physiological range in blood, with a sensitivity of 11.25 kΩ μM − 1 .

An improved anion-exchange high-performance liquid chromatography method for measuring oxidized form of LDLs in human plasma

Circulating oxidized low-density lipoproteins (LDLs) (ox-LDLs) could be a sensitive marker to predict future cardiovascular events. However, a method to evaluate oxidized forms of LDLs systemically in human plasma is not yet established. In this study, we developed a novel and convenient high-performance liquid chromatography (HPLC) method for measuring ox-LDL levels in humans. Human plasma lipoproteins were separated by a modified HPLC method using a diethylaminoethyl-type anion-exchange gel column with stepwise elution. Ox-LDLs were detected by postcolumn reaction with a reagent containing cholesterol esterase and cholesterol oxidase. Particle size of each LDL fraction separated by HPLC was determined in 61 healthy subjects. Our HPLC method separated LDLs into three fractions, which were designated as LDL-1, LDL-2 and LDL-3, on the basis of their negative charges, with LDL-3 the most strongly retained fraction migrating fastest in the anodic direction, a property that reflects the net negative charge of the molecule. Western blot analysis revealed that apolipoprotein B100 in LDL-3 fraction was the most fragmented and oxidatively modified. When LDLs were oxidized in vitro by Cu2+ or 2,2-azo-bis (2-aminopropane)-2HCl or modified by various aldehydes, all of the LDL fractions migrated at the position of LDL-3. Further, among three fractions, particle size was smallest in LDL-3 fraction. Here, we developed a convenient HPLC method and identified LDL-3 as oxidized LDL fractions, although ox-LDLs were present in LDL-2 fraction, albeit lesser concentrations than in LDL-3 subfraction. Measuring ox-LDL levels in human plasma by this method may be useful to evaluate atherosclerotic disorders.

Model of human low-density lipoprotein and bound receptor based on CryoEM

Human plasma low-density lipoproteins (LDL), a risk factor for cardiovascular disease, transfer cholesterol from plasma to liver cells via the LDL receptor (LDLr). Here, we report the structures of LDL and its complex with the LDL receptor extracellular domain (LDL.LDLr) at extracellular pH determined by cryoEM. Difference imaging between LDL.LDLr and LDL localizes the site of LDLr bound to its ligand. The structural features revealed from the cryoEM map lead to a juxtaposed stacking model of cholesteryl esters (CEs). High density in the outer shell identifies protein-rich regions that can be accounted for by a single apolipoprotein (apo B-100, 500 kDa) leading to a model for the distribution of its alpha-helix and beta-sheet rich domains across the surface. The structural relationship between the apo B-100 and CEs appears to dictate the structural stability and function of normal LDL.