Autoantibody response to chromatographic fractions from oxidized LDL in unstable angina patients and healthy controls

Levels of autoantibodies to oxidized low-density lipoprotein (oxLDL) have been correlated to atherosclerosis; however, contradictory results have been shown. To better understand the role of autoantibodies to oxLDL in atherogenesis, and their potential to predict risk of developing coronary artery disease we investigated the antibody response of unstable angina (UA) patients and healthy controls against chromatographic separated fractions of oxLDL. Five major peaks were detected after chromatographic separation of oxLDL and 10 fractions were collected. Surprisingly, when the response to high molecular weight fractions was analysed, we observed a significant increase in the levels of autoantibodies in controls compared to UA. In contrast, when the autoantibody response to intermediate and low molecular weight fractions was analysed, we observed that the UA group showed consistently higher levels compared with controls. Our data demonstrates that within oxLDL there are major fractions that can be recognized by autoantibodies from either UA patients or healthy individuals, and that the use of total oxLDL as an antigen pool may mask the presence of some antigenic molecules and their corresponding antibodies. Further studies are needed, but the analysis of antibody profiles may indeed open up a novel approach for evaluation and prevention against atherosclerosis.

Preparation of recoverable Ru catalysts for liquid-phase oxidation and hydrogenation reactions

We here report the synthesis, characterization and catalytic performance of new supported Ru(III) and Ru(0) catalysts. In contrast to most supported catalysts, these new developed catalysts for oxidation and hydrogenation reactions were prepared using nearly the same synthetic strategy, and are easily recovered by magnetic separation from liquid phase reactions. The catalysts were found to be active in both forms, Ru(III) and Ru(0), for selective oxidation of alcohols and hydrogenation of olefins, respectively. The catalysts operate under mild conditions to activate molecular oxygen or molecular hydrogen to perform clean conversion of selected substrates. Aryl and alkyl alcohols were converted to aldehydes under mild conditions, with negligible metal leaching. If the metal is properly reduced, Ru(0) nanoparticles immobilized on the magnetic support surface are obtained, and the catalyst becomes active for hydrogenation reactions. (c) 2009 Elsevier B.V. All rights reserved.

A combination of techniques to evaluate photodynamic efficiency of photosensitizers

Photodynamic therapy, used mainly for cancer treatment and microorganisms inaction, is based on production of reactive oxygen species by light irradiation of a sensitizer. Hematoporphyrin derivatives as Photofrin (R) (PF) Photogem (R) (PG) and Photosan (R) (PF), and chlorin-c6-derivatives as Photodithazine (R)(PZ), have suitable sensitizing properties. The present study provides a way to make a fast previous evaluation of photosensitizers efficacy by a combination of techniques: a) use of brovine serum albumin and uric acid as chemical dosimeters; b) photo-hemolysis of red blood cells used as a cell membrane interaction model, and c) octanol/phosphate buffer partition to assess the relative lipophilicity of the compounds. The results suggest the photodynamic efficient rankings PZ > PG >= PF > PS. These results agree with the cytotoxicity of the photosensitizers as well as to chromatographic separation of the HpDs, both performed in our group, showing that the more lipophilic is the dye, the more acute is the damage to the RBC membrane and the oxidation of indol, which is immersed in the hydrophobic region of albumin.

Evidence of fluoride oxidation for development of the electrochemically-induced crystallization observed in lead oxyfluoroborate glasses

This work reports on a distinct experimental procedure conceived to closely approach the question of development of crystallization in lead oxyfluoroborate glasses in the presence of an electric field. After proposing earlier that this phenomenon should involve occurrence of redox-type electrochemical reactions occurring at the electrodes. it was in fact recently shown that a direct contact of the glasses with both the cathode and anode revealed essential, provided that crystallization did not develop when ions migration to these electrodes became frustrated. The present study demonstrates that. even in Pt,Ag/Glass/YSZ:PbF(2)/Ag,Pt-type electrochemical cells subjected to electric field action, where YSZ:PbF(2) represents composite-like mixtures (formed by Y(2)O(3)-doped ZrO(2) and PbF(2)) placed between the glass and anode. crystallization was observable in given cases. In summary, supported by (micro)structural and electrical characterizations, clear evidence is provided here that, besides Pb(2+) reduction at the cathode, crystallization really involves simultaneous F(-) oxidation at the anode, completing thus the whole redox electrochemical reaction so far postulated. In these cases, F(-) migration to the anode was achievable following PbF(2) percolative-like paths through the YSZ:PbF(2) mixtures. (C) 2010 Elsevier B.V. All rights reserved.

Lipid hydroperoxide-induced and hemoglobin-enhanced oxidative damage to colon cancer cells

Epidemiological studies have indicated that Western diets are related to an increase in a series of malignancies. Among the compounds that are credited for this toxic effect are heme and lipid peroxides. We evaluated the effects of hemoglobin (Hb) and linoleic acid hydroperoxides (LAOOH) on a series of toxicological endpoints, such as cytotoxicity, redox status, lipid peroxidation, and DNA damage. We demonstrated that the preincubation of SW480 cells with Hb and its subsequent exposure to LAOOH (Hb + LAOOH) led to an increase in cell death, DCFH oxidation, malonaldehyde formation, and DNA fragmentation and that these effects were related to the peroxide group and the heme present in Hb. Furthermore, Hb and LAOOH alone exerted a toxic effect on the endpoints assayed only at concentrations higher than 100 mu M. We were also able to show that SW480 cells presented a higher level of the modified DNA bases 8-oxo-7,8-dihydro-2`-deoxyguanosine and 1,N(2)-etheno-2`-deoxyguanosine compared to the control. Furthermore, incubations with Hb led to an increase in intracellular iron levels, and this high level of iron correlated with DNA oxidation, as measured as EndoIII- and Fpg-sensitive sites. Thus, Hb from either red meat or bowel bleeding could act as an enhancer of fatty acid hydroperoxide genotoxicity, which contributes to the accumulation of DNA lesions in colon cancer cells. (C) 2011 Elsevier Inc. All rights reserved.

Oxidation and nitration of ribonuclease and lysozyme by peroxynitrite and myeloperoxidase

In spite of the many studies on protein modifications by reactive species, knowledge about the products resulting from the oxidation of protein-aromatic residues, including protein-derived radicals and their stable products, remains limited. Here, we compared the oxidative modifications promoted by peroxynitrite and myeloperoxidase/hydrogen peroxide/nitrite in two model proteins, ribonuclease (6Tyr) and lysozyme (3Tyr/6Trp). The formation of protein-derived radicals and products was higher at pH 5.4 and 7.4 for myeloperoxidase and peroxynitrite, respectively. The main product was 3-nitro-Tyr for both proteins and oxidants. Lysozyme rendered similar yields of nitro-Trp, particularly when oxidized by peroxynitrite. Hydroxylated and dimerized products of Trp and Tyr were also produced, but in lower yields. Localization of the main modified residues indicates that peroxynitrite decomposes to radicals within the proteins behaving less specifically than myeloperoxidase. Nitrogen dioxide is emphasized as an important protein modifier. (C) 2009 Elsevier Inc. All rights reserved.

Characterization of O(2) ((1)Delta(g))-Derived Oxidation Products of Tryptophan: A Combination of Tandem Mass Spectrometry Analyses and Isotopic Labeling Studies

The fragmentation mechanisms of singlet oxygen [O(2) ((1)Delta(g))]-derived oxidation products of tryptophan (W) were analyzed using collision-induced dissociation coupled with (18)O-isotopic labeling experiments and accurate mass measurements. The five identified oxidized products, namely two isomeric alcohols (trans and cis WOH), two isomeric hydroperoxides (trans and cis WOOH), and N-formylkynurenine (FMK), were shown to share some common fragment ions and losses of small neutral molecules. Conversely, each oxidation product has its own fragmentation mechanism and intermediates, which were confirmed by (18)O-labeling studies. Isomeric WOH lost mainly H(2)O + CO, while WOOH showed preferential elimination of C(2)H(5)NO(3) by two distinct mechanisms. Differences in the spatial arrangement of the two isomeric WOHs led to differences in the intensities of the fragment ions. The same behavior was also found for trans and cis WOOH. FMK was shown to dissociate by a diverse range of mechanisms, with the loss of ammonia the most favored route. MS/MS analyses, (18)O-labeling, and H(2)(18)O experiments demonstrated the ability of FMK to exchange its oxygen atoms with water. Moreover, this approach also revealed that the carbonyl group has more pronounced oxygen exchange ability compared with the formyl group. The understanding of fragmentation mechanisms involved in O(2) ((1)Delta(g))-mediated oxidation of W provides a useful step toward the structural characterization of oxidized peptides and proteins. (J Am Soc Mass Spectrom 2009, 20, 188-197) (C) 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry

1,4-Diamino-2-butanone, a wide-spectrum microbicide, yields reactive species by metal-catalyzed oxidation

The alpha-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB`s cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37 degrees C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other alpha-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml(-1)) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(center dot), and those with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(center dot) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 mu M) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity. (C) 2011 Elsevier Inc. All rights reserved.

Singlet oxygen reacts with 2 `,7 `-dichlorodihydrofluorescein and contributes to the formation of 2 `,7 `-dichlorofluorescein

There are controversial reports in the literature concerning the reactivity of singlet oxygen ((1)O(2)) with the redox probe 2`,7`-dichlorodihydrofluorescein (DCFH). By carefully preparing solutions in which (1)O(2) is quantitatively generated in the presence of DCFH, we were able to show that the formation rate of the fluorescent molecule derived from DCFH oxidation, which is 2`,7`-dichlorofluorescein (DCF), increases in D(2)O and decreases in sodium azide, proving the direct role of (1)O(2) in this process. We have also prepared solutions in which either (1)O(2) or dication (MB(center dot 2+)) and semi-reduced (MB(center dot)) radicals of the sensitizer and subsequently super-oxide radical (O(2)(center dot-)) are generated. The absence of any effect of SOD and catalase ruled out the DCFH oxidation by O(2)(center dot-), indicating that both (1)O(2) and MB(center dot 2+) react with DCFH. Although the formation of DCF was 1 order of magnitude larger in the presence of MB(center dot 2+) than in the presence of (1)O(2), considering the rate of spontaneous decays of these species in aqueous solution, we were able to conclude that the reactivity of (1)O(2) with DCFH is actually larger than that of MB(center dot 2+). We conclude that DCFH can continue to be used as a probe to monitor general redox misbalance induced in biologic systems by oxidizing radicals and (1)O(2).

Tryptophan oxidation by singlet molecular oxygen [O-2 ((1)Delta(g))]: Mechanistic studies using O-18-labeled hydroperoxides, mass spectrometry, and light emission measurements

Proteins have been considered important targets for reactive oxygen species. Indeed, tryptophan (W) has been shown to be a highly susceptible amino acid to many oxidizing agents, including singlet molecular oxygen [O-2 ((1)Delta(g))]. In this study, two cis- and trans-tryptophan hydroperoxide (WOOH) isomers were completely characterized by HPLC/mass spectrometry and NMR analyses as the major W-oxidation photoproducts. These photoproducts underwent thermal decay into the corresponding alcohols. Additionally, WOOHs were shown to decompose under heating or basification, leading to the formation of N-formylkynurenine (FMK). Using O-18-labeled hydroperoxides ((WOOH)-O-18-O-18), it was possible to confirm the formation of two oxygen-labeled FMK molecules derived from (WOOH)-O-18-O-18 decomposition. This result demonstrates that both oxygen atoms in FMK are derived from the hydroperoxide group. In addition, these reactions are chemiluminescent (CL), indicating a dioxetane cleavage pathway. This mechanism was confirmed since the CL spectrum of the WOOH decomposition matched the FMK fluorescence spectrum, unequivocally identifying FMK as the emitting species.

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 +/- 0.2) x 10(7) M(-1) s(-1)) and peroxynitrite ((3.7 +/- 0.4) x 10(5) M(-1) s(-1)) at pH 7.4 and 25 degrees C. (C) 2011 Elsevier Inc. All rights reserved.

Saccharomyces cerevisiae coq10 null mutants are responsive to antimycin A

Deletion of COQ10 in Saccharomyces cerevisiae elicits a respiratory defect characterized by the absence of cytochrome c reduction, which is correctable by the addition of exogenous diffusible coenzyme Q(2). Unlike other coq mutants with hampered coenzyme Q(6) (Q(6)) synthesis, coq10 mutants have near wild-type concentrations of Q(6). In the present study, we used Q-cycle inhibitors of the coenzyme QH(2)-cytochrome c reductase complex to assess the electron transfer properties of coq10 cells. Our results show that coq10 mutants respond to antimycin A, indicating an active Q-cycle in these mutants, even though they are unable to transport electrons through cytochrome c and are not responsive to myxothiazol. EPR spectroscopic analysis also suggests that wild-type and coq10 mitochondria accumulate similar amounts of Q(6) semiquinone, despite a lower steady-state level of coenzyme QH(2)-cytochrome c reductase complex in the coq10 cells. Confirming the reduced respiratory chain state in coq10 cells, we found that the expression of the Aspergillus fumigatus alternative oxidase in these cells leads to a decrease in antimycin-dependent H(2)O(2) release and improves their respiratory growth.

Polymethine cyanine dyes in beta-cyclodextrin solution: multiple equilibria and chemical oxidation

Absorption and fluorescence spectroscopy, electrochemical techniques, and semiempirical calculations were employed to characterize the multiple complexation equilibria between two polymethine cyanine dyes (IR-786 and Indocyanine green-ICG, 5) and beta-cyclodextrin (beta-CD, L), as well as the chemical reactivity of the complexed and uncomplexed species against the oxidizing agents hypochlorite (HC) and hydrogen peroxide (HP). IR-786 dimerization is favored with the increase in beta-CD concentration in the form of (SL)(2) complexes. In the case of ICG, free dimers (D) and SL complexes are favored. Both IR-786 and ICG react and discolor in the presence of HC and HP. For IR-786, the reaction with HP and HC proceeds with observed rate constants of 10(-3) and 0.28 s(-1) and second-order rate constants (k(2)) of similar to 10(-3) and 10(4) M(-1) s(-1), respectively. The intermediate species observed in the bleaching reactions of IR-786 and ICG were shown, by cyclic voltammetry and VIS absorption, to result from one electron oxidation. IR-786 complexed with beta-CD is protected against bleaching in the presence of HP and HC by factors of 20 and 4, respectively. This protection was not observed in ICG complexes. Superdelocalizability profile of both dyes and frontier orbital analysis indicates that beta-CD does not protect ICG from oxidation by HP or HC, whereas the 2:2 IR-786/beta-Cd complex is able to avoid the oxidation of IR-786. We concluded that the decrease in the chemical reactivity of the dyes against oxidant agents in the presence of beta-CD is due to the formation of (SL)(2) complexes. Copyright (C) 2010 John Wiley & Sons, Ltd.

Detection and Characterization of Cholesterol-Oxidized Products Using HPLC Coupled to Dopant Assisted Atmospheric Pressure Photoionization Tandem Mass Spectrometry

Oxidation of cholesterol (Ch) by a variety of reactive oxygen species gives rise mainly to hydroperoxides and aldehydes. Despite the growing interest in Ch-oxidized products, the detection and characterization of these products is still a matter of concern. In this work, the main Ch-oxidized products, namely, 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide (7 alpha-OOH), 3 beta-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 alpha-hydroperoxide (6 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH), and 3 beta-hydroxy-5 beta-hydroxy-B-norcholestane-6 beta-carboxaldehyde (ChAld), were detected in the same analysis using high-performance liquid chromatography (HPLC) coupled to dopant assisted atmospheric pressure photoionization tandem mass spectrometry. The use of selected reaction monitoring mode (SRM) allowed a sensitive detection of each oxidized product, while the enhanced product ion mode (EPI) helped to improve the confidence of the analyses. Isotopic labeling experiments enabled one to elucidate mechanistic features during fragmentation processes. The characteristic fragmentation pattern of Ch-oxidized products is the consecutive loss of 1120 molecules, yielding cationic fragments at m/z 401, 383, and 365. Homolytic scissions of the peroxide bond are also seen. With (18)O-labeling approach, it was possible to establish a fragmentation order for each isomer. The SRM transitions ratio along with EPI and (18)O-labeled experiments give detailed information about differences for water elimination, allowing a proper discrimination between the isomers:Phis is of special interest considering the emerging role of Ch-oxidized products in the development of diseases.

Effect of flavonoids on 2 `-deoxyguanosine and DNA oxidation caused by singlet molecular oxygen

Antioxidant potential is generally investigated by assaying the ability of a compound to protect biological systems from free radicals. However, non-radical reactive oxygen species can also be harmful. Singlet molecular oxygen ((1)O(2)) is generated by energy transfer to molecular oxygen. The resulting (1)O(2) is able to oxidize the nucleoside 2`-deoxyguanosine (dGuo), which leads to the formation of 8-oxo-7,8-dihydro-2`-deoxyguanosine (8-oxodGuo) and spiroiminodihydantoin 2`-deoxyribonucleoside diastereomers (dSp) in an aqueous solution. The main objective of the present study was to verify whether the presence of flavonoids (flavone, apigenin, quercetin, morin and catechin) at different concentrations could protect dGuo from (1)O(2) damage. Of the tested flavonoids, flavone possessed antioxidant activity, as determined by a decrease in the formation of both products. Apigenin, morin, quercetin and catechin all increased the formation of 8-oxodGuo at a concentration of 100 mu M. The quantification of plasmid strand breaks after treatment with formamidopyrimidine-DNA glycosylase showed that flavone protected and quercetin and catechin enhanced DNA oxidation. Our results show that compounds, such as flavonoids, may affect the product distribution of (1)O(2)-mediated oxidation of dGuo, and, in particular, high concentrations of flavonoids with hydroxyl groups in their structure lead to an increase in the formation of the mutagenic lesion 8-oxodGuo. (C) 2010 Elsevier Ltd. All rights reserved.