A central role for intermolecular dityrosine cross-linking of fibrinogen in high molecular weight advanced oxidation protein product (AOPP) formation

Background

Advanced oxidation protein products (AOPPs) are dityrosine cross-linked and carbonyl-containing protein products formed by the reaction of plasma proteins with chlorinated oxidants, such as hypochlorous acid (HOCl). Most studies consider human serum albumin (HSA) as the main protein responsible for AOPP formation, although the molecular composition of AOPPs has not yet been elucidated. Here, we investigated the relative contribution of HSA and fibrinogen to generation of AOPPs.

Methods

AOPP formation was explored by SDS-PAGE, under both reducing and non-reducing conditions, as well as by analytical gel filtration HPLC coupled to fluorescence detection to determine dityrosine and pentosidine formation.

Results

Following exposure to different concentrations of HOCl, HSA resulted to be carbonylated but did not form dityrosine cross-linked high molecular weight aggregates. Differently, incubation of fibrinogen or HSA/fibrinogen mixtures with HOCl at concentrations higher than 150 μM induced the formation of pentosidine and high molecular weight (HMW)-AOPPs (> 200 kDa), resulting from intermolecular dityrosine cross-linking. Dityrosine fluorescence increased in parallel with increasing HMW-AOPP formation and increasing fibrinogen concentration in HSA/fibrinogen mixtures exposed to HOCl. This conclusion is corroborated by experiments where dityrosine fluorescence was measured in HOCl-treated human plasma samples containing physiological or supra-physiological fibrinogen concentrations or selectively depleted of fibrinogen, which highlighted that fibrinogen is responsible for the highest fluorescence from dityrosine.

Conclusions

A central role for intermolecular dityrosine cross-linking of fibrinogen in HMW-AOPP formation is shown.

General significance

These results highlight that oxidized fibrinogen, instead of HSA, is the key protein for intermolecular dityrosine formation in human plasma.     

Investigation of protein oxidation and lipid peroxidation in patients with rheumatoid arthritis

We aimed to determine the importance of neutrophil activation and the source of oxidative stress in the pathogenesis of rheumatoid arthritis (RA) by quantification of advanced oxidation protein products (AOPP) and total thiol levels as markers of oxidative protein damage, malondialdehyde (MDA) levels as a marker of lipid peroxidation and myeloperoxidase (MPO) activity as a marker of neutrophil activation in patients with RA. Fifty-seven rheumatoid arthritis patients were included in the study and sub-grouped according to disease activity (active, n = 31; inactive, n = 26) and compared with healthy controls (n = 25). Serum MPO activity, AOPP, MDA, and thiol levels were measured by an enzymic spectrophotometric method. Serum MPO activity (p < 0.001), AOPP (p < 0.001), MDA (p < 0.001) and levels of thiol (p < 0.002), were higher in the patient group than the controls. Active and inactive RA groups were compared with the control group and there were significant differences between each parameter. MPO activity, AOPP, MDA and thiol levels were significantly higher in both active and inactive RA patients than the controls. On the other hand, when a comparison was made between active and the inactive stage, a statistically significant difference was present only in MDA (p < 0.05) and AOPP levels (p < 0.05). There was also a significant positive correlation between all parameters. These data strongly suggest that neutrophils, which constitute the most important source of chlorinated oxidants due to their high MPO content, may be involved in serum AOPP formation and therefore the production of a novel class of pro-inflammatory mediators of oxidative stress in RA patients and that protein oxidation could play an important role in the pathogenesis of RA as does lipid peroxidation.     

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.     

Detection of advanced oxidation protein products in patients with chronic kidney disease by a novel monoclonal antibody

Advanced oxidation protein products (AOPP) as a biomarker of oxidative stress has been demonstrated in chronic kidney disease (CKD) patients; however, current methods to detect the accumulation of AOPP in serum and in tissues are limited and unreliable. This study generated a monoclonal antibody (mAb) designated 3F2, that reacts specifically with hypochlorous acid (HOCl)-modified proteins, but not with the native forms or with other types of oxidative modifications. Notably, mAb 3F2 recognizes the AOPP deposited in renal tissues of AOPP-treated rats and of patients with different kinds of CKD. Moreover, this mAb can almost completely inhibit the production of reactive oxygen species in RAW264.7 cells induced by AOPP (p < 0.001). In conclusion, mAb 3F2 can be used to detect AOPP specifically in serum and in tissues, and this antibody can potentially provide an important tool and new insight into research on diseases related to oxidative stress.     

Hypohalous acid-modified human serum albumin induces neutrophil NADPH oxidase activation,degranulation, and shape change

Halogenated lipids, proteins, and lipoproteins formed in reactions with myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) and hypobromous acid (HOBr) can contribute to the regulation of functional activity of cells and serve as mediators of inflammation. Human serum albumin (HSA) is the major plasma protein target of hypohalous acids. This study was performed to assess the potency of HSA modified by HOCl (HSA–Cl) and HOBr (HSA–Br) to elicit selected neutrophil responses. HSA–Cl/Br were found to induce neutrophil degranulation, generation of reactive oxygen intermediates, shape change, and actin cytoskeleton reorganization. Thus HSA–Cl/Br can initially act as a switch and then as a feeder of the “inflammatory loop” under oxidative stress. In HSA–Cl/Br-treated neutrophils, monoclonal antibodies against CD18, the β subunit of β₂ integrins, reduced the production of superoxide anion radicals and hydrogen peroxide as well as MPO exocytosis, suggesting that CD18 contributed to neutrophil activation. HSA–Cl/Br-induced neutrophil responses were also inhibited by genistein, a broad-specificity tyrosine kinase inhibitor, and wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, supporting the notion that activation of both tyrosine kinase and PI3K may play a role in neutrophil activation by HSA modified in MPO-dependent reactions. These results confirm the hypothesis that halogenated molecules formed in vivo via MPO-dependent reactions can be considered as a new class of biologically active substances potentially able to contribute to activation of myeloid cells in sites of inflammation and serve as inflammatory response modulators.     

Detection of advanced oxidation protein products in patients with chronic kidney disease by a novel monoclonal antibody

Abstract

Advanced oxidation protein products (AOPP) as a biomarker of oxidative stress has been demonstrated in chronic kidney disease (CKD) patients; however, current methods to detect the accumulation of AOPP in serum and in tissues are limited and unreliable. This study generated a monoclonal antibody (mAb) designated 3F2, that reacts specifically with hypochlorous acid (HOCl)-modified proteins, but not with the native forms or with other types of oxidative modifications. Notably, mAb 3F2 recognizes the AOPP deposited in renal tissues of AOPP-treated rats and of patients with different kinds of CKD. Moreover, this mAb can almost completely inhibit the production of reactive oxygen species in RAW264.7 cells induced by AOPP (p < 0.001). In conclusion, mAb 3F2 can be used to detect AOPP specifically in serum and in tissues, and this antibody can potentially provide an important tool and new insight into research on diseases related to oxidative stress.     

The structure and function of oxidized albumin in hemodialysis patients: Its role in elevated oxidative stress via neutrophil burst

Oxidized albumin is a reliable marker of oxidative stress in hemodialysis (HD) patients. However, oxidized albumin in vivo and its possible clinical significance has been rarely investigated. In the present study, the qualitative modification of albumin in HD patients (n = 20) was examined and their results were compared with healthy age-matched controls (n = 10). The increase in plasma protein carbonyl levels in HD patients was largely due to an increase in oxidized albumin. Human serum albumin (HSA) of HD patients, HSA of HD patients (HD-HSA) and normal subjects (Normal-HSA) were purified on a blue Sepharose CL-6B column. Spectroscopic analysis confirmed that the HD-HSA samples contained higher levels of carbonyls than Normal-HSA. An HPLC analysis also suggested that the state of the purified HSA used throughout the experiments accurately reflects the redox state of albumin in blood. HD-HSA was found to have a decreased the antioxidant activity, and was able to trigger the oxidative burst of human neutrophils, compared to Normal-HSA. HD-HSA was conformationally altered, with its hydrophobic regions more exposed and to have a negative charge. In binding experiments, HD-HSA showed impaired Site II-ligand binding capabilities. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in HD patients.     

Hypochlorite modification of sphingomyelin generates chlorinated lipid species that induce apoptosis and proteome alterations in dopaminergic PC12 neurons in vitro

Recent observations link myeloperoxidase (MPO) activation to neurodegeneration. In multiple sclerosis MPO is present in areas of active demyelination where the potent oxidant hypochlorous acid (HOCl), formed by MPO from H₂O₂ and chloride ions, could oxidatively damage myelin-associated lipids. The purpose of this study was (i) to characterize reaction products of sphingomyelin (SM) formed in response to modification by HOCl, (ii) to define the impact of exogenously added SM and HOCl-modified SM (HOCl-SM) on viability parameters of a neuronal cell line (PC12), and (iii) to study alterations in the PC12 cell proteome in response to SM and HOCl-SM. MALDI-TOF-MS analyses revealed that HOCl, added as reagent or generated enzymatically, transforms SM into chlorinated species. On the cellular level HOCl-SM but not SM induced the formation of reactive oxygen species. HOCl-SM induced severely impaired cell viability, dissipation of the mitochondrial membrane potential, and activation of caspase-3 and DNA damage. Proteome analyses identified differential expression of specific subsets of proteins in response to SM and HOCl-SM. Our results demonstrate that HOCl modification of SM results in the generation of chlorinated lipid species with potent neurotoxic properties. Given the emerging connections between the MPO–H₂O₂–chloride axis and neurodegeneration, this chlorinating pathway might be implicated in neuropathogenesis.     

Comparison of low-density lipoprotein modification by myeloperoxidase-derived hypochlorous and hypobromous acids.

Myeloperoxidase (MPO), a heme enzyme secreted by activated phagocytes, catalyzes the oxidation of halides to hypohalous acids. At plasma concentrations of halides, hypochlorous acid (HOCl) is the major strong oxidant produced. In contrast, the related enzyme eosinophil peroxidase preferentially generates hypobromous acid (HOBr). Since reagent and MPO-derived HOCl converts low-density lipoprotein (LDL) to a potentially atherogenic form, we investigated the effects of HOBr on LDL modification. Compared to HOCl, HOBr caused 2-3-fold greater oxidation of tryptophan and cysteine residues of the protein moiety (apoB) of LDL and 4-fold greater formation of fatty acid halohydrins from the lipids in LDL. In contrast, HOBr was 2-fold less reactive than HOCl with lysine residues and caused little formation of N-bromamines. Nevertheless, HOBr caused an equivalent increase in the relative electrophoretic mobility of LDL as HOCl, which was not reversed upon subsequent incubation with ascorbate, in contrast to the shift in mobility caused by HOCl. Similar apoB modifications were observed with HOBr generated by MPO/H(2)O(2)/Br(-). In the presence of equivalent concentrations of Cl(-) and Br(-), modifications of LDL by MPO resembled those seen in the presence of Br(-) alone. Interestingly, even at physiological concentrations of the two halides (100 mM Cl(-), 100 microM Br(-)), MPO utilized a portion of the Br(-) to oxidize apoB cysteine residues. MPO also utilized the pseudohalide thiocyanate to oxidize apoB cysteine residues. Our data show that even though HOBr has different reactivities than HOCl with apoB, it is able to alter the charge of LDL, converting it into a potentially atherogenic particle.     

Immunohistochemical evidence for the myeloperoxidase/H2O2/halide system in human atherosclerotic lesions: colocalization of myeloperoxidase and hypochlorite-modified proteins.

The 'oxidation theory' of atherosclerosis proposes that oxidation of low density lipoprotein (LDL) contributes to atherogenesis. Although the precise mechanisms of in vivo oxidation are widely unknown, increasing evidence suggests that myeloperoxidase (MPO, EC 1.11.1.7), a protein secreted by activated phagocytes, generates modified/oxidized (lipo)proteins via intermediate formation of hypochlorous acid (HOCl). In vitro generation of HOCl transforms lipoproteins into high uptake forms for macrophages giving rise to cholesterol-engorged foam cells. To identify HOCl-modified-epitopes in human plaque tissues we have raised monoclonal antibodies (directed against human HOCl-modified LDL) that do not cross-react with other LDL modifications, i.e. peroxynitrite-LDL, hemin-LDL, Cu2+-oxidized LDL, 4-hydroxynonenal-LDL, malondialdehyde-LDL, glycated-LDL, and acetylated-LDL. The antibodies recognized a specific epitope present on various proteins after treatment with OCl- added as reagent or generated by the MPO/H2O2/halide system. Immunohistochemical studies revealed pronounced staining for HOCl-modified-epitopes in fibroatheroma (type V) and complicated (type VI) lesions, while no staining was observed in aortae of lesion-prone location (type I). HOCl-oxidation-specific epitopes are detected in cells in the majority of atherosclerotic plaques but not in control segments. Staining was shown to be inside and outside monocytes/macrophages, endothelial cells, as well as in the extracellular matrix. A similar staining pattern using immunohistochemistry could be obtained for MPO. The colocalization of immunoreactive MPO and HOCl-modified-epitopes in serial sections of human atheroma (type IV), fibroatheroma (type V) and complicated (type VI) lesions provides further convincing evidence for MPO/H2O2/halide system-mediated oxidation of (lipo)proteins under in vivo conditions. We propose that MPO could act as an important link between the development of atherosclerotic plaque in the artery wall and chronic inflammatory events.     

High plasma thiocyanate levels modulate protein damage induced by myeloperoxidase and perturb measurement of 3-chlorotyrosine

Smokers have an elevated risk of atherosclerosis but the origin of this elevated risk is incompletely defined, though increasing evidence supports a role for the oxidant-generating enzyme myeloperoxidase (MPO). In previous studies we have demonstrated that smokers have elevated levels of thiocyanate ions (SCN(-)), relative to nonsmokers, and increased thiol oxidation, as SCN(-) is a favored substrate for MPO, and the resulting hypothiocyanous acid (HOSCN) targets thiol groups rapidly and selectively. In this study we show that increased HOSCN formation by MPO diminishes damage to nonthiol targets on both model proteins and human plasma proteins. Thus high SCN(-) levels protect against HOCl- and MPO-mediated damage to methionine, tryptophan, lysine, histidine, and tyrosine residues on proteins. Furthermore, levels of the HOCl-mediated marker compound 3-chlorotyrosine and the cross-linked product dityrosine are decreased. Plasma protein 3-chlorotyrosine levels induced by HOCl exposure in nonsmokers are elevated over the levels detected in smokers when exposed to identical oxidative insult (P<0.05), and a strong inverse correlation exists between plasma SCN(-) levels and 3-chlorotyrosine concentrations (r=0.6182; P<0.0001). These correlations were also significant for smokers (r=0.2724; P<0.05) and nonsmokers (r=0.4141; P<0.01) when analyzed as individual groups. These data indicate that plasma SCN(-) levels are a key determinant of the extent and type of protein oxidation induced by MPO on isolated and plasma proteins and that smoking status and resulting high SCN(-) levels can markedly modulate the levels of the widely used biomarker compound 3-chlorotyrosine.     

High plasma thiocyanate levels in smokers are a key determinant of thiol oxidation induced by myeloperoxidase

Smokers have an elevated risk of atherosclerosis but the origins of this elevated risk are incompletely defined, though evidence supports an accumulation of the oxidant-generating enzyme myeloperoxidase (MPO) in the inflamed artery wall. We hypothesized that smokers would have a high level of thiocyanate (SCN(-)), a preferred substrate for MPO, which in turn would predispose to thiol oxidation, an established independent risk factor for atherosclerosis. In this study it is shown that on exposure to MPO/H(2)O(2), thiols on plasma proteins from nonsmokers were increasingly oxidized with increasing added SCN(-) concentrations. Plasma from smokers contained significantly higher endogenous levels of SCN(-) than that from nonsmokers (131±31 vs 40±24 μM, P<0.0001). When plasma from smokers and nonsmokers was exposed to MPO/H(2)O(2)-stimulated oxidation, a strong positive correlation (r=0.8139, P<0.0001) between the extent of thiol oxidation and the plasma SCN(-) concentrations was observed. Computational calculations indicate a changeover from HOCl to HOSCN as the major MPO-generated oxidant in plasma, with increasing SCN(-) levels. These data indicate that plasma SCN(-) levels are a key determinant of the extent of thiol oxidation on plasma proteins induced by MPO, and implicate HOSCN as an important mediator of inflammation-induced oxidative damage to proteins in smokers.     

Hydrogen sulphide: a novel inhibitor of hypochlorous acid-mediated oxidative damage in the brain?

Hydrogen sulphide (H(2)S) is a cytotoxic gas that has recently been proposed as a novel neuromodulator. Endogenous levels of H(2)S in the brain range between 50 and 160 microM, and considerably lower H(2)S levels are reported in the brains of Alzheimer's disease (AD) patients. Levels of myeloperoxidase (MPO), an enzyme that catalyses the formation of the oxidant hypochlorous acid (HOCl), are elevated in the prefrontal cortex, hippocampal microglia, and neurons of AD patients where MPO co-localised with beta-amyloid plaques. Recently 3-chlorotyrosine, a bio-marker for MPO activity (and HOCl production), was shown to be elevated threefold in hippocampal proteins from AD patients. Since H(2)S and HOCl are important mediators in brain function and disease, we investigated the effects of H(2)S on HOCl-mediated damage to bio-molecules and to cultured human SH-SY5Y cells. H(2)S significantly inhibited HOCl-mediated inactivation of alpha(1)-antiproteinase and protein oxidation to a comparable extent to reduced glutathione. H(2)S also inhibited HOCl-induced cytotoxicity, intracellular protein oxidation, and lipid peroxidation in SH-SY5Y cells. These data suggest that H(2)S has the potential to act as an inhibitor of HOCl-mediated processes in vivo and that the potential antioxidant action of H(2)S deserves further study, especially since extracellular GSH levels in the brain are very low.     

Antioxidant binding of caeruloplasmin to myeloperoxidase: myeloperoxidase is inhibited, but oxidase, peroxidase and immunoreactive properties of caeruloplasmin remain intact

The neutrophil enzyme myeloperoxidase (MPO) purposefully makes hypochlorous acid (HOCl) as part of the cells defence against microbial infections. During cell lysis, however, MPO will be released into the extracellular environment where production of HOCl, a powerful oxidant, will lead to molecular damage. Extracellular MPO binds to the copper-containing protein caeruloplasmin (Cp) and prevents MPO making HOCl. Cp has several important antioxidant functions in extracellular fluids associated with its ability to catalyse oxidation of ferrous ions and to remove peroxides. The binding of MPO to Cp did not inhibit these important extracellular antioxidant activities of Cp, but in so doing it provided additional antioxidant protection against formation of HOCl.     

Antioxidant binding of caeruloplasmin to myeloperoxidase: Myeloperoxidase is inhibited,but oxidase,peroxidase and immunoreactive properties of caeruloplasmin remain intact

The neutrophil enzyme myeloperoxidase (MPO) purposefully makes hypochlorous acid (HOCl) as part of the cells defence against microbial infections. During cell lysis, however, MPO will be released into the extracellular environment where production of HOCl, a powerful oxidant, will lead to molecular damage. Extracellular MPO binds to the copper-containing protein caeruloplasmin (Cp) and prevents MPO making HOCl. Cp has several important antioxidant functions in extracellular fluids associated with its ability to catalyse oxidation of ferrous ions and to remove peroxides. The binding of MPO to Cp did not inhibit these important extracellular antioxidant activities of Cp, but in so doing it provided additional antioxidant protection against formation of HOCl.     

Myeloperoxidase-induced Genomic DNA-centered Radicals

Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis.     

Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100

Oxidation of LDL by the myeloperoxidase (MPO)-H₂O₂-chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modified LDL and at revealing posttranslational modifications on apoB-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry, we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently reflected by local structural changes in MPO observed by circular dichroism. Using MS, we further analyzed in vitro modifications of apoB-100 by hypochlorous acid (HOCl) generated by the MPO-H₂O₂-chloride system or added as a reagent. A total of 97 peptides containing modified residues could be identified. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H₂O₂-chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confirm that our in vitro findings are also relevant in vivo. We show that several HOCl-mediated modifications of apoB-100 identified in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modified LDL. In conclusion, these data emphasize the specificity of MPO to oxidize LDL.     

Impact of HDL oxidation by the myeloperoxidase system on sterol efflux by the ABCA1 pathway

Protein oxidation by phagocytic white blood cells is implicated in tissue injury during inflammation. One important target might be high-density lipoprotein (HDL), which protects against atherosclerosis by removing excess cholesterol from artery wall macrophages. In the human artery wall, cholesterol-laden macrophages are a rich source of myeloperoxidase (MPO), which uses hydrogen peroxide for oxidative reactions in the extracellular milieu. Levels of two characteristic products of MPO-chlorotyrosine and nitrotyrosine-are markedly elevated in HDL from human atherosclerotic lesions. Here, we describe how MPO-dependent chlorination impairs the ability of apolipoprotein A-I (apoA-I), HDL's major protein, to transport cholesterol by the ATP-binding cassette transporter A1 (ABCA1) pathway. Faulty interactions between apoA-I and ABCA1 are involved. Tandem mass spectrometry and investigations of mutated forms of apoA-I demonstrate that tyrosine residues in apoA-I are chlorinated in a site-specific manner by chloramine intermediates on suitably juxtaposed lysine residues. Plasma HDL isolated from subjects with coronary artery disease (CAD) also contains higher levels of chlorinated and nitrated tyrosine residues than HDL from healthy subjects. Thus, the presence of chlorinated HDL might serve as a marker of CAD risk. Because HDL damaged by MPO in vitro becomes dysfunctional, inhibiting MPO in vivo might be cardioprotective.     

Detection and characterization of N-alpha-chloramines by electrospray tandem mass spectrometry

Hypochlorous acid (HOCl) is a major product of activated neutrophils and may be important in antimicrobial activities of cells by oxidation or chlorination of susceptible amino acids. Three major peaks separated using C18 reverse phase-high-performance liquid chromatography RP-HPLC after incubation of leucine enkephalin (LeuEnk) with HOCl. Electrospray mass spectrometry showed masses of m/z 556.2, 590.2, and 624.4 corresponding to unmodified LeuEnk and peptides altered by addition of one or two chlorines (Cl). Formation of stable N-alpha-chloramines was indicated because the chlorinated peptides were readily reduced with the physiological reductants glutathione and ascorbic acid to LeuEnk (m/z 556.2) within 10 min. Sequence-specific ions observed in product ion spectra of single-charged monochlorinated and dichlorinated peptides were consistent with modification of the N-terminal amine. There was no evidence for chlorination of the Tyr aromatic ring in any spectra. Similar RP-HPLC profiles were obtained after oxidation of des-Tyr1-LeuEnk (GGFL) with the masses of the major products being m/z 393.3, 427.2, and 461.1. These were identified as unmodified GGFL, N-alpha-Cl-GGFL, and N-alpha-Cl2-GGFL based on comparison of tandem mass spectra. Oxidation of Met and formation of disulfide dimers was observed after incubation of either N-alpha-Cl-LeuEnk or N-alpha-Cl2-LeuEnk with a protein, indicating that both peptide N-alpha-chloramines were able to readily modify sulfur-containing amino acids within proteins. These data indicate initial formation of stable N-alpha-chorinated peptides after incubation with HOCl and suggest that N-alpha-chlorinated peptides may exist for some hours in the absence of physiological reducing agents or sulfur-containing amino acids.     

The importance of oxidative stress in patients with chronic renal failure whose hypertension is treated with peritoneal dialysis

Increased oxidative stress is a well-known phenomenon in dialysis patients. However, the contribution of hypertension to the oxidative stress in peritoneal dialysis patients has not yet been assessed. The present study aimed to investigate if hypertension had an additional effect on oxidative stress in peritoneal dialysis patients. A total of 50 patients treated with peritoneal dialysis were divided into two groups: The patients with mean of last three blood pressure results as 135/90 mmHg and above were considered hypertensive, the patients with lower blood pressure were considered normotensive. The control group included 25 healthy individuals. Serum malondialdehyde (MDA), advanced oxidation protein product (AOPP), myeloperoxidase (MPO), catalase (CAT) and glutathione peroxidase (GSH-Px) levels were measured in all groups. MDA level, an indicator of lipid peroxidation, was significantly higher in the hypertensive group compared to the control group, while the increase in the normotensive group was not significant. However, the difference between the hypertensive and normotensive groups was significant. The levels of AOPP, an indicator of protein oxidation level, and MPO, an indicator of neutrophil activation, were not different between the groups, while the activities of antioxidant CAT and GSH-Px decreased in both normotensive and hypertensive groups compared to the control group, and there was no significant difference between the patient groups. This study shows that both normotensive and hypertensive peritoneal dialysis patients have increased-oxidative stress and decreased antioxidant levels and hypertension might have an additional effect on oxidative stress by increasing MDA level in peritoneal dialysis patients.