Approaches for the analysis of chlorinated lipids

Leukocytes are key cellular mediators of human diseases through their role in inflammation. Identifying unique molecules produced by leukocytes may provide new biomarkers and mechanistic insights into the role of leukocytes in disease. Chlorinated lipids are generated as a result of myeloperoxidase-containing leukocyte-derived hypochlorous acid targeting the vinyl ether bond of plasmalogens. The initial product of this reaction is α-chlorofatty aldehyde. α-Chlorofatty aldehyde is both oxidized to α-chlorofatty acid and reduced to α-chlorofatty alcohol by cellular metabolism. This review focuses on the separation techniques and quantitative analysis for these chlorinated lipids. For α-chlorofatty acid, the negative charge of carboxylic acids is exploited to detect the chlorinated lipid species of these acids by electrospray ionization mass spectrometry in the negative ion mode. In contrast, α-chlorofatty aldehyde and α-chlorofatty alcohol are converted to pentafluorobenzyl oxime and pentafluorobenzoyl ester derivatives, which are detected by negative ion chemical ionization mass spectrometry. These two detection methods coupled with the use of stable isotope internal standards and either liquid chromatography or gas chromatography provide highly sensitive analytical approaches to measure these novel lipids.     

Strategies for the analysis of chlorinated lipids in biological systems

Myeloperoxidase-derived HOCl reacts with the vinyl ether bond of plasmalogens yielding α-chlorofatty aldehydes. These chlorinated aldehydes can be purified using thin-layer chromatography, which is essential for subsequent analysis of extracts from some tissues such as myocardium. The α-chlorofatty aldehyde 2-chlorohexadecanal (2-ClHDA) is quantified after conversion to its pentafluorobenzyl oxime derivative using gas chromatography–mass spectrometry and negative-ion chemical ionization detection. 2-ClHDA accumulates in activated human neutrophils and monocytes, as well as in atherosclerotic lesions and infarcted myocardium. Metabolites of 2-ClHDA have also been identified, including the oxidation product, 2-chlorohexadecanoic acid (2-ClHA), and the reduction product, 2-chlorohexadecanol. 2-ClHA can be quantified using LC–MS with selected reaction monitoring (SRM) detection. 2-ClHA can be ω-oxidized by hepatocytes and subsequently β-oxidized from the ω-end, leading to the production of the dicarboxylic acid, 2-chloroadipic acid. This dicarboxylic acid is excreted in the urine and can also be quantified using LC–MS methods with SRM detection. Quantitative analyses of these novel chlorinated lipids are essential to identify the role of these lipids in leukocyte-mediated injury and disease.     

Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid.

Stimulated neutrophils produce hypochlorous acid (HOCl) via the myeloperoxidase-catalyzed reaction of hydrogen peroxide with chloride. The reactions of HOCl with oleic, linoleic, and arachidonic acids both as free fatty acids or bound in phosphatidylcholine have been studied. The products were identified by gas chromatography-mass spectrometry of the methylated and trimethylsilylated derivatives. Oleic acid was converted to the two 9,10-chlorohydrin isomers in near stoichiometric yield. Linoleic acid, at low HOCl:fatty acid ratios, yielded predominantly a mixture of the four possible monochlorohydrin isomers. Bischlorohydrins were also formed, in increasing amounts at higher HOCl concentrations. Arachidonic acid gave a complex mixture of mono- and bischlorohydrins, the relative proportions depending on the amount of HOCl added. Linoleic acid appears to be slightly more reactive than oleic acid with HOCl. Reactions of oleic and linoleic acids with myeloperoxidase, hydrogen peroxide, and chloride gave chlorohydrin products identical to those with HOCl. Lipid chlorohydrins have received little attention as products of reactions of neutrophil oxidants. They are more polar than the parent fatty acids, and if formed in cell membranes could cause disruption to membrane structure. Since cellular targets for HOCl appear to be membrane constituents, chlorohydrin formation from unsaturated lipids could be significant in neutrophil-mediated cytotoxicity.     

Hypochlorous acid as a precursor of free radicals in living systems

Hypochlorous acid (HOCl) is produced in the human body by the family of mammalian heme peroxidases, mainly by myeloperoxidase, which is secreted by neutrophils and monocytes at sites of inflammation. This review discusses the reactions that occur between HOCl and the major classes of biologically important molecules (amino acids, proteins, nucleotides, nucleic acids, carbohydrates, lipids, and inorganic substances) to form free radicals. The generation of such free radical intermediates by HOCl and other reactive halogen species is accompanied by the development of halogenative stress, which causes a number of socially important diseases, such as cardiovascular, neurodegenerative, infectious, and other diseases usually associated with inflammatory response and characterized by the appearance of biomarkers of myeloperoxidase and halogenative stress. Investigations aimed at elucidating the mechanisms regulating the activity of enzyme systems that are responsible for the production of reactive halogen species are a crucial step in opening possibilities for control of the development of the body’s inflammatory response.     

The vinyl ether linkages of plasmalogens are favored targets for myeloperoxidase-derived oxidants: a kinetic study

Plasmalogens, which contain a vinyl ether bond, are major phospholipids of the plasma membranes of endothelial and vascular smooth muscle cells and cardiac myocytes. These lipids, in contrast to other phospholipids, have been reported to be targets of HOCl/HOBr generated by myeloperoxidase, with elevated levels of the products of these reactions (alpha-chloro/alpha-bromo aldehydes and unsaturated lysophospholipids) having been detected in human atherosclerotic lesions. The reason(s) for the targeting of this lipid class, over other phospholipids, is poorly understood, and is examined here. It is shown that HOCl and HOBr react with a model vinyl ether (ethylene glycol vinyl ether) 200-300-fold faster ( k = 1.6 x 10 (3) and 3.5 x 10 (6) M (-1) s (-1), respectively) than with aliphatic alkenes (models of phospholipids). True plasmalogens react ca. 20-fold slower than the models. Chloramines and bromamines (from reaction of HOCl/HOBr with primary amines and alpha-amino groups) also react with vinyl ethers, unlike aliphatic alkenes, with k = 10 (-3)-10 (2) M (-1) s (-1) for chloramines (with the His side chain chloramine being the most reactive, k = 172 M (-1) s (-1)) and k = 10 (3)-10 (4) M (-1) s (-1) for bromamines. The bromamine rate constants are typically 10 (5)-10 (6) larger than those of the chloramines. Intermolecular vinyl ether oxidation by phospholipid headgroup bromamines can also occur. These kinetic data indicate that plasmalogens are significantly more susceptible to oxidation than the aliphatic alkenes of phospholipids, thereby rationalizing the detection of products from the former, but not the latter, in human atherosclerotic lesions.     

The reactions of hypochlorous acid, the reactive oxygen species produced by myeloperoxidase, with lipids

Myeloperoxidase (MPO), an abundant enzyme in phagocytes, has been implicated in the pathogenesis of various inflammatory diseases including atherosclerosis. The major oxidant produced by MPO, hypochlorous acid (HOCl), is able to modify a great variety of biomolecules by chlorination and/or oxidation. In this paper the reactions of lipids (preferentially unsaturated fatty acids and cholesterol) with either reagent HOCl or HOCl generated by the MPO-hydrogen peroxide-chloride system are reviewed. One of the major issues has been whether the reaction of HOCl with lipids of low density lipoprotein (LDL) yields predominantly chlorohydrins or lipid hydroperoxides. Electrospray mass spectrometry provided direct evidence that chlorohydrins rather than peroxides are the major products of HOCl- or MPO-treated LDL phosphatidylcholines. Nevertheless lipid peroxidation is a possible alternative reaction of HOCl with polyunsaturated fatty acids if an additional radical source such as pre-formed lipid hydroperoxides is available. In phospholipids carrying a primary amino group such as phosphatidylethanolamine chloramines are the preferred products compared to chlorohydrins. Cholesterol can be converted by HOCl to great variety of oxysterols besides three isomers of chlorohydrins. For the situation in vivo it appears that the type of reaction occurring between HOCl and lipids would very much depend on the circumstances, e.g. the pH and the presence of radical initiators. The biological effects of lipid chlorohydrins are not yet well understood. It has been shown that chlorohydrins of both unsaturated fatty acids as well as of cholesterol may cause lysis of target cells, possibly by disruption of membrane structures.     

Selective plasmenylcholine oxidation by hypochlorous acid: formation of lysophosphatidylcholine chlorohydrins

The plasmalogen sn-1 vinyl ether bond is targeted by hypochlorous acid (HOCl) produced by activated phagocytes. In the present study, the attack of the plasmalogen sn-1 vinyl ether bond by HOCl is shown to be preferred compared to the attack of double bonds present in the sn-2 position aliphatic chain (sn-2 alkenes) of both plasmenylcholine and phosphatidylcholine. Lysophosphatidylcholine (LPC) is a product from the initial HOCl attack of plasmenylcholine and the sn-2 alkene bonds present in this LPC product are secondary targets of HOCl leading to the production of LPC-chlorohydrins (ClOH). The aliphatic ClOH was demonstrated in both the positive and negative ion mode using collisionally-activated dissociation (CAD) of the molecular ion of LPC-ClOH. Furthermore, HOCl treatment of endothelial cells led to the preferential attack of plasmalogens in comparison to that of diacyl choline glycerophospholipids. Taken together, plasmenylcholine is oxidized preferentially over phosphatidylcholine and leads to the production of LPC-ClOH.     

Chlorination of pyridinium compounds. Possible role of hypochlorite, N-chloramines, and chlorine in the oxidation of pyridinoline cross-links of articular cartilage collagen type II during acute inflammation

Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation. To evaluate their potential to oxidize the pyridinoline (Pyd) cross-links found in collagen types I and II, we reacted hydrogen peroxide (H(2)O(2)), hypochlorous acid/hypochlorite (HOCl/OCl(-)), and singlet oxygen (O(2)((1)delta g)) with the Pyd substitutes, pyridoxamine dihydrochloride and vitamin B(6), which share the same chemical structure and spectral properties of Pyd cross-links. Neither H(2)O(2) (125-500 microm) nor O(2)((1)delta g) (10-25 microm) significantly changed the spectral properties of pyridoxamine or vitamin B(6). Reaction of HOCl/OCl(-) (12.5-50 microm) with pyridoxamine at pH 7.2 resulted in a concentration-dependent appearance of two new absorbance peaks and a decrease in fluorescence at 400 nm (excitation 325 nm). The new absorbance peaks correlated with the formation of an N-chloramine and the product of its subsequent reaction with pyridoxamine. In contrast, the extent to which HOCl reacted with vitamin B(6), which lacks a primary amine group, was variable at this pH. At lysosomal pH 5.5, Cl(2)/HOCl/OCl(-) reacted with both pyridoxamine and vitamin B(6). Four of the chlorinated products of this reaction were identified by gas chromatography-mass spectrometry and included 3-chloropyridinium, an aldehyde, and several chlorinated products with disrupted rings. To evaluate the effects of Cl(2)/HOCl/OCl(-) on Pyd cross-links in collagen, we exposed bone collagen type I and articular cartilage type II to HOCl. Treatment of either collagen type with HOCl at pH 5. 0 or 7.2 resulted in the oxidation of amine groups and, for collagen type II, the specific decrease in Pyd cross-link fluorescence, suggesting that during inflammation both oxidations may be used by neutrophils and monocytes to promote the loss of matrix integrity.     

Arthromyces ramosus peroxidase produces two chlorinating species

We previously reported that the hemes of horseradish peroxidase (HRP) and Arthromyces ramosus peroxidase (ARP) undergo vinyl and meso-carbon modifications when the enzymes oxidize chloride ion. Here we demonstrate for ARP that, although both modifications exhibit the same pH profile with an optimum at approximately pH 4.0, monochlorodimedone suppresses the vinyl but not meso-carbon modifications. Furthermore, meso-chlorination occurs when ARP reacts with exogenous HOCl, implicating an Fe(III)-O-Cl intermediate in the reaction. These results establish that (a) the chloro species involved in meso-modification differs from that which reacts with the vinyl groups, (b) equilibration of the vinyl modifying species (HOCl) into the medium occurs more rapidly than vinyl group modification, and (c) the oxidation of chloride by ARP produces two reactive species: HOCl, which adds to the heme vinyl but not meso-positions, and a distinct second species that adds to the meso-carbon.     

Chlorinative stress in age-related diseases: a literature review

Aging is an agglomerate of biological long-lasting processes that result being inevitable. Main actors in this scenario are both long-term inflammation and oxidative stress. It has been proved that oxidative stress induce alteration in proteins and this fact itself is critically important in the pathophysiological mechanisms leading to diseases typical of aging. Among reactive species, chlorine ones such as hypochlorous acid (HOCl) are cytotoxic oxidants produced by activated neutrophils during chronic inflammation processes. HOCl can also cause damages by reacting with biological molecules. HOCl is generated by myeloperoxidase (MPO) and augmented serum levels of MPO have been described in acute and chronic inflammatory conditions in cardiovascular patients and has been implicated in many inflammatory diseases such as atherosclerosis, neurodegenerative conditions, and some cancers. Due to these data, we decided to conduct an up-to-date review evaluating chlorinative stress effects on every age-related disease linked; potential anti-oxidant countermeasures were also assessed. Results obtained associated HOCl generation to the aging processes and confirmed its connection with diseases like neurodegenerative and cardiovascular pathologies, atherosclerosis and cancer; chlorination was mainly linked to diseases where molecular (protein) alteration constitute the major suspected cause: i.e. inflammation, tissue lesions, DNA damages, apoptosis and oxidative stress itself. According data collected, a healthy lifestyle together with some dietary suggestion and/or the administration of nutracetical antioxidant integrators could balance the effects of chlorinative stress and, in some cases, slow down or prevent the onset of age-releated diseases.     

次氯酸与不饱和脂肪酸氧化机制和转化产物的理论研究

摘要 目的：揭示次氯酸与不饱和脂肪酸的氧化反应机制及转化产物。方法：运用Gaussian 16软件包,采用密度泛函方法M06-2X(D3),结合6-31+G(d)基组,在SMD液相水模型水平下进行计算。结果：次氯酸与单不饱和脂肪酸油酸的氧化反应是先形成氯鎓离子中间体,氯鎓离子再与水分子反应生成氯醇,第一步氯鎓离子的形成是控速步骤,其反应活化自由能~8 kcal/mol。环氧化合物和短链的醛是两种转化产物,前者由氯醇脱氯化氢而来,而后者由环氧化合物和氯醇通过系列与次氯酸根的反应而得到,生成它们的控速步骤的反应活化自由能分别为23 和24 kcal/mol。选取两个乙基为取代基的乙烯为油酸模型,其与次氯酸反应的活化自由能仅比油酸高1 kcal/mol。计算得到次氯酸与亚油酸、顺-9,反-11 亚油酸、梓树酸和花生四烯酸模型氧化反应生成氯醇的活化自由能分别是~10、13、16和14 kcal/mol。结论：氯鎓离子中间体机制是次氯酸与不饱和脂肪酸氧化反应的主要机制,反应的活化自由能通常低于15 kcal/mol,意味着此氧化反应动力学上容易发生。氧化产物氯醇能转化为环氧化合物和短链的醛,但活化自由能较高,约23和24 kcal/mol。选取距离双键3个碳以内的结构为不饱和脂肪酸模型,它能够很好地反映不饱和脂肪酸的反应活性。     

Glutathione oxidation by hypochlorous acid in endothelial cells produces glutathione sulfonamide as a major product but not glutathione disulfide

Treatment of cells with hypochlorous acid (HOCl) at sublethal doses causes a concentration-dependent loss in reduced glutathione (GSH) levels. We have investigated the products of the reaction of HOCl with GSH in human umbilical vein endothelial cells. Despite a complete loss of GSH, there were only very small increases in intracellular and extracellular glutathione disulfide and glutathione sulfonic acid after exposure to HOCl. (35)S labeling of the GSH pool showed only a minimal increase in protein-bound GSH, suggesting that S-thiolation was not a major contributor to HOCl-mediated loss of GSH in endothelial cells. Rather, the products of the reaction were mostly exported from cells and included a peak that co-eluted with the cyclic sulfonamide that is a product of the reaction of GSH with reagent HOCl. Evidence of this species in endothelial cell supernatants after HOCl treatment was also obtained using electrospray mass spectrometry. In conclusion, exposure to HOCl causes the irreversible loss of cellular GSH with the formation of novel products that are rapidly exported from the cell, and resynthesis of GSH will be required to restore levels. The loss of GSH would alter the redox state of the cell and compromise its defenses against further oxidative stress.     

Pathways of phospholipid oxidation by HOCl in human LDL detected by LC-MS

A wealth of evidence now indicates that low-density lipoprotein (LDL) must be modified to promote atherosclerosis, and that this may involve oxidants released by phagocytes. Many studies of oxidative damage in atherosclerosis previously have concentrated on damage by nonhalogenated oxidants, but HOCl is a highly toxic oxidant produced by myeloperoxidase in phagocytes, which is also likely to be important in the disease pathogenesis. Currently some controversy exists over the products resulting from reaction of HOCl with LDL lipids, in particular regarding whether predominantly chlorohydrins or lipid peroxides are formed. In this study LC-MS of phosphatidylcholines in human LDL treated either with HOCl or the myeloperoxidase system was used as a specific method to detect chlorohydrin and peroxide formation simultaneously, and with comparable sensitivity. Chlorohydrin products from lipids containing oleic, linoleic and arachidonic acids were detected, but no hydroperoxides of linoleoyl or arachidonoyl lipids could be observed. This study provides the first direct evidence that lipid chlorohydrins rather than peroxides are the major products of HOCl- or myeloperoxidase-treated LDL phospholipids. This in turn provides important information required for the study of oxidative damage in vivo which will allow the type and source of oxidants involved in the pathology of atherosclerosis to be investigated.     

The reaction of HOCl and cyanocobalamin: corrin destruction and the liberation of cyanogen chloride

Overproduction of hypochlorous acid (HOCl) has been associated with the development of a variety of disorders such as inflammation, heart disease, pulmonary fibrosis, and cancer through its ability to modify various biomolecules. HOCl is a potent oxidant generated by the myeloperoxidase-hydrogen peroxide-chloride system. Recently, we have provided evidence to support the important link between higher levels of HOCl and heme destruction and free iron release from hemoglobin and RBCs. Our current findings extend this work and show the ability of HOCl to mediate the destruction of metal-ion derivatives of tetrapyrrole macrocyclic rings, such as cyanocobalamin (Cobl), a common pharmacological form of vitamin B12. Cyanocobalamin is a water-soluble vitamin that plays an essential role as an enzyme cofactor and antioxidant, modulating nucleic acid metabolism and gene regulation. It is widely used as a therapeutic agent and supplement, because of its efficacy and stability. In this report, we demonstrate that although Cobl can be an excellent antioxidant, exposure to high levels of HOCl can overcome the beneficial effects of Cobl and generate proinflammatory reaction products. Our rapid kinetic, HPLC, and mass spectrometric analyses showed that HOCl can mediate corrin ring destruction and liberate cyanogen chloride (CNCl) through a mechanism that initially involves α-axial ligand replacement in Cobl to form a chlorinated derivative, hydrolysis, and cleavage of the phosphonucleotide moiety. Additionally, it can liberate free Co, which can perpetuate metal-ion-induced oxidant stress. Taken together, these results are the first report of the generation of toxic molecular products through the interaction of Cobl with HOCl.     

Human neutrophils employ the myeloperoxidase/hydrogen peroxide/chloride system to oxidatively damage apolipoprotein A-I.

The structural integrity of apolipoprotein A-I (apo A-I) is critical to the physiological function of high-density lipoprotein (HDL). Oxidized lipoproteins are thought to be of central importance in atherogenesis, and oxidation products characteristic of myeloperoxidase, a heme protein secreted by activated phagocytes, have been detected in human atherosclerotic tissue. At plasma concentrations of halide ion, hypochlorous acid is a major product of the myeloperoxidase-hydrogen peroxide-chloride system. We therefore investigated the effects of activated human neutrophils, a potent source of myeloperoxidase and hydrogen peroxide, on the protein and lipid components of HDL. Both free and HDL-associated apo A-I exposed to activated human neutrophils underwent extensive degradation as monitored by RP-HPLC and Western blotting with a polyclonal antibody to apo A-I. Replacement of the neutrophils with reagent HOCl resulted in comparable damage (at molar oxidant : HDL subclass 3 ratio = 100) as observed in the presence of activated phagocytes. Apo A-I degradation by activated neutrophils was partially inhibited by the HOCl scavenger methionine, by the heme inhibitor azide, by chloride-free conditions, by the peroxide scavenger catalase, and by a combination of superoxide dismutase (SOD)/catalase, implicating HOCl in the cell-mediated reaction. The addition of a protease inhibitor (3,4-dichloroisocoumarin) further reduced the extent of apo A-I damage. In contrast to the protein moiety, there was little evidence for oxidation of unsaturated fatty acids or cholesterol in HDL3 exposed to activated neutrophils, suggesting that HOCl was selectively damaging apo A-I. Our observations indicate that HOCl generated by myeloperoxidase represents one pathway for protein degradation in HDL3 exposed to activated phagocytes.     

Myeloperoxidase-generated oxidants and atherosclerosis

Atherosclerosis is a chronic inflammatory process where oxidative damage within the artery wall is implicated in the pathogenesis of the disease. Mononuclear phagocytes, an inflammatory cell capable of generating a variety of oxidizing species, are early components of arterial lesions. Their normal functions include host defense and surveillance through regulated generation of diffusible radical species, reactive oxygen or nitrogen species, and HOCl (hypochlorous acid). However, under certain circumstances an excess of these oxidizing species can overwhelm local antioxidant defenses and lead to oxidant stress and oxidative tissue injury, processes implicated in the pathogenesis of atherosclerosis. This review focuses on oxidation reactions catalyzed by myeloperoxidase (MPO), an abundant heme protein secreted from activated phagocytes which is present in human atherosclerotic lesions. Over the past several years, significant evidence has accrued demonstrating that MPO is one pathway for protein and lipoprotein oxidation during the evolution of cardiovascular disease. Multiple distinct products of MPO are enriched in human atherosclerotic lesions and LDL recovered from human atheroma. However, the biological consequences of these MPO-catalyzed reactions in vivo are still unclear. Here we discuss evidence for the occurrence of MPO-catalyzed oxidation reactions in vivo and the potential role MPO plays in both normal host defenses and inflammatory diseases like atherosclerosis.     

Chlorinative stress: An under appreciated mediator of neurodegeneration?

Oxidative stress has been implicated as playing a role in neurodegenerative disorders, such as ischemic stroke, Alzheimer's, Huntington's, and Parkinson's disease. Persuasive evidences have shown that microglial-mediated oxidative stress contributes significantly to cell loss and accompanying cognitive decline characteristic of the diseases. Based on the facts that (i) levels of catalytically active myeloperoxidase are elevated in diseased brains and (ii) myeloperoxidase polymorphism is associated with the risk of developing neurodegenerative disorders, HOCl as a major oxidant produced by activated phagocytes in the presence of myeloperoxidase is therefore suggested to be involved in neurodegeneration. Its association with neurodegeneration is further showed by elevated level of 3-chlorotyrosine (bio-marker of HOCl in vivo) in affected brain regions as well as HOCl scavenging ability of neuroprotectants, desferrioxamine and uric acid. In this review, we will summary the current understanding concerning the association of HOCl and neuronal cell death where production of HOCl will lead to further formation of reactive nitrogen and oxygen species. In addition, HOCl also causes tissue destruction and cellular damage leading cell death.     

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.     

Hypochlorous acid generates N epsilon-(carboxymethyl)lysine from Amadori products

Since the accumulation of N^ε-(carboxymethyl)lysine (CML), a major antigenic advanced glycation end product, is implicated in tissue disorders in hyperglycemia and inflammation, the identification of the pathway of CML formation will provide important information regarding the development of potential therapeutic strategies for these complications. The present study was designed to measure the effect of hypochlorous acid (HOCl) on CML formation from Amadori products. The incubation of glycated human serum albumin (glycated-HSA), a model of Amadori products, with HOCl led to CML formation, and an increasing HOCl concentration and decreasing pH, which mimics the formation of these products in inflammatory lesions. CML formation was also observed when glycated-HSA was incubated with activated neutrophils, and was completely inhibited in the presence of an HOCl scavenger. These data demonstrated that HOCl-mediated CML formation from Amadori products plays a role in CML formation and tissue damage at sites of inflammation.     

Oxidation of flavonoids by hypochlorous acid: reaction kinetics and antioxidant activity studies

Flavonoids, plant polyphenols, ubiquitous components of human diet, are excellent antioxidants. Hypochlorous acid (HOCl), produced by activated neutrophils, is highly reactive chlorinating and oxidizing species. It has been reported earlier that flavonoids are chlorinated by HOCl. Here we show that flavonoids from flavonol subclass are also oxidized by HOCl, but only if the latter is in a large molar excess (≥?10). The kinetics of this reaction was studied by stopped-flow spectrophotometry, at different pH. We found that flavonols were oxidized by HOCl with the rate constants of the order of 10⁴–10⁵ M^?1 s^?1 at pH 7.5. Antioxidant activity of HOCl-modified flavonoids was measured by 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) method. Slightly higher antioxidant activity, compared to parent compounds, was observed for flavonols after their reaction with equimolar or moderate excess of HOCl whereas flavonols treated with high molar excess of HOCl exhibited decrease in antioxidant activity. The mechanism of flavonoid reaction with HOCl at physiological pH is proposed, and biological consequences of this reaction are discussed.