Inhibition of Low-Density Lipoprotein Peroxidation by BHA Use: Fluorimetric Assay

Abstract

In the presence of free radicals, low-density lipoproteins (LDL) undergo oxidation that leads to the formation of lipoperoxides, which cause various damaging effects. Starting from the classical oxygen radical absorption capacity (ORAC) method, a modified method was formulated to develop a laboratory model that is able to deliver information about the use of polyphenols as nutrient supplements. The aim of this work was to establish the possible inhibition of LDL peroxidation by two different antioxidants, Trolox and BHA, and to assess if the effect is either of prevention or repair. 2,2,-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) was used as free radical generator, and fluorescein was used as fluorescent probe. It was noticed that the radical scavenging effect decreases in the order BHA to Trolox to LDL itself. Trolox shows a higher efficacy against the free radicals generated by AAPH than against lipoperoxides, whereas BHA is more efficient against lipoperoxides.     

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.     

Minimizing tocopherol-mediated radical phase transfer in low-density lipoprotein oxidation with an amphiphilic unsymmetrical azo initiator

The antioxidant alpha-tocopherol (alpha-TOH) has been found to act as a pro-oxidant under many in vitro conditions. The observed tocopoherol-mediated peroxidation (TMP) is dependent on two primary factors. (1) Chain transfer: alpha-TO. radical reacts with lipid to form lipid peroxyl radicals. (2) Phase transfer: alpha-TOH can transport radical character into the lipoprotein. Given the limitations of existing initiators, there is a need for new compounds that avoid the requirement for alpha-TOH to act as a phase-transfer agent. We report here a study showing that the new unsymmetrical azo compound, C-8, initiates LDL lipid peroxidation without requirement for alpha-TOH. This initiator provides a steady source of free amphiphilic peroxyl radicals that efficiently initiates oxidation of alpha-TOH-depleted LDL at a rate comparable to that reported for the very reactive hydroxyl radical (.OH). With other initiators tested, unsymmetrical C-12 and C-16 and symmetrical C-0 and MeOAMVN, alpha-TOH-depleted LDL displayed significant resistance to oxidation. Results indicate that the amphiphilic nature of the unsymmetrical initiators increases their partitioning into lipoprotein depending on the hydrocarbon chain length, and the symmetrical azo initiators C-0 and MeOAMVN primarily remain in the aqueous phase. Evidence suggests that even when the phase-transfer activity of alpha-TOH is limited, with the use of an initiator such as C-8, the mechanism of peroxidation remains controlled by TMP chain-transfer activity.     

Simultaneous production of superoxide radical and singlet oxygen by sulphonated chloroaluminum phthalocyanine incorporated in human low-density lipoproteins: implications for photodynamic therapy

Sulfonated chloroaluminum phthalocyanines have been studied for their use in the photodynamic therapy (PDT) of tumors. Plasma low-density lipoproteins (LDL) are important carriers of phthalocyanines in the blood, but on exposure to visible light, phthalocyanine-loaded LDL undergo an oxidation process that propagates to erythrocytes. We attempted to identify the reactive species involved in LDL and erythrocyte oxidation by means of electron paramagnetic resonance (EPR) spectroscopy in the presence of 2,2,6,6-tetramethyl-4-piperidone (TEMP) and the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Irradiation of phthalocyanine-loaded LDL in the presence of DMPO resulted in the formation of a four-line EPR spectrum with relative intensity of 1:2:2:1 (a(N) = a(H) = 14.8 G), characteristic of DMPO-hydroxyl radical spin adduct. This signal was sensitive to superoxide dismutase and slightly sensitive to catalase, but a mixture of the two enzymatic activities was the most efficient in promoting a decrease in the intensity of the EPR signal. In the presence of erythrocytes, an increase in the quartet intensity for a hematocrit of 1% and 4% was observed, decreasing for higher erythrocyte concentrations. The irradiation of phthalocyanine-loaded LDL in the presence of TEMP resulted in the formation of a nitroxide radical, 2,2,6,6-tetramethyl-4-piperidone-N-oxyl radical, intensity of which was sensitive to histidine, a singlet oxygen ((1)O(2)) quencher. Under both incubation conditions, with DMPO and TEMP, the formation of the respective EPR signals required the sensitizer (phthalocyanine), light and oxygen. Overall, the results are compatible with the simultaneous formation of superoxide anion and (1)O(2), implying that Type-I and Type-II mechanisms of photochemistry are simultaneously operative in phthalocyanine-loaded LDL. However, for a constant LDL/phthalocyanine ratio, the formation of oxygen free radicals shows a biphasic behavior with the concentration of LDL increasing and reaching a plateau, whereas the formation of (1)O(2) increases linearly with LDL concentration. Erythrocytes at high (physiological) concentrations induced a decrease in the intensity of both EPR signals. The physiological relevance of these findings in the framework of PDT is briefly discussed.     

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.     

The contrasting kinetics of peroxidation of vitamin E-containing phospholipid unilamellar vesicles and human low-density lipoprotein

It is well established that alpha-tocopherol, TocH, is an outstanding lipid-soluble, peroxyl radical trapping antioxidant in homogeneous systems. It is also well established that TocH functions as a prooxidant in human low-density lipoprotein, LDL, subjected to attack by peroxyl radicals generated in the aqueous phase by, for example, thermal decomposition of the azo compound, ABAP. This tocopherol-mediated peroxidation, TMP, of LDL involves a three-step chain reaction, one step being hydrogen atom abstraction from the LDL lipids by the tocopheroxyl radical, Toc*. The occurrence of TMP has been attributed to three factors, (i) translocation by TocH of radical character from the aqueous phase into LDL lipid, (ii) isolation of the water-insoluble Toc* in the LDL particle in which it is formed for times sufficient to permit it to react with the lipid, and (iii) the small lipid volume of LDL which ensures that no particle can contain more than a single radical for a significant length of time. This consensus view of TMP implies that it should occur in any TocH-containing dispersion of small lipid particles. However, the present examination of the kinetics of the ABAP-initiated peroxidation of small unilamellar vesicles, SUVs, made from palmitoyllinoleoylphosphatidylcholine and cholesterol with a composition designed to mimic the surface coat of LDL, has shown that TocH functions as an antioxidant in such systems and that TMP does not occur under conditions where it would have occurred if the particles had been LDL. Several possible reasons for the kinetic differences between SUVs and LDL have been considered and ruled out by experiment. It is concluded that TMP can occur in LDL because these particles contain a lipid core in which the Toc* radical "hides" for much of its lifetime well away from the peroxyl radicals in the aqueous phase. In contrast, because SUVs have no lipid core, the Toc(*) radical is always "exposed" and available to aqueous peroxyl radicals with which it reacts rapidly and is destroyed before it can abstract a hydrogen atom from the lipid.     

Colloidal catalysts based on iron(III) oxides. 2. Peculiarities of catalyzed oxidation of palm oil

The catalytic activity of an iron(III)-oxide-based colloidal catalyst with respect to oxidation of palm oil with atmospheric oxygen at 60°C is investigated. In a microheterogeneous system of palm oil containing phospholipids and large amounts of ??-tocopherol and ??-carotene, oxidation in the presence of the catalyst proceeds by a latent radical mechanism. This mechanism manifests itself as oxygen uptake against a background of almost constant concentrations of natural antioxidants, i.e., ??-tocopherol and ??-carotene. Using a model reaction of oxidation of an individual natural olefin, limonene, it is shown that, in the presence of the catalyst and trace amounts of hydroperoxide, oxidation of the hydrocarbon occurs through the chain free-radical mechanism. The presence of a phospholipid (egg lecithin) dramatically decelerates the catalytic oxidation of limonene and makes the process proceed through the latent radical mechanism, the rate of which is unaffected by oil-soluble inhibitors.     

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.     

Kinetic modelling of the thermal oxidation of polyisoprene elastomers. Part 1: Unvulcanized unstabilized polyisoprene

The thermal oxidation of an unvulcanized, unstabilized polyisoprene rubber (IR) has been studied in the 40-140 °C temperature range. Ageing was monitored by FTIR determination of double bonds and carbonyl groups, mass uptake measurement, and weight average molar mass determination. A mechanistic scheme based on the standard scheme for radical chain oxidation, but taking into account the diversity of initiation processes and the existence of inter- and intramolecular radical additions to double bonds, was built. The kinetic model derived from this scheme is composed of seven differential equations to be solved in discrete thickness layers to take into account the kinetic control by oxygen diffusion. This system was numerically solved using a Matlab program dedicated to stiff systems of differential equations. The elementary rate constants and other kinetic parameters were then determined from experimental data, using an inverse approach. A set of physically reasonable parameter values was obtained, thus allowing us to envisage lifetime predictions at low temperature (long term). The results led to observations difficult to make from classical analytical studies, for instance the predominance of bimolecular hydroperoxide decomposition among other initiation modes or the competition between intermolecular hydrogen abstraction and intramolecular addition of peroxy radicals to double bonds.     

Oxidation of Low-Density Lipoproteins in the Presence of a Fullerene-Containing Silica Gel

The results of a study on the oxidation of low-density lipoproteins (LDLs) in the presence of a fullerene-containing silica gel in the sorbent phase (a solid stationary phase) and in the phase of an LDL solution (a liquid mobile phase) are presented. It was found that the main distinctive feature of the process of LDL oxidation in the presence of a fullerene-containing silica gel consists in its heterogeneity; that is, the oxidation simultaneously occurs in both of the phases. In this case, the effect of the oxidation process in a solid phase on the kinetics in a liquid phase can be described in terms of the nonuniform distribution of reactant concentrations in the phases of the system and the formation of a donor–acceptor complex. It was found that the concentration of oxidation products is an exponential function of LDL concentration; this fact is indicative of a free-radical chain mechanism of the LDL oxidation reaction in the presence of the sorbent. This relationship was described by an empirical equation, which is the same for either of the phases, and the parameters of this equation have precise physical meanings. It was found from calculations that both the formation of donor–acceptor complexes and the nonuniform distribution of LDL in the two-phase system are responsible for a considerable difference between the kinetics of sorbent-initiated LDL oxidation in the sorbent phase and the solution phase.     

Fluorescence spectroscopic study of hypericin-photosensitized oxidation of low-density lipoproteins

By means of UV-VIS absorption and fluorescence spectroscopy, we demonstrate that the photosensitizer hypericin (Hyp) interacts nonspecifically with low-density lipoproteins (LDL), most probably with the lipid fraction of LDL. The molar ratio of monomeric Hyp binding to nonoxidized LDL and mildly oxidized LDL is 30:1. Increasing the Hyp concentration further leads to the formation of Hyp aggregates inside the LDL molecule. We also demonstrate that photoactivated Hyp oxidizes LDL in a light dose and excitation wavelength dependent manner. The level of oxidation of LDL depends on the amount of Hyp inside the LDL molecule. The maximum of the photosensitized oxidation of the LDL by Hyp is achieved for a 30:1 molar ratio, which corresponds to the maximum concentration of monomeric form of Hyp in LDL.     

SENSIBILISIERTE PHOTOOXIDATION DURCH METHYLENBLAU, THIOPYRONIN UND PYRONIN — II. PHYSIKOCHEMISCHE GRUNDLAGEN DER PHOTODYNAMISCHEN WIRKUNG VON THIOPYRONIN

Abstract— The physical-chemical properties of thiopyronine which result in its outstanding photodynamic effectiveness (in comparison to methylene blue and pyronine) are: (1) High velocity of free radical generation (kinetics followed by spectroscopy after flash photolysis). (2) High velocity of reoxidation of free radical (rate constant determined by kinetic spectroscopy under aerobic conditions). (3) Fairly positive reduction potential of oxidized free radical (from polarographic analysis of redox behavior). (4) Irreversible oxidation behavior of leuco-thiopyronin in contrast to leuco-methylene blue (the structure of leuco-thiopyronin was elucidated by NMR-analysis). (5) Fairly high equilibrium constant for complex formation with DN A (determination by high sensitive pulse-polarography).
From the sedimentation behavior and T-jump relaxation of in vitro photodynamically treated DNA it is shown that not only does guanine photooxidation take place but also single- and double-strand breaks can occur. The mechanism of this phenomenon is not yet elucidated.     

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.     

Applications on the monitoring of oxidative modification of LDL by capillary electrophoresis: a comparison with spectrophotometer assay

The aim of this work is the application of Stocks and Miller capillary electrophoresis (CE) method in order to evaluate the human LDL susceptibility to Cu²⁺-induced oxidation. Lipid peroxidation determines a change in the relative electrophoretic mobility (REM) of lipoprotein that can be monitored by capillary electrophoresis using uncoated fused silica capillaries and tricine-methylglucamine as electrophoretic buffer.We have evaluated the differences in the susceptibility to oxidation of LDL subjected to different preparations (dialysis or gel filtration, after ultracentrifugation, to remove EDTA), and different storage (4 °C for 1 week or lyophilization) by measuring REM and lipid hydroperoxides (ROOH) with a spectrophotometer assay. Our results indicate that gel filtration is a more convenient procedure than dialysis for the isolation of LDL and that lyophilised samples are less prone to oxidation than those stored at 4 °C. Moreover, REM appears to be a more sensitive and easier method than spectrophotometer assay both to monitor the oxidative modification of LDL and to evaluate oxidative state of native LDL.     

MODIFICATION OF ε-AMINO GROUP OF LYSINES, CHOLESTEROL OXIDATION AND OXIDIZED LIPID-APOPROTEIN CROSS-LINK FORMATION BY PORPHYRIN-PHOTOSENSITIZED OXIDATION OF HUMAN LOW DENSITY LIPOPROTEINS

Abstract— When incorporated into LDL, protoporphyrin and the porphyrin mixture constituting the trade mark Photofrin II photosensitize peroxidations of cholesterol and lipids. Not only 5a-hydroperoxicholesterol, the specific product of cholesterol oxidation by ¹O₂ but also the epimeric 6-hydroperoxicholesterols are produced as shown by HPLC. In addition to malonaldehyde-like substances, the formation of 4-hydroxynonenal, a highly reactive and toxic aldehyde resulting from lipid peroxide breakdown is detected. These products are also formed by dark radical chain reactions of lipid photoperoxides induced by photosensitization. The involvement of lipid photoperoxidation in LDL apoprotein modification is demonstrated by following the derivatization of e-NH₂ groups of Lys residues which are necessary to binding to the LDL receptor. As a result, photosensitized LDL cannot bind to their receptors on fibroblasts. Lys residues are not sensitive to direct photodynamic reaction as confirmed by delipidation of LDL and solubilization of apoLDL in 1% SDS which totally inhibit Lys derivatization without affecting the degradation of Trp residues susceptible to the photodynamic attack. Another consequence of lipid peroxidation at the protein level is the formation of cross-links between apoLDL and photooxidized lipids as shown using LDL loaded with radioactive arachidonic acid. On the other hand, cholesterol photoperoxidation does not lead to protein-oxidized cholesterol cross-links. These reactions between peroxidized lipids and LDL proteins are also responsible for the formation of lipofuscin-like fluorescent pigments encountered in all aging processes. The biological and biomedical consequences of these results are discussed.     

SENSIBILISIERTE PHOTOOXYDATION DURCH METHYLENBLAU THIOPYRONIN UND PYRONIN-III. MITTEILUNG: ÜBER DEN MECHANISMUS DER PHOTOSENSIBILISIERTEN OXYDATION DES GUANOSINS DURCH THIOPYRONIN

Abstract— The thiopyronin-sensitized photooxidation of guanosine was investigated, using flash photolysis techniques. The reaction kinetics of three short-lived intermediates (the triplet state, the reduced radical, and the oxidized radical) were followed by spectroscopy. The influence of guanosine concentration on the reaction rate shows that only the oxidized radical (reaction 7) of thiopyronin is affected by guanosine. This suggests that the primary step in the photodynamic destruction of DNA is the oxidation of guanosine by the oxidized radical of thiopyronin (reaction 9). This mechanism is discussed in terms of the redox potentials of the donor and the acceptor for three different cases: (1) where the oxidation potential of the donor is more negative than that of the triplet state, the acceptor being the triplet state ( F^T ); (2) where the oxidation potential of the donor is between the potentials of the triplet state and the oxidized radical, the acceptor being the oxidized radical (F_ox); and (3) where the oxidation potential of the donor is more positive than the reduction potential of both the triplet state and the oxidized radical; in this case no electron exchange takes place. The thiopyronin-guanosine system is an example of the second case.     

Neighboring pyrrolidine amide participation in thioether oxidation. Methionine as a "hopping" site

Methionine residues have been shown to function as efficient "hopping" sites in long-range electron transfer in model polyprolyl peptides. We suggest that a key to this ability of methionine is stabilization of the transient sulfur radical cation by neighboring proline amide participation. That is, in a model system a neighboring pyrrolidine amide lowers the oxidation potential of the thioether by over 0.5 V by formation of a two-center three-electron SO bond.     

Hydrogen bonding between histidine and lignin model compounds or redox mediators as calculated with the DFT method. Effects on the ease of oxidation

Using the Density Functional Theory method, the effect of hydrogen bonding between imidazole (IM) and ten benzyl alcohol derivatives (BA) on the ionization potentials of the latter is calculated. IM is used as a model for histidine, which is found in the reaction sites of laccases and lignin peroxidases, and the BA-derivatives serve as lignin model compounds. A marked decrease ([similar]15 kcal mol(-1)) is found for the IP's of the BA-derivatives when paired with IM. This should facilitate the one-electron oxidation of BA in the reaction site of the enzyme. The same effect is found for the known redox mediators violuric acid, 1-hydroxybenzotriazole and N-hydroxyacetanilide which are assumed to enter the reaction site of the enzymes. Furthermore, upon one-electron oxidation the strength of the H-bond from BA to IM is considerably increased and in the case of the mediators this effect is so pronounced that the relevant proton shifts from them to IM. If this occurs in the active site of the enzyme then the oxidized redox mediators are released into the aqueous phase in their neutral form rather than as radical cations (deprotonation of the radical cations). The oxidation power of the neutral radical mediators, however, is too low to initialize oxidation of lignin. A more likely reaction pathway is oxidation of the substrates via hydrogen abstraction. The pertinent bond dissociation energies are similar for the BA-derivatives and the redox mediators, which in principle allows the reaction to occur.     

Advances in the non-linear optical investigation of lyotropic-like low-density human lipoproteins in the native and oxidised states

The Z-Scan optical technique was recently used to measure the non-linear optical response of human lipoproteins, in particular low-density lipoprotein (LDL) particles, regarding their oxidative (atherogenic) state. The higher is the oxidation degree of the LDL particle, the smaller is their non-linear optical response. The main physical process involved in this phenomenon is the increase in the thermal diffusivity of the aqueous LDL solution, as a function of the oxidation degree. The responsible for the heat diffusion across the sample are the hydroperoxides generated by the oxidation. Other human lipoproteins were shown to present non-linear optical responses in the thermal timescale regime. The non-linear optical response of LDL solutions may be used in the development of new tools to quantify the atherogenic particles in the human blood.