Cyclic diarylheptanoids inhibit cell-mediated low-density lipoprotein oxidation

Two cyclic diarylheptanoids, garugamblin-3 (1) and acerogenin L (2), isolated from the MeOH extract of the fruits of Alnus japonica Steud., inhibited human low-density lipoprotein (LDL) oxidation in the thiobarbituric acid-reactive substance assay with IC50 values of 2.9 and 1.7 microM, respectively, and they also inhibited cell-mediated LDL oxidation more than five times more strongly than that of a well-known antioxidant, probucol, at a concentration of 10 microM. 1 had no effect on the anti-atherogenesis in low-density lipoprotein receptor- deficient mice.     

Purification of monoclonal antibodies against the low-density lipoprotein receptor by preparative isotachophoresis

A preparative free-flow isotachophoretic method for the purification of monoclonal antibodies from mouse ascites fluid and tissue culture media is described. This high-resolution method allows the direct separation of monoclonal antibodies from antibody-containing tissue culture media or ascites fluid and gives a better separation from the major contaminant protein fractions and a higher recovery of the monoclonal antibodies than anion-exchange chromatography. The purification can run continuously and without any time-consuming regeneration procedures; the monoclonal antibody is obtained under mild conditions in a small electrolyte volume.     

Probabilistic kinetic model of slow oxidation of low-density lipoprotein. 3. Hydroperoxide-free initiation

A theoretical model is presented that explains slow copper-induced oxidation of low-density lipoprotein in the systems free of seeded hydroperoxydes. The model is based on the probabilistic kinetic theory, modified to take into account different radical generation rates by oxidized and reduced forms of the metal ions. It is shown that the initiation and progression of the LDL oxidation can take place in any LDL dispersion by metal-induced oxidation of alpha-tocopherol, one of the constituents of LDL, and formation of slowly reacting alpha-tocopheroxyl radical. Selected values of several adjustable parameters define actual temporal profiles of the quantities defining the oxidation process.     

Cyclic diarylheptanoids inhibit cell mediated low-density lipoprotein oxidation

Two cyclic diarylheptanoids, garugamblin-3 (1) and acerogenin L (2), isolated from the MeOH extract of the fruits of Alnus japonica Steud., inhibited human low-density lipoprotein (LDL) oxidation in the thiobarbituric acid-reactive substance assay with IC(50) values of 2.9 and 1.7 microM, respectively, and they also inhibited cell-mediated LDL oxidation more than 5 times more strongly than that of a well-known antioxidant, probucol, at a concentration of 10 microM. Compound (1) had no effect on the anti-atherogenesis in low-density lipoprotein receptor-deficient mice.     

Charge density profiling of circulating human low-density lipoprotein particles by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) has been utilized to profile the low-density (LDL) particles in human blood serum in this study. A 5 mM sodium phosphate buffer, pH 7.40, was chosen as the most suitable CE buffer and an extensive ultrafiltration (UF) procedure was applied to purify the LDL sample. Two LDL particle species, LDL with lower mobility and LDL- with higher mobility were observed. The electropherograms were highly reproducible with good precision of effective mobilities, corrected peak areas (CPAs) and CPA ratio of LDL-/LDL. LDL particles shown on the electropherogram were also characterized by several procedures. The applications of Sigma HDL cholesterol reagent and CE on-line 2-propanol precipitation indicated that the two particle species shown in the electropherogram belong to LDL. The LDL particles were found to associate with the buoyant LDL fraction and the LDL- particles associate with the dense LDL fraction. This study utilizes CZE for the profiling of LDL isoforms and provides a new analytical method for the resolution of LDL subspecies. It demonstrates a high-mobility LDL particle which circulates in healthy subjects and diminishes in atherosclerotic patients. Diminution of the high-mobility LDL subspecies may be linked to minimal formation of arterial plaque in atherosclerotic patients.     

Trout Erythrocyte Membranes. Thermoresistivity and thermally induced structural transitions

Differential scanning microcalorimetry and equilibrium thermohemolysis procedure were used to study the effect of acclimation temperature on thermally induced transitions and thermoresistivity of fish (trout) erythrocyte membranes. Strong correlation has been found between the rates and activation energies of erythrocyte thermohemolysis and acclimation temperature. Transition temperatures of five thermodynamically irreversible and one partially reversible transitions at about 87°C as well as the overall shape of microcalorimetric curves of the erythrocyte ghosts do not vary with acclimation temperature. The results suggest an essential conservation of phospholipid microenvironment of membrane skeleton proteins despite the compensatory response in lipid composition of erythrocyte membrane bilayer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fabrication of a label-free electrochemical immunosensor of low-density lipoprotein

The silver chloride@polyaniline (PANI) core-shell nanocomposites (AgCl@PANI) combined with Au nanoparticles (AuNPs) were used to prepare the AuNPs-AgCl@PANI hybrid material. A novel sensitive label-free low-density lipoprotein (LDL) electrochemical biosensor was fabricated by adsorption of antibody to apolipoprotein B-100 (aopB-100) on an AuNPs-AgCl@PANI-modified glassy carbon (GC) electrode. The hybrid material could provide surface for high antibody loading due to its large surface-to-volume ratio. Since each LDL has an apoB-100 on its phospholipids coat, they could be bonded to the electrode surface through the specific antibody-antigen reaction. Electrochemical impedance spectroscopy (EIS) was used to characterize the recognition of LDL. The negative charges carried by LDL phospholipids coat would block the electron transfer of the [Fe(CN)6]3-/4- redox couple severely. In addition, the conductivity of LDL is very poor, so small amounts of LDL on the electrode could result in great change in the electron-transfer resistance (Ret). The biosensor exhibited a highly sensitive response to LDL with a detection limit of 0.34 pg/mL, and some factors that would affect the performance of the biosensor were studied, such as incubation time and temperature.     

Quantitative studies on the peroxidation of human low-density lipoprotein initiated by superoxide and by charged and neutral alkylperoxyl radicals.

Rates of peroxidation of human LDL and rates of consumption of the LDL's alpha-tocopherol (TocH) have been measured at 37 degrees C. Peroxidation was initiated by radicals generated in the aerated aqueous phase at known rates by thermal decomposition of appropriate precursors: superoxide (O2(*-)/HOO(*)) from a hyponitrite and alkylperoxyls (ROO(*), two positively charged, one negatively charged and one neutral) from azo compounds. The efficiencies of escape from the solvent cage of the geminate pair of neutral carbon-centered radicals was found to be 0.1, but it was 0.5 for the three charged radicals, a result attributed to radical/radical Coulombic repulsion within the cage. All four alkylperoxyls initiated and terminated tocopherol-mediated peroxidation (TMP) with about equal efficiency and essentially all of these radicals that were generated were consumed in these two reactions. TMP is a radical chain process, and when initiated by the alkylperoxyls, the rate of LDL peroxidation was faster in the early stages while TocH was present than later, after all of this "antioxidant" had been consumed. In contrast, only about 3-4% of the generated superoxide radicals reacted in any measurable fashion with TocH-containing LDL at pH's from 7.6 to 6.5 and peroxidation was much slower than with a similar rate of generation of alkylperoxyls. After all the TocH had been consumed, LDL peroxidation was negligible at pH 7.6 and 7.4, but at pH 6.8 and 6.5, the peroxidation rates showed a large increase over the rates while the TocH had been present. That is, endogenous TocH behaves as an antioxidant in LDL subjected to attack by the physiologically relevant superoxide radical, whereas TocH behaves as a prooxidant in LDL subjected to attack by the probably far less physiologically important alkylperoxyls. Rates of LDL peroxidation initiated by superoxide increased as the pH was decreased, and the results are consistent with the initiation of peroxidation of fresh LDL occurring via H-atom abstraction from TocH by HOO(*) to form the Toc(*) radical and termination by reaction of O2(*-) with Toc(*), a process that occurs partly by addition leading to TocH consumption and partly by electron plus proton transfer leading to the regeneration of TocH.     

Effect of oxidation of low-density lipoprotein on drug binding affinity studied by high performance frontal analysis-capillary electrophoresis.

The effect of oxidation of low-density lipoprotein (LDL) on the enantioselective drug binding affinity was investigated using high performance frontal analysis--capillary electrophoresis (HPFA-CE). Verapamil and nilvadipine enantiomers were used as the chiral model drugs. LDL was oxidized with copper sulfate for 0, 0.5, 1, 2, and 12 h at 37 degrees C. The HPFA-CE method enabled microdetermination of unbound drug concentrations in native and oxidized LDL solutions. It was found that the bindings between LDL and the model drugs were not enantioselective at any oxidation stage. The total binding affinity (nK) between LDL and verapamil enantiomers was increased by 3.3-, 4.6-, 7.0-, and 19-fold after 0.5, 1, 2, and 12 h oxidation, respectively, whereas the nK value between nilvadipine and LDLwas increased by 1.3-, 1.4-, 1.4-, and 1.7-fold in the same reaction times, respectively. These results indicate that the LDL oxidation enhances the drug binding affinity, and the affinity of verapamil is increased more sensitively than that of nilvadipine. The nK value of each model drug increased steeply after the first 2 h oxidation, followed by the gradual increase after the next 10 h oxidation. It is considered that the net increase in the negative charges and/or the formation of hydroperoxides in the first 2 h oxidation enhances the drug-LDL binding more significantly than the formation of aldehydes or Schiff bases in the following 10 h oxidation.     

Identification and analysis of stereoselective drug interactions with low-density lipoprotein by high-performance affinity chromatography

Columns containing immobilized low-density lipoprotein (LDL) were prepared for the analysis of drug interactions with this agent by high-performance affinity chromatography (HPAC). R/S-Propranolol was used as a model drug for this study. The LDL columns gave reproducible binding to propranolol over 60 h of continuous use in the presence of pH 7.4 0.067 M potassium phosphate buffer. Experiments conducted with this type of column through frontal analysis indicated that two types of interactions were occurring between R-propranolol and LDL, while only a single type of interaction was observed between S-propranolol and LDL. The first type of interaction, which was seen for both enantiomers, involved non-saturable binding; this interaction had an overall affinity (nK _a) of 1.9 (±0.1) × 10⁵ M⁻¹ for R-propranolol and 2.7 (±0.2) × 10⁵ M⁻¹ for S-propranolol at 37 °C. The second type of interaction was observed only for R-propranolol and involved saturable binding that had an association equilibrium constant (K _a) of 5.2 (±2.3) × 10⁵ M⁻¹ at 37 °C. Similar differences in binding behavior were found for the two enantiomers at 20 °C and 27 °C. This is the first known example of stereoselective binding of drugs by LDL or other lipoproteins. This work also illustrates the ability of HPAC to be used as a tool for characterizing mixed-mode interactions that involve LDL and related binding agents.     

Cyclodextrin-micellar electrokinetic chromatography of apolipoproteins on human very low-density lipoprotein

The apolipoproteins (APOs) of human very low-density lipoprotein (VLDL) were investigated by an optimized cyclodextrin-micellar electrokinetic chromatography (CD-MEKC) method. The separation buffer consisted of 20 mM sodium phosphate, 40 mM bile salts (50% sodium cholate and 50% sodium deoxycholate), 25 mM carboxymethyl-β-cyclodextrin (CM-β-CD) (pH 7.0). For CD-MEKC separation, a sample injection time of 12 s, a separation voltage of 15 KV, and a capillary temperature of 15°C were chosen. The optimal CD-MEKC method showed good resolution and repeatability for VLDL APOs. Identification and quantitation of VLDL APOs CI, CIII, and E were based on comparison with human APO standards. Good linear relationships with correlation coefficient (R²) 0.99 were obtained for APOs CI, CIII, and E standards. For these three APOs, the linear ranges were within 0.01-0.54 mg/mL, and the concentration limits of detection (LODs) were lower than 0.02 mg/mL. Moreover, VLDL APOs from four uremic patients and four healthy subjects were compared. The uremic and healthy CD-MEKC profiles showed dramatic difference. The levels of APO CIII were significantly higher for two patients, and the level of APO E was significantly higher for one patient. This study might be helpful for following the disease development of uremia and cardiovascular disease (CVD) in the future.     

Structural alteration of hybrid supramolecular capsule induced by guest encapsulation

Heterofunctionalized C(2v) symmetrical cavitand 1 with 4-pyridylethynyl and 3-carbamoylphenyl groups in alternating arrangement was designed and synthesized. A 1:1 mixture of the cavitand 1 and a cis-coordinated palladium(II) or platinum(II) complex self-assembled into a hybrid supramolecular capsule via both metal-ligand coordination bonds and hydrogen bonds. Formation of the capsular assembly was confirmed by NMR spectroscopy and mass spectrometry. The hybrid capsule encapsulated the appropriate guest, the molecular sizes of which fit the size of the capsular cavity. Structural alteration of the hybrid capsule was induced by the guest encapsulation. A C(2h) structure for the encapsulation complex was assigned by 2D NMR spectra analysis. Thermodynamic and kinetic properties of the guest encapsulation were investigated. The kinetics of in/out guest exchange was strongly influenced by hydrogen bonding in the hybrid capsule.     

Inhibition of the binding of low-density lipoprotein to its cell surface receptor in human fibroblasts by positively charged proteins.

A group of proteins and polyamino acids with positively charged domains were shown to inhibit the binding of 125I-LDL to its receptor on the surface of human fibroblasts. The list of inhibitory proteins included platelet factor 4 (which has a cluster of lysine residues at its carboxyl terminus), two lysine-rich histones, poly-L-lysines of chain length greater than 4, and protamine. These proteins were effective in the concentration range of 5--10 microgram/ml. Two other positively charged proteins, lysozyme and avidin, did not inhibit 125I-LDL binding. Kinetic studies suggested that protamine was not acting simply as a competitive inhibitor with regard to the LDL receptor. In light of previous data showing that polyanions such as heparin and polyphosphates also inhibit 125-I-LDL binding to its cell surface receptor, the current findings suggest that charge interactions are important in this binding reaction. In a related series of studies, a number of glycoproteins and their asialo derivatives as well as a number of sugar phosphates failed to inhibit 125I-LDL binding to its receptor in fibroblasts.     

Effect of sphingomyelinase-mediated generation of ceramide on aggregation of low-density lipoprotein

This study addresses the response-to-retention hypothesis, which states that the subendothelial retention of atherogenic lipoproteins is the necessary and sufficient condition for the initiation of atherosclerosis. Here we focus on the relationship between the generation of ceramide in the low-density lipoprotein (LDL) phospholipid monolayer and the resulting aggregation of LDL particles. This study provides the first measurement of neutral, Mg (2+)-dependent Sphingomyelinase (Smase)-mediated ceramide formation from LDL-sphingomyelin and does so for a range of enzyme concentrations (0-0.22 units Smase/mL). The kinetics of ceramide generation was measured using a fluorescence assay for the above enzyme concentrations with a fixed substrate concentration (0.33 mg LDL/mL). The kinetics of LDL aggregate formation was measured by dynamic light scattering (DLS, method of cumulants) for identical enzyme concentrations. Ceramide concentration profiles were fit with a modification of the Michaelis-Menten model ( k a = 1.11 x 10 (-1) microM (-1) min (-1), k -a = 6.54 x 10 (2) microM (-1) min (-1), k 1 = 3.33 x 10 (1) microM (-1) min (-1), k -1 = 1.41 x 10 (-2) min (-1), k cat = 8.05 x 10 (1) min (-1), K M = 2.418 microM, k deact = 4.66 x 10 (-2) microM (-1) min (-1)) that accounts for the effects of enzyme attachment to the LDL monolayer and for deactivation of Smase due to product inhibition. LDL aggregation is described by a mass action model as explained in previous studies. A key result of this work is the finding that LDL aggregate size depends directly on ceramide concentration and is independent of enzyme concentration. This study demonstrates how principles of colloid science are relevant to important biomedical problems.     

Substructure of human erythrocyte spectrin.

The human erythrocyte structural protein spectrin and its subunits I, II were isolated in the presence of Na-dodecyl-sulfate by gel filtration and preparative gel electrophoresis. After removal of the detergent, spectrin alpha-helical content is comparable to spectrin isolated without detergent. Subunits I and II formed single bands in isoelectric focusing (pI = 5.6) and in Ornstein-Davis disc gel electrophoresis systems, indicating the individual subunits are homogenous in nature. The molecular weights of the subunits I and II, determined by Ferguson plot, are 237,500 and 238,600, respectively, which is in good agreement with values obtained by the standard SDS gel relative mobility method. Limited tryptic digestion of spectrin and two-dimensional peptide maps of the individual subunits cleaved by S-cyanylation reaction showed dissimilar patterns, suggesting differences in primary structure between the two subunits.     

Subcellular alterations induced by UV-oxidized low-density lipoproteins in epithelial cells can be counteracted by alpha-tocopherol

Oxidized LDL (ox-LDL) have been involved in the pathogenesis of several human diseases including dermatological pathologies. Oxidative modification of low-density lipoproteins (LDL) is accompanied by both extensive degradation of its polyunsaturated fatty acids and production of lipoperoxides. These highly reactive products induce an intracellular oxidative stress with a variety of cytotoxic effects. In order to evaluate cellular damage induced by oxidative stress in epidermal cells, a human epidermoid carcinoma cell line in culture (A 431) was used as experimental model. Cell treatment with UV-oxidized LDL resulted in cytostatic and cytotoxic effects characterized by morphological and functional alterations: inhibition of cell proliferation, modifications of cytoskeleton network, microtubular derangement, loss of cell–cell and cell–substrate contacts, cell detachment and cell death by apoptosis. The ox-LDL-induced alterations were almost completely prevented by pre-incubating cells with α-tocopherol. The results presented here could be of relevance for a better comprehension of the pathogenic mechanisms of several human diseases, including dermatological pathologies, and could indicate that antioxidants such as α-tocopherol could represent an important therapeutic challenge in the maintenance of cell and tissue homeostasis in the long run.     

Rapid method for measuring plasma low-density lipoprotein turnover using high-performance gel exclusion chromatography

High-performance gel exclusion chromatography using flow-through radioactivity monitoring was employed for rapid measurement of low-density lipoprotein (LDL) turnover. Iodinated LDL was injected into five fasting rabbits. Serial blood samples were obtained and small aliquots (0.1-0.2 ml) were injected into the chromatographic system using 0.2 M Tris-acetate buffer (pH 7.0). Radioactivity in lipoproteins was measured by an on-line gamma counter (Flo-One) attached to the high-performance liquid chromatograph and in a regular gamma counter after collecting the fractions. Sequential ultracentrifugation was also used to separate lipoproteins, and the radioactivity in each fraction was measured. The Flo-One method was faster; however, the efficiency of Flo-One varied with the amount of radioactivity and therefore it was necessary to inject the same amount of radioactivity to get comparable values.     

High level of low-density lipoprotein receptors enhance hypericin uptake by U-87 MG cells in the presence of LDL

The dependence of the uptake of hypericin (Hyp) by human glioma U-87 MG cells on the level of expression of low-density lipoprotein (LDL) receptors has been studied in this work. A special role of the LDL receptor-pathway for Hyp delivery to U-87 MG cells in the presence of LDL was revealed by the substantial increase of Hyp uptake in the situation, when the number of LDL receptors on the cell surface was elevated. Moreover, the colocalization experiments showed the lysosomal localization of Hyp following the uptake and that the concentration of Hyp in these organelles was enhanced in the cells with elevated number of LDL receptors when the incubation medium contained LDL. Both these findings suggest that LDL and LDL receptor-pathway play an important role in the delivery and accumulation of Hyp into the cells.