Bilirubin scavenges chloramines and inhibits myeloperoxidase-induced protein/lipid oxidation in physiologically relevant hyperbilirubinemic serum

Hypochlorous acid (HOCl), an oxidant produced by myeloperoxidase (MPO), induces protein and lipid oxidation, which is implicated in the pathogenesis of atherosclerosis. Individuals with mildly elevated bilirubin concentrations (i.e., Gilbert syndrome; GS) are protected from atherosclerosis, cardiovascular disease, and related mortality. We aimed to investigate whether exogenous/endogenous unconjugated bilirubin (UCB), at physiological concentrations, can protect proteins/lipids from oxidation induced by reagent and enzymatically generated HOCl. Serum/plasma samples supplemented with exogenous UCB (≤250 µM) were assessed for their susceptibility to HOCl and MPO/H₂O₂/Cl⁻ oxidation, by measuring chloramine, protein carbonyl, and malondialdehyde (MDA) formation. Serum/plasma samples from hyperbilirubinemic Gunn rats and humans with GS were also exposed to MPO/H₂O₂/Cl⁻ to: (1) validate in vitro data and (2) determine the relevance of endogenously elevated UCB in preventing protein and lipid oxidation. Exogenous UCB dose-dependently (P<0.05) inhibited HOCl and MPO/H₂O₂/Cl⁻-induced chloramine formation. Albumin-bound UCB efficiently and specifically (3.9–125 µM; P<0.05) scavenged taurine, glycine, and N-α-acetyllysine chloramines. These results were translated into Gunn rat and GS serum/plasma, which showed significantly (P<0.01) reduced chloramine formation after MPO-induced oxidation. Protein carbonyl and MDA formation was also reduced after MPO oxidation in plasma supplemented with UCB (P<0.05; 25 and 50 µM, respectively). Significant inhibition of protein and lipid oxidation was demonstrated within the physiological range of UCB, providing a hypothetical link to protection from atherosclerosis in hyperbilirubinemic individuals. These data demonstrate a novel and physiologically relevant mechanism whereby UCB could inhibit protein and lipid modification by quenching chloramines induced by MPO-induced HOCl.     

Impact of myeloperoxidase-derived oxidants on the product profile of human 5-lipoxygenase

Human 5-lipoxygenase (5-LOX) oxidizes arachidonic acid to 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HpETE) and leukotriene (LT) A₄. In neutrophils, LTA₄ is further converted to the potent chemoattractant LTB₄. These cells also contain the heme enzyme myeloperoxidase (MPO), which produces several potent oxidants such as hypochlorous acid (HOCl), which are involved in pathogen defense and immune regulation. Here, we addressed the question whether MPO-derived oxidants are able to affect the activity of 5-LOX and the product profile of this enzyme. Human 5-LOX was incubated with increasing amounts of HOCl or HOBr. Afterward, arachidonic acid metabolites of 5-LOX were analyzed by reverse-phase high-performance liquid chromatography as well as by liquid chromatography–electrospray ionization–tandem mass spectrometry. The incubation of 5-LOX with the MPO-derived oxidants significantly changed the product profile of 5-LOX. Thereby, HOCl and HOBr increased the ratio of 5-H(p)ETE to 6-trans-LTB₄ in a concentration-dependent manner. At low oxidant concentrations, there was a strong decrease in the yield of 6-trans-LTB₄, whereas 5-HpETE did not change or increased. Additionally, the formation of 8-HpETE and 12-HpETE by 5-LOX rose slightly with increasing HOCl and HOBr. Comparable results were obtained with the MPO–H₂O₂–Cl⁻ system when glucose oxidase and glucose were applied as a source of H₂O₂. This was necessary because of a strong impairment of 5-LOX activity by H₂O₂. In summary, MPO-derived oxidants showed a considerable impact on 5-LOX, impairing the epoxidation of 5-HpETE, whereas the hydroperoxidation of arachidonic acid was unaffected. Apparently, this was caused by an oxidative modification of critical amino acid residues of 5-LOX. Further work is necessary to assess the specific type and position of oxidation in the substrate-binding cavity of 5-LOX and to specify whether this interaction between 5-LOX and MPO-derived oxidants also takes place in stimulated neutrophils.     

Carbamoylated free amino acids in uremia: HOCl generates volatile protein modifying and cytotoxic oxidant species from N-carbamoyl-threonine but not threonine

N-carbamoylation is the non-enzymatic reaction of cyanate with amino groups. Due to urea-formed cyanate in uremic patients beside carbamoylated proteins also free amino acid carbamoylation has been detected, a modification which has been linked to disturbed protein synthesis as NH₂-derivatisation interferes with peptide bond formation. HOCl the product of the activated MPO/H₂O₂/Cl⁻ system is known to react with the NH₂-group of free amino acids to form chloramines which could exert some protective effect against protein modification and cytotoxicity induced by HOCl. As N-carbamoylation may inhibit formation of chloramines we have used N-carbamoyl-threonine as a model amino acid to study its ability to limit the reactivity of HOCl with proteins (LDL and human serum albumin) and cells (THP-1 monocytes and coronary artery endothelial cells). The data indicate that N-carbamoylation completely abolished the protein- and cell-protective effect of threonine against HOCl attack. In contrast to threonine the reaction of HOCl with carbamoyl-threonine resulted in the formation of volatile oxidant species with protein modifying and cytotoxic potential. The volatile lipophilic inorganic monochloramine (NH₂Cl) was identified as a breakdown product of this reaction.     

Inhibition of Lung Fluid Clearance and Epithelial Na+ Channels by Chlorine, Hypochlorous Acid, and Chloramines

We investigated the mechanisms by which chlorine (Cl₂) and its reactive byproducts inhibit Na⁺-dependent alveolar fluid clearance (AFC) in vivo and the activity of amiloride-sensitive epithelial Na⁺ channels (ENaC) by measuring AFC in mice exposed to Cl₂ (0–500 ppm for 30 min) and Na⁺ and amiloride-sensitive currents (I_Na and I_amil, respectively) across Xenopus oocytes expressing human α-, β-, and γ-ENaC incubated with HOCl (1–2000 μm). Both Cl₂ and HOCl-derived products decreased AFC in mice and whole cell and single channel I_Na in a dose-dependent manner; these effects were counteracted by serine proteases. Mass spectrometry analysis of the oocyte recording medium identified organic chloramines formed by the interaction of HOCl with HEPES (used as an extracellular buffer). In addition, chloramines formed by the interaction of HOCl with taurine or glycine decreased I_Na in a similar fashion. Preincubation of oocytes with serine proteases prevented the decrease of I_Na by HOCl, whereas perfusion of oocytes with a synthetic 51-mer peptide corresponding to the putative furin and plasmin cleaving segment in the γ-ENaC subunit restored the ability of HOCl to inhibit I_Na. Finally, I_Na of oocytes expressing wild type α- and γ-ENaC and a mutant form of βENaC (S520K), known to result in ENaC channels locked in the open position, were not altered by HOCl. We concluded that HOCl and its reactive intermediates (such as organic chloramines) inhibit ENaC by affecting channel gating, which could be relieved by proteases cleavage.     

Kinetic analysis of the role of histidine chloramines in hypochlorous acid mediated protein oxidation

Hypochlorous acid (HOCl) is a powerful oxidant generated from H(2)O(2) and chloride ions by the heme enzyme myeloperoxidase (MPO) released from activated leukocytes. In addition to its potent antibacterial effects, excessive HOCl production can lead to host tissue damage, with this implicated in human diseases such as atherosclerosis, cystic fibrosis, and arthritis. HOCl reacts rapidly with biological materials, with proteins being major targets. Chlorinated amines (chloramines) formed from Lys and His side chains and alpha-amino groups on proteins are major products of these reactions; these materials are however also oxidants and can undergo further reactions. In this study, the kinetics of reaction of His side-chain chloramines with other protein components have been investigated by UV/visible spectroscopy and stopped flow methods at pH 7.4 and 22 degrees C, using the chloramines of the model compound 4-imidazoleacetic acid and N-alpha-acetyl-histidine. The second-order rate constants decrease in a similar order (Cys > Met > disulfide bonds > Trp approximately alpha-amino > Lys > Tyr > backbone amides > Arg) to the corresponding reactions of HOCl, but are typically 5-25 times slower. These rate constants are consistent with His side-chain chloramines being important secondary oxidants in HOCl-mediated damage. These studies suggest that formation and subsequent reactions of His side-chain chloramines may be responsible for the targeted secondary modification of selected protein residues by HOCl that has previously been observed experimentally and highlight the importance of chloramine structure on their subsequent reactivity.     

Formation of reactive sulfite-derived free radicals by the activation of human neutrophils: an ESR study

The objective of this study was to determine the effect of (bi)sulfite (hydrated sulfur dioxide) on human neutrophils and the ability of these immune cells to produce reactive free radicals due to (bi)sulfite oxidation. Myeloperoxidase (MPO) is an abundant heme protein in neutrophils that catalyzes the formation of cytotoxic oxidants implicated in asthma and inflammatory disorders. In this study sulfite (^?SO₃^?) and sulfate (SO₄^??) anion radicals are characterized with the ESR spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the reaction of (bi)sulfite oxidation by human MPO and human neutrophils via sulfite radical chain reaction chemistry. After treatment with (bi)sulfite, phorbol 12-myristate 13-acetate-stimulated neutrophils produced DMPO–sulfite anion radical, –superoxide, and –hydroxyl radical adducts. The last adduct probably resulted, in part, from the conversion of DMPO–sulfate to DMPO–hydroxyl radical adduct via a nucleophilic substitution reaction of the radical adduct. This anion radical (SO₄^??) is highly reactive and, presumably, can oxidize target proteins to protein radicals, thereby initiating protein oxidation. Therefore, we propose that the potential toxicity of (bi)sulfite during pulmonary inflammation or lung-associated diseases such as asthma may be related to free radical formation.     

Inhibition of myeloperoxidase: Evaluation of 2H-indazoles and 1H-indazolones

Myeloperoxidase (MPO) produces hypohalous acids as a key component of the innate immune response; however, release of these acids extracellularly results in inflammatory cell and tissue damage. The two-step, one-pot Davis–Beirut reaction was used to synthesize a library of 2H-indazoles and 1H-indazolones as putative inhibitors of MPO. A structure–activity relationship study was undertaken wherein compounds were evaluated utilizing taurine-chloramine and MPO-mediated H₂O₂ consumption assays. Docking studies as well as toxicophore and Lipinski analyses were performed. Fourteen compounds were found to be potent inhibitors with IC₅₀ values <1 μM, suggesting these compounds could be considered as potential modulators of pro-oxidative tissue injury pertubated by the inflammatory MPO/H₂O₂/HOCl/HOBr system.     

Chloramines and hypochlorous acid oxidize erythrocyte peroxiredoxin 2

Peroxiredoxin 2 (Prx2) is an abundant thiol protein that is readily oxidized in erythrocytes exposed to hydrogen peroxide. We investigated its reactivity in human erythrocytes with hypochlorous acid (HOCl) and chloramines, relevant oxidants in inflammation. Prx2 was oxidized to a disulfide-linked dimer by HOCl, glycine chloramine (GlyCl), and monochloramine (NH₂Cl) in a dose-dependent manner. In the absence of added glucose, Prx2 and GSH showed similar sensitivities. Second-order rate constants for the reactions of Prx2 with NH₂Cl and GlyCl were 1.5 × 10⁴ and 8 M⁻¹ s⁻¹, respectively. The NH₂Cl value is 10 times higher than that for GSH, whereas Prx2 is 30 times less sensitive than GSH to GlyCl. Thus, the relative sensitivity of Prx2 to GlyCl is greater in the erythrocyte. Oxidation of erythrocyte Prx2 and GSH was less in the presence of glucose, probably because of recycling. High doses of NH₂Cl resulted in incomplete regeneration of reduced Prx2, suggesting impairment of the recycling mechanism. Our results show that, although HOCl and chloramines are less selective than H₂O₂, they nevertheless oxidize Prx2. Exposure to these inflammatory oxidants will result in Prx2 oxidation and could compromise the erythrocyte's ability to resist damaging oxidative insult.     

Reevaluation of the rate constants for the reaction of hypochlorous acid (HOCl) with cysteine,methionine, and peptide derivatives using a new competition kinetic approach

Activated white cells use oxidants generated by the heme enzyme myeloperoxidase to kill invading pathogens. This enzyme utilizes H₂O₂ and Cl⁻, Br⁻, or SCN⁻ to generate the oxidants HOCl, HOBr, and HOSCN, respectively. Whereas controlled production of these species is vital in maintaining good health, their uncontrolled or inappropriate formation (as occurs at sites of inflammation) can cause host tissue damage that has been associated with multiple inflammatory pathologies including cardiovascular diseases and cancer. Previous studies have reported that sulfur-containing species are major targets for HOCl but as the reactions are fast the only physiologically relevant kinetic data available have been extrapolated from data measured at high pH (>10). In this study these values have been determined at pH 7.4 using a newly developed competition kinetic approach that employs a fluorescently tagged methionine derivative as the competitive substrate (k(HOCl + Fmoc-Met), 1.5×10⁸ M⁻¹ s⁻¹). This assay was validated using the known k(HOCl + NADH) value and has allowed revised k values for the reactions of HOCl with Cys, N-acetylcysteine, and glutathione to be determined as 3.6×10⁸, 2.9×10⁷, and 1.24×10⁸ M⁻¹ s⁻¹, respectively. Similar experiments with methionine derivatives yielded k values of 3.4×10⁷ M⁻¹ s⁻¹ for Met and 1.7×10⁸ M⁻¹ s⁻¹ for N-acetylmethionine. The k values determined here for the reaction of HOCl with thiols are up to 10-fold higher than those previously determined and further emphasize the critical importance of reactions of HOCl with thiol targets in biological systems.     

Inhibition of myeloperoxidase- and neutrophil-mediated oxidant production by tetraethyl and tetramethyl nitroxides

The powerful oxidant HOCl (hypochlorous acid and its corresponding anion, ⁻OCl) generated by the myeloperoxidase (MPO)–H₂O₂–Cl⁻ system of activated leukocytes is strongly associated with multiple human inflammatory diseases; consequently there is considerable interest in inhibition of this enzyme. Nitroxides are established antioxidants of low toxicity that can attenuate oxidation in animal models, with this ascribed to superoxide dismutase or radical-scavenging activities. We have shown (M.D. Rees et al., Biochem. J. 421, 79–86, 2009) that nitroxides, including 4-amino-TEMPO (4-amino-2,2,6,6-tetramethylpiperidin-1-yloxyl radical), are potent inhibitors of HOCl formation by isolated MPO and activated neutrophils, with IC₅₀ values of ~1 and ~6 µM respectively. The utility of tetramethyl-substituted nitroxides is, however, limited by their rapid reduction by biological reductants. The corresponding tetraethyl-substituted nitroxides have, however, been reported to be less susceptible to reduction. In this study we show that the tetraethyl species were reduced less rapidly than the tetramethyl species by both human plasma (89–99% decreased rate of reduction) and activated human neutrophils (62–75% decreased rate). The tetraethyl-substituted nitroxides retained their ability to inhibit HOCl production by MPO and activated neutrophils with IC₅₀ values in the low-micromolar range; in some cases inhibition was enhanced compared to tetramethyl substitution. Nitroxides with rigid structures (fused oxaspiro rings) were, however, inactive. Overall, these data indicate that tetraethyl-substituted nitroxides are potent inhibitors of oxidant formation by MPO, with longer plasma and cellular half-lives compared to the tetramethyl species, potentially allowing lower doses to be employed.     

Selenium-containing amino acids are targets for myeloperoxidase-derived hypothiocyanous acid: determination of absolute rate constants and implications for biological damage

Elevated MPO (myeloperoxidase) levels are associated with multiple human inflammatory pathologies. MPO catalyses the oxidation of Cl-, Br- and SCN- by H2O2 to generate the powerful oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) respectively. These species are antibacterial agents, but misplaced or excessive production is implicated in tissue damage at sites of inflammation. Unlike HOCl and HOBr, which react with multiple targets, HOSCN targets cysteine residues with considerable selectivity. In the light of this reactivity, we hypothesized that Sec (selenocysteine) residues should also be rapidly oxidized by HOSCN, as selenium atoms are better nucleophiles than sulfur. Such oxidation might inactivate critical Sec-containing cellular protective enzymes such as GPx (glutathione peroxidase) and TrxR (thioredoxin reductase). Stopped-flow kinetic studies indicate that seleno-compounds react rapidly with HOSCN with rate constants, k, in the range 2.8×10(3)-5.8×10(6) M-1·s-1 (for selenomethionine and selenocystamine respectively). These values are ~6000-fold higher than the corresponding values for H2O2, and are also considerably larger than for the reaction of HOSCN with thiols (16-fold for cysteine and 80-fold for selenocystamine). Enzyme studies indicate that GPx and TrxR, but not glutathione reductase, are inactivated by HOSCN in a concentration-dependent manner; k for GPx has been determined as ~5×105 M-1·s-1. Decomposed HOSCN did not induce inactivation. These data indicate that selenocysteine residues are oxidized rapidly by HOSCN, with this resulting in the inhibition of the critical intracellular Sec-dependent protective enzymes GPx and TrxR.     

Loss of 3-chlorotyrosine by inflammatory oxidants: implications for the use of 3-chlorotyrosine as a bio-marker in vivo

Activated neutrophils generate the potent oxidant hypochlorous acid (HOCl) from the enzyme myeloperoxidase (MPO). A proposed bio-marker for MPO-derived HOCl in vivo is 3-chlorotyrosine, elevated levels of which have been measured in several human inflammatory pathologies. However, it is unlikely that HOCl is produced as the sole oxidant at sites of chronic inflammation as other reactive species are also produced during the inflammatory response. The work presented shows that free and protein bound 3-chlorotyrosine is lost upon addition of the pro-inflammatory oxidants, HOCl, peroxynitrite, and acidified nitrite. Furthermore, incubation of 3-chlorotyrosine with activated RAW264.7 macrophages or neutrophil-like HL-60 cells resulted in significant loss of 3-chlorotyrosine. Therefore, at sites of chronic inflammation where there is concomitant ONOO⁻ and HOCl formation, it is possible measurement of 3-chlorotyrosine may represent an underestimate of the true extent of tyrosine chlorination. This finding could account for some of the discrepancies reported between 3-chlorotyrosine levels in tissues in the literature.     

Thiocyanate supplementation decreases atherosclerotic plaque in mice expressing human myeloperoxidase

Abstract

Elevated levels of the heme enzyme myeloperoxidase (MPO) are associated with adverse cardiovascular outcomes. MPO predominantly catalyzes formation of the oxidants hypochlorous acid (HOCl) from Cl^?, and hypothiocyanous acid (HOSCN) from SCN^?, with these anions acting as competitive substrates. HOSCN is a less powerful and more specific oxidant than HOCl, and selectively targets thiols; such damage is largely reversible, unlike much HOCl-induced damage. We hypothesized that increased plasma SCN^?, and hence HOSCN formation instead of HOCl, may decrease artery wall damage. This was examined using high-fat fed atherosclerosis-prone LDLR^–/– mice transgenic for human MPO, with and without SCN^? (10 mM) added to drinking water. Serum samples, collected fortnightly, were analyzed for cholesterol, triglycerides, thiols, MPO, and SCN^?; study-long exposure was calculated by area under the curve (AUC). Mean serum SCN^? concentrations were elevated in the supplemented mice (200–320 μM) relative to controls (< 120 μM). Normalized aortic root plaque areas at sacrifice were 26% lower in the SCN^?-supplemented mice compared with controls (P = 0.0417), but plaque morphology was not appreciably altered. Serum MPO levels steadily increased in mice on the high-fat diet, however, comparison of SCN^?-supplemented versus control mice showed no significant changes in MPO protein, cholesterol, or triglyceride levels; thiol levels were decreased in supplemented mice at one time-point. Plaque areas increased with higher cholesterol AUC (r = 0.4742; P = 0.0468), and decreased with increasing SCN^? AUC (r = ? 0.5693; P = 0.0134). These data suggest that increased serum SCN^? levels, which can be achieved in humans by dietary manipulation, may decrease atherosclerosis burden.     

Protein thiol oxidation and formation of S-glutathionylated cyclophilin A in cells exposed to chloramines and hypochlorous acid

Neutrophil oxidants, including the myeloperoxidase products, HOCl and chloramines, have been linked to endothelial dysfunction in inflammatory diseases such as atherosclerosis. As they react preferentially with sulfur centers, thiol proteins are likely to be cellular targets. Our objectives were to establish whether there is selective protein oxidation in vascular endothelial cells treated with HOCl or chloramines, and to identify sensitive proteins. Cells were treated with HOCl, glycine chloramine and monochloramine, reversibly oxidized cysteines were labeled and separated by 1D or 2D SDS-PAGE, and proteins were characterized by mass spectrometry. Selective protein oxidation was observed, with chloramines and HOCl causing more changes than H(2)O(2). Cyclophilin A was one of the most sensitive targets, particularly with glycine chloramine. Cyclophilin A was also oxidized in Jurkat T cells where its identity was confirmed using a knockout cell line. The product was a mixed disulfide with glutathione, with glutathionylation at Cys-161. Glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxins and cofilin were also highly sensitive to HOCl/chloramines. Cyclophilins are becoming recognized as redox regulatory proteins, and glutathionylation is an important mechanism for redox regulation. Cells lacking Cyclophilin A showed more glutathionylation of other proteins than wild-type cells, suggesting that cyclophilin-regulated deglutathionylation could contribute to redox changes in HOCl/chloramine-exposed cells.     

Selenium compounds have disparate abilities to impose oxidative stress and induce apoptosis

The cancer chemopreventive effect of selenium cannot be fully accounted for by the role of selenium as a component of the antioxidant enzyme glutathione peroxidase, which suggests that chemoprevention occurs by another mechanism. Several studies have shown that thiol oxidation and free radical generation occur as a consequence of selenium catalysis and toxicity. In the present study, we evaluated three different selenium compounds; selenite, selenocystamine, and selenomethionine to determine the relative importance of the prooxidative effects of these compounds with regard to their ability to induce apoptosis. The experimental results suggest that, in addition to supporting an increased activity of glutathione peroxidase, an antioxidant function that the three selenium compounds did with equal efficacy, catalytic selenite, and selenocystamine generated 8-hydroxydeoxyguanosine DNA adducts, induced apoptosis and were found to be cytotoxic in mouse keratinocytes. The noncatalytic selenomethionine was not cytotoxic, did not generate 8-hydroxydeoxyguanosine adducts and did not induce cellular apoptosis at any of the selenium concentrations studied. In keratinocytes, apoptosis may be initiated by superoxide (O₂^•−) and oxidative free radicals that are generated by selenite and selenocystamine, but not by selenomethionine.     

Hypochlorous acid-modified low-density lipoprotein inactivates the lysosomal protease cathepsin B: protection by ascorbic and lipoic acids

Abstract

Unregulated uptake of oxidized LDL by the scavenger receptor (s) of macrophages is thought to be an early event in atherosclerotic lesion development. Accumulation of oxidized LDL within macrophages may result from resistance of the modified LDL to enzymatic hydrolysis or from direct inactivation of lysosomal enzymes by reactive LDL-associated moieties. Since HOCl-modified LDL has been detected in vivo, the effects of HOCl-modified LDL on the activities of the cysteine protease cathepsin B and the aspartyl protease cathepsin D were investigated. LDL (0.5 mg protein/ml), which had been exposed to HOCl (25–200 µM), caused rapid dose-dependent inactivation of cathepsin B, but not of cathepsin D. Exposure of LDL to HOCl results primarily in the formation of LDL-associated chloramines, and the model chloramine N^-acetyl-lysine chloramine also caused dose-dependent inactivation of cathepsin B. Incubation of HOCl-modified LDL with ascorbic and lipoic acids (25–200 µM) resulted in dose-dependent reduction of LDL-associated chloramines and concomitant protection against cathepsin B inactivation. Thus, the data indicate that HOCl-modified LDL inactivates cathepsin B by a chloramine-dependent mechanism, most likely via oxidation of the enzyme's critical cysteine residue. Furthermore, small molecule antioxidants, such as ascorbic and lipoic acids, may be able to inhibit this potentially proatherogenic process by scavenging LDL-associated chloramines.     

Identification of a Novel Selenium-containing Compound,Selenoneine, as the Predominant Chemical Form of Organic Selenium in the Blood of Bluefin Tuna

A novel selenium-containing compound having a selenium atom in the imidazole ring, 2-selenyl-N_α,N_α,N_α-trimethyl-l-histidine, 3-(2-hydroseleno-1H-imidazol-5-yl)-2-(trimethylammonio)propanoate, was identified from the blood and other tissues of the bluefin tuna, Thunnus orientalis. The selenium-containing compound was purified from the tuna blood in several chromatographic steps. High resolution mass spectrometry and nuclear magnetic resonance spectroscopy showed that the exact mass of the [M+H]⁺ ion of the compound was 533.0562 and the molecular formula was C₁₈H₂₉N₆O₄Se₂. Its gross structure was assigned as the oxidized dimeric form of an ergothioneine selenium analog in which the sulfur of ergothioneine is replaced by selenium. Therefore, we named this novel selenium-containing compound “selenoneine.” By speciation analysis of organic selenium compounds using liquid chromatography inductively coupled plasma mass spectrometry, selenoneine was found widely distributed in various tissues of the tuna, with the highest concentration in blood; mackerel blood contained similar levels. Selenoneine was measurable at 2–4 orders of magnitude lower concentration in a limited set of tissues from squid, tilapia, pig, and chicken. Quantitatively, selenoneine is the predominant form of organic selenium in tuna tissues.     

High plasma thiocyanate levels are associated with enhanced myeloperoxidase-induced thiol oxidation and long-term survival in subjects following a first myocardial infarction

Abstract

Elevated levels of myeloperoxidase (MPO) are associated with poor cardiovascular outcomes. MPO uses H₂O₂ to generate oxidants including HOCl and HOSCN, from chloride and thiocyanate (SCN^?) ions, respectively. SCN^? is the preferred substrate. Elevation of this anion decreases HOCl generation and increases HOSCN formation, a thiol-specific oxidant. Such changes are of potential relevance to people with elevated SCN^? levels, such as smokers. In this retrospective study, we examined whether elevated plasma MPO and SCN^? levels increased thiol oxidation as a result of increased HOSCN formation, and impacted on long-term survival in 176 subjects (74 non-smokers, 46 smokers, and 56 previous smokers) hospitalized after a first myocardial infarction. Plasma thiols were not significantly altered in smokers compared to non-smokers or past smokers. However, significant positive correlations were detected between SCN^? levels and MPO-induced thiol loss in the total population (r = 0.19, P = 0.020) and smokers alone (r = 0.58, P < 0.0001). Twelve-year all-cause mortality data indicate that above median MPO is significantly associated with higher mortality, but below-median MPO and above-median SCN^? results in increased survival, compared to below-median SCN^?. Cox proportional hazard analysis showed a significant decrease in mortality for each 1 μM increase in SCN^? (0.991; P = 0.040). Subject age was, as expected, a strong predictor of subject survival. Overall these data suggest that subjects with below-median MPO and above-median SCN^? have better long-term survival, and that elevated plasma levels of SCN^? might be protective in at least some populations.     

Hypochlorous Acid Converts the ��-Glutamyl Group of Glutathione Disulfide to 5-Hydroxybutyrolactam, a Potential Marker for Neutrophil Activation

In healthy cells, glutathione disulfide (GSSG) is rapidly reduced back to glutathione (GSH) by glutathione reductase to maintain redox status. The ratio of GSH/GSSG has been used as an indicator of oxidative stress. However, hypochlorous acid (HOCl) generated by the myeloperoxidase-H₂O₂-Cl⁻ system of neutrophils converts GSH to irreversible oxidation products. Although several such products have been identified, yields of these compounds are very low in biological systems, and they cannot account quantitatively for thiol loss. In the current studies, we use liquid chromatography-mass spectrometry (LC-MS) to demonstrate that HOCl and chloramines oxidize GSSG to two irreversible products in high yield. The products, termed M-45 and M-90, are, respectively, 45 or 90 atomic mass units lighter than GSSG. The reaction pathway involves chloramine and aldehyde intermediates, and converts the γ-glutamyl residues of GSSG to 5-hydroxybutyrolactam. Importantly, M-45 and M-90 were resistant to reduction by glutathione reductase. Moreover, the monohydroxylbutyrolactam M-45 accounted for >90% of the endogenous GSH oxidation products generated by activated neutrophils. Because the reaction pathway involves chlorinating intermediates, hydroxylbutyrolactams are likely to be specific products of HOCl, which is generated only by myeloperoxidase. Therefore, our observations implicate M-45 as a potential biomarker for myeloperoxidase activity in vivo.Glutathione (GSH), a tripeptide synthesized in the cytosol from glutamate, cysteine, and glycine, is the predominant antioxidant in mammalian cells. Its concentration ranges from millimolar inside cells to micromolar in plasma (1, 2). In many cells, GSH accounts for >90% of total nonprotein thiol (3, 4). The free thiol group in GSH is responsible for biological activity. As a nucleophilic scavenger, GSH can directly react with electrophilic substances, such as reactive oxygen/nitrogen species, or be oxidized by GSH peroxidase to glutathione disulfide (GSSG). Therefore, it is essential for maintaining intracellular redox status and defending against oxidative injury. Under normal circumstances, GSSG is rapidly reduced back to GSH by glutathione reductase and NADPH. Thus, most of the GSH remains in the reduced form. Under oxidative stress, however, GSH is converted to GSSG, which potentially accumulates (2, 5). Indeed, the GSH/GSSG ratio has been used to evaluate oxidative stress in biological systems. Alterations of this ratio associate with a variety of diseases, including atherosclerosis, cancer, and human immunodeficiency virus infection (6–10).One important source of oxidative stress in humans is myeloperoxidase (MPO),² a heme protein expressed by neutrophils, monocytes, and certain populations of macrophages (11–13). Activation of these inflammatory white blood cells results in the secretion of MPO, which uses hydrogen peroxide (H₂O₂, produced by NADPH oxidase) and chloride anion to generate hypochlorous acid (HOCl) (14). HOCl rapidly reacts with a wide range of functional groups (15–19). At physiological pH, thiol groups and free amino groups are its main targets, and the initial products are oxidized thiols and chloramines.HOCl generates other products in addition to GSSG when it reacts with GSH. Chesney et al. (20) suggested that it oxidizes GSH to a higher oxidation state than the disulfide form because the molar ratio of HOCl consumed to GSH oxidized was 4:1 instead of 1:1 in Escherichia coli. Winterbourn (21) reported that approximately half of the GSH oxidized by HOCl could not be regenerated. These researchers have identified glutathione sulfonamide (GSA), glutathione thiosulfonate, and dehydroglutathione as irreversible higher oxidation products (22, 23). Their observations suggest that the formation of higher order GSSG oxidation products might account in part for the irreversible loss of GSH induced by HOCl. However, activated neutrophils (the source of MPO and therefore of HOCl) generate only low yields of these higher oxidation products, suggesting that the major products of GSH oxidation by MPO remain to be identified.The disulfide and α-amino groups of GSSG are also potential targets of HOCl (17). Disulfides can be oxidized to sulfonic acid via a sulfenyl chloride intermediate (16). α-Amino groups yield chloramines, which undergo decarboxylation, intramolecular H-abstraction, or other reaction pathways to form various products, such as aldehydes and carboxymethyllysine (16, 24). These reactions may be biologically relevant, because carboxymethyllysine production is impaired in mice deficient in the phagocyte NADPH oxidase (25). These observations suggest that GSSG is a potential scavenger of HOCl. Indeed, GSSG reportedly competes for HOCl with its rate constant expected to be 2 × 10⁵ m⁻¹ s⁻¹ (26, 27). Studies from Bast et al. (28) demonstrated that GSSG protects acetylcholinesterase from oxidative inactivation by HOCl. Nagy and Ashby (29) studied the kinetics and mechanism of GSSG oxidation by HOCl. They proposed that HOCl generates the bis-N-chloro-γ-l-glutamyl derivative of GSSG. These studies suggest that GSSG itself may function as an antioxidant.In the current study, we investigated the reaction of GSSG with HOCl and other oxidants. Using liquid chromatography in concert with mass spectrometry (LC-MS), we identified two groups of novel oxidation products, which we termed M-45 and M-90. We characterized their structures and potential reaction pathways. Our results indicate that HOCl and chloramines oxidize the γ-glutamyl moiety of GSSG to 5-hydroxybutyrolactam in high yield.     

A revision of the structure of cucurbitacin S from Bryonia dioica

¹H NMR studies at 400 MHz on cucurbitacin S and its derivatives suggest that the structure of cucurbitacin S previously isolated from Bryonia dioica should be revised from (22S)-22, 25-anhydro-2,16α,22,25-tetrahydroxy-3,11,23-trioxo-cucurbita-1,5-diene to (24S)-16,24-anhydro- 2,16α,24,25-tetrahydroxy-3,11,22-trioxo-cucurbita-1,5-diene.