Elevated seminal plasma myeloperoxidase is associated with a decreased sperm concentration in young men.

Myeloperoxidase is a major neutrophil protein which generates oxidants that are highly reactive, and if present in seminal fluid, could be potentially damaging to spermatozoa. We recruited young males aged 18-35 years, unscreened for fertility status, for a pilot study measuring seminal plasma myeloperoxidase. On three occasions, over a 3-month period, we measured parameters of semen quality and correlated these with seminal myeloperoxidase protein and activity. After baseline measurement, participants were supplemented daily with 250 mg of vitamin C, a potent scavenger of reactive oxygen species with antiinflammatory activities. Seminal plasma from eight of the 12 participants had measurable concentrations of myeloperoxidase protein, across a broad range (15-250 ng/mL). Median myeloperoxidase protein concentrations were ~45-fold higher in semen samples with low vs. high sperm concentrations. Seminal plasma myeloperoxidase protein concentration was inversely correlated with the percentage of rapidly motile spermatozoa assessed by computer-assisted sperm analysis, and the total number of spermatozoa per ejaculate, but positively correlated with sperm maturity, measured by DNA staining ability. We measured an inverse correlation between semen vitamin C concentration and seminal plasma myeloperoxidase protein concentration, although vitamin C supplementation had no effect on semen quality. Our pilot data suggest that high concentrations of myeloperoxidase were present in the seminal plasma of many of our young participants, and that this may be associated with decreases in semen quality. A larger study is required to confirm these findings.

Red wine antioxidants bind to human lipoproteins and protect them from metal ion-dependent and -independent oxidation.

Plant-derived polyphenols may exert beneficial effects on atherosclerosis and cardiovascular diseases, in part, because of their antioxidant properties. In this study we compared the effects of unbound (free) and lipoprotein-associated red wine components on in vitro antioxidant protection of human low-density lipoprotein (LDL). Preincubation of LDL (1 mg protein/mL) with 0-2.5% (v/v) red wine for 3 h at 37 degrees C followed by gel filtration to remove unbound red wine components resulted in a dose-dependent, up to 4-fold increase in LDL-associated antioxidant capacity (measured as Trolox equivalents). Similar results were obtained with high-density lipoprotein (HDL) and bovine serum albumin (BSA). Furthermore, LDL was subjected to oxidation by copper and aqueous peroxyl radicals (2,2'-azobis[2-amidinopropane] dihydrochloride, AAPH). Under both types of oxidative stress, LDL-associated and free red wine components significantly decreased oxidation of the lipoprotein's protein moiety (assessed by tryptophan fluorescence) and lipid moiety (assessed by thiobarbituric acid-reactive substances and conjugated dienes). Similar protective effects of red wine components were observed against HDL oxidation. In contrast, red wine exerted a pro-oxidant effect on copper-induced oxidation of BSA tryptophan residues, while protecting them from AAPH-induced oxidation. Ascorbate strongly enhanced the protective effect of red wine against copper-induced LDL oxidation, and had an additive effect against AAPH-induced oxidation. Our data indicate that red wine components bind to LDL and HDL and protect these lipoproteins from metal ion-dependent and -independent protein and lipid oxidation.

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.

Ldl modified by hypochlorous acid is a potent inhibitor of lecithin-cholesterol acyltransferase activity.

Modification of low density lipoprotein (LDL) by myeloperoxidase-generated HOCl has been implicated in human atherosclerosis. Incubation of LDL with HOCl generates several reactive intermediates, primarily N-chloramines, which may react with other biomolecules. In this study, we investigated the effects of HOCl-modified LDL on the activity of lecithin-cholesterol acyltransferase (LCAT), an enzyme essential for high density lipoprotein maturation and the antiatherogenic reverse cholesterol transport pathway. We exposed human LDL (0.5 mg protein/mL) to physiological concentrations of HOCl (25 to 200 micromol/L) and characterized the resulting LDL modifications to apolipoprotein B and lipids; the modified LDL was subsequently incubated with apolipoprotein B-depleted plasma (density >1.063 g/mL fraction), which contains functional LCAT. Increasing concentrations of HOCl caused various modifications to LDL, primarily, loss of lysine residues and increases in N-chloramines and electrophoretic mobility, whereas lipid hydroperoxides were only minor products. LCAT activity was extremely sensitive to HOCl-modified LDL and was reduced by 23% and 93% by LDL preincubated with 25 and 100 micromol/L HOCl, respectively. Addition of 200 micromol/L ascorbate or N-acetyl derivatives of cysteine or methionine completely prevented LCAT inactivation by LDL preincubated with

Fatty acid chlorohydrins and bromohydrins are cytotoxic to human endothelial cells.

Reaction of unsaturated lipids with the hypohalous acids (hypochlorous acid and hypobromous acid) results in the addition of the halide (X) across double bonds to form halohydrins (-CH2CH(OH)CH(X)CH2-). These modified lipids could be potentially destabilising to cell membranes due to their increased polarity. We have investigated the effect of pre-formed halohydrins on human umbilical vein endothelial cells (HUVEC) by incubating cultured cells with oleic acid micelles containing chlorohydrins or bromohydrins. Cell detachment and necrotic death were observed with increasing doses of halohydrins, whereas the cells were unaffected by equivalent doses of oleic acid. Bromohydrins caused more lysis than did chlorohydrins at equivalent doses. Complete lysis was seen with 200 microM fatty acid/chlorohydrin micelles and with 50 microM fatty acid/bromohydrin micelles. Chlorohydrin uptake was much less than the oleic acid control whereas bromohydrins were incorporated into the endothelial cells similarly to oleic acid. This difference or the bulkier nature of the bromohydrins could account for their increased toxicity. This study has demonstrated the potential toxicity of the halohydrins, and implications for their formation in inflammation are discussed.

Relative reactivities of N-chloramines and hypochlorous acid with human plasma constituents.

Hypochlorous acid (HOCl), the major strong oxidant produced by the phagocyte enzyme myeloperoxidase, reacts readily with free amino groups to form N-chloramines. Since different N-chloramines have different stabilities and reactivities depending on their structures, we investigated the relative reactivities of three model N-chloramines and HOCl with human plasma constituents. TheN-chloramines studied were N(alpha)-acetyl-lysine chloramine (LysCA, a model of protein-associated N-chloramines), taurine chloramine (TaurCA, the primary N-chloramine produced by activated neutrophils), and monochloramine (MonoCA, a lipophilic N-chloramine). Addition of these chlorine species (100--1000 microM each) to plasma resulted in rapid loss of thiols, with the extent of thiol oxidation decreasing in the order TaurCA = LysCA > MonoCA = HOCl. The single reduced thiol of albumin was the major target. Loss of plasma ascorbate also occurred, with the extent decreasing in the order HOCl > LysCA > TaurCA > MonoCA. Experiments comparing equimolar albumin thiols and ascorbate showed that while HOCl caused equivalent loss of thiols and ascorbate, theN-chloramines reacted preferentially with thiols. The chlorine species also inactivated alpha(1)-antiproteinase, implicating oxidation of methionine residues, and ascorbate provided variable protection depending on the chlorine species involved. Together, our data indicate that in biological fluids N-chloramines react more readily with protein thiols than with methionine residues or ascorbate, and thus may cause biologically relevant, selective loss of thiol groups.

The nitric oxide congener nitrite inhibits myeloperoxidase/H2O2/ Cl- -mediated modification of low density lipoprotein.

Nitric oxide, a pivotal molecule in vascular homeostasis, is converted under aerobic conditions to nitrite. Recent studies have shown that myeloperoxidase (MPO), an abundant heme protein released by activated leukocytes, can oxidize nitrite (NO(2-)) to a radical species, most likely nitrogen dioxide. Furthermore, hypochlorous acid (HOCl), the major strong oxidant generated by MPO in the presence of physiological concentrations of chloride ions, can also react with nitrite, forming the reactive intermediate nitryl chloride. Since MPO and MPO-derived HOCl, as well as reactive nitrogen species, have been implicated in the pathogenesis of atherosclerosis through oxidative modification of low density lipoprotein (LDL), we investigated the effects of physiological concentrations of nitrite (12.5-200 microm) on MPO-mediated modification of LDL in the absence and presence of physiological chloride concentrations. Interestingly, nitrite concentrations as low as 12.5 and 25 microm significantly decreased MPO/H2O2)/Cl- -induced modification of apoB lysine residues, formation of N-chloramines, and increases in the relative electrophoretic mobility of LDL. In contrast, none of these markers of LDL atherogenic modification were affected by the MPO/H2O2/NO2-) system. Furthermore, experiments using ascorbate (12.5-200 microm) and the tyrosine analogue 4-hydroxyphenylacetic acid (12.5-200 microm), which are both substrates of MPO, indicated that nitrite inhibits MPO-mediated LDL modifications by trapping the enzyme in its inactive compound II form. These data offer a novel mechanism for a potential antiatherogenic effect of the nitric oxide congener nitrite.

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

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 HOCI-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 microM), 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(alpha)-acetyl-lysine chloramine also caused dose-dependent inactivation of cathepsin B. Incubation of HOCl-modified LDL with ascorbic and lipoic acids (25-200 microM) 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 pro-atherogenic process by scavenging LDL-associated chloramines.

Vitamin C suppresses oxidative lipid damage in vivo, even in the presence of iron overload.

Ascorbate is a strong antioxidant; however, it can also act as a prooxidant in vitro by reducing transition metals. To investigate the in vivo relevance of this prooxidant activity, we performed a study using guinea pigs fed high or low ascorbate doses with or without prior loading with iron dextran. Iron-loaded animals gained less weight and exhibited increased plasma beta-N-acetyl-D-glucosaminidase activity, a marker of tissue lysosomal membrane damage, compared with control animals. The iron-loaded animals fed the low ascorbate dose had decreased plasma alpha-tocopherol levels and increased plasma levels of triglycerides and F(2)-isoprostanes, specific and sensitive markers of in vivo lipid peroxidation. In contrast, the two groups of animals fed the high ascorbate dose had significantly lower hepatic F(2)-isoprostane levels than the groups fed the low ascorbate dose, irrespective of iron load. These data indicate that 1) ascorbate acts as an antioxidant toward lipids in vivo, even in the presence of iron overload; 2) iron loading per se does not cause oxidative lipid damage but is associated with growth retardation and tissue damage, both of which are not affected by vitamin C; and 3) the combination of iron loading with a low ascorbate status causes additional pathophysiological changes, in particular, increased plasma triglycerides.

Potential antiatherogenic mechanisms of ascorbate (vitamin C) and alpha-tocopherol (vitamin E).

The premise that oxidative stress, among several other factors, plays an important role in atherogenesis implies that the development and progression of atherosclerosis can be inhibited by antioxidants. In this minireview we discuss several mechanisms by which the antioxidants ascorbate (vitamin C) and alpha-tocopherol (vitamin E) may protect against atherosclerosis. These mechanisms include inhibition of LDL oxidation and inhibition of leukocyte adhesion to the endothelium and vascular endothelial dysfunction. Overall, ascorbate appears to be more effective than alpha-tocopherol in mitigating these pathophysiological processes, most likely as a result of its abilities to effectively scavenge a wide range of reactive oxygen and nitrogen species and to regenerate alpha-tocopherol, and possibly tetrahydrobiopterin, from its radical species. In contrast, alpha-tocopherol can act either as an antioxidant or a pro-oxidant to inhibit or facilitate, respectively, lipid peroxidation in LDL. However, this pro-oxidant activity of alpha-tocopherol is prevented by ascorbate acting as a coantioxidant. Therefore, an optimum vitamin C intake or body status may help protect against atherosclerosis and its clinical sequelae, whereas vitamin E may only be effective in combination with vitamin C.

Oxidation of LDL by myeloperoxidase and reactive nitrogen species: reaction pathways and antioxidant protection.

Oxidative modification of low density lipoprotein (LDL) appears to play an important role in atherogenesis. Although the precise mechanisms of LDL oxidation in vivo are unknown, several lines of evidence implicate myeloperoxidase and reactive nitrogen species, in addition to ceruloplasmin and 15-lipoxygenase. Myeloperoxidase generates a number of reactive species, including hypochlorous acid, chloramines, tyrosyl radicals, and nitrogen dioxide. These reactive species oxidize the protein, lipid, and antioxidant components of LDL. Modification of apolipoprotein B results in enhanced uptake of LDL by macrophages with subsequent formation of lipid-laden foam cells. Nitric oxide synthases produce nitric oxide and, under certain conditions, superoxide radicals. Numerous other sources of superoxide radicals have been identified in the arterial wall, including NAD(P)H oxidases and xanthine oxidase. Nitric oxide and superoxide readily combine to form peroxynitrite, a reactive nitrogen species capable of modifying LDL. In this review, we examine the reaction pathways involved in LDL oxidation by myeloperoxidase and reactive nitrogen species and the potential protective effects of the antioxidant vitamins C and E.

Vitamin C protects against and reverses specific hypochlorous acid- and chloramine-dependent modifications of low-density lipoprotein.

Activated phagocytes produce the highly reactive oxidant hypochlorous acid (HOCl) via the myeloperoxidase-catalysed reaction of hydrogen peroxide with chloride ions. HOCl reacts readily with a number of susceptible targets on apolipoprotein B-100 of low-density lipoprotein (LDL), resulting in uncontrolled uptake of HOCl-modified LDL by macrophages. We have investigated the effects of vitamin C (ascorbate), an effective water-soluble antioxidant, on the HOCl- and chloramine-dependent modification of LDL. Co-incubation of vitamin C (25-200 microM) with LDL resulted in concentration-dependent protection against HOCl (25-200 microM)-mediated oxidation of tryptophan and lysine residues, formation of chloramines and increases in the relative electrophoretic mobility of LDL. Vitamin C also partially protected against oxidation of cysteine residues by HOCl, and fully protected against oxidation of these residues by the low-molecular-mass chloramines, N(alpha)-acetyl-lysine chloramine and taurine chloramine, and to a lesser extent monochloramine (each at 25-200 microM). Further, we found that HOCl (25-200 microM)-dependent formation of chloramines on apolipoprotein B-100 was fully reversed by 200 microM vitamin C; however, the loss of lysine residues and increase in relative electrophoretic mobility of LDL were only partially reversed, and the loss of tryptophan and cysteine residues was not reversed. Time-course experiments showed that the reversal by vitamin C of HOCl-dependent modifications became less efficient as the LDL was incubated for up to 4 h at 37 degrees C. These data show that vitamin C not only protects against, but also reverses, specific HOCl- and chloramine-dependent modifications of LDL. As HOCl-mediated LDL modifications have been strongly implicated in the pathogenesis of atherosclerosis, our data indicate that vitamin C could contribute to the anti-atherogenic defence against HOCl.

Myeloperoxidase binds to low-density lipoprotein: potential implications for atherosclerosis.

Myeloperoxidase (MPO), an abundant heme enzyme released by activated phagocytes, catalyzes the formation of a number of reactive species that can modify low-density lipoprotein (LDL) to a form that converts macrophages into lipid-laden or 'foam' cells, the hallmark of atherosclerotic lesions. Since MPO has been shown to bind to a number of different cell types, we investigated binding of MPO to LDL. Using the precipitation reagents phosphotungstate or isopropanol, MPO co-precipitated with LDL, retaining its catalytic activity. The association of MPO with LDL was confirmed using native gel electrophoresis. MPO was also found to co-precipitate with apolipoprotein B-100-containing lipoproteins in whole plasma. No precipitation of MPO was observed in lipoprotein-deficient plasma, and there was a dose-dependent increase in precipitation following addition of LDL to lipoprotein-deficient plasma. Binding of MPO to LDL could potentially enhance site-directed oxidation of the lipoprotein and limit scavenging of reactive oxygen species by antioxidants.

Toward a new recommended dietary allowance for vitamin C based on antioxidant and health effects in humans.

The current recommended dietary allowance (RDA) for vitamin C for adult nonsmoking men and women is 60 mg/d, which is based on a mean requirement of 46 mg/d to prevent the deficiency disease scurvy. However, recent scientific evidence indicates that an increased intake of vitamin C is associated with a reduced risk of chronic diseases such as cancer, cardiovascular disease, and cataract, probably through antioxidant mechanisms. It is likely that the amount of vitamin C required to prevent scurvy is not sufficient to optimally protect against these diseases. Because the RDA is defined as "the average daily dietary intake level that is sufficient to meet the nutrient requirement of nearly all healthy individuals in a group," it is appropriate to reevaluate the RDA for vitamin C. Therefore, we reviewed the biochemical, clinical, and epidemiologic evidence to date for a role of vitamin C in chronic disease prevention. The totality of the reviewed data suggests that an intake of 90-100 mg vitamin C/d is required for optimum reduction of chronic disease risk in nonsmoking men and women. This amount is about twice the amount on which the current RDA for vitamin C is based, suggesting a new RDA of 120 mg vitamin C/d.

Increased prevalence of antithyroid antibodies identified in women with recurrent pregnancy loss but not in women undergoing assisted reproduction.

OBJECTIVE: To assess the prevalence of antibodies to thyroglobulin and thyroid peroxidase (or microsomal) in women with recurrent pregnancy loss and women undergoing assisted reproductive techniques (ART) compared with healthy controls. DESIGN: Retrospective, two-centered study. SETTING: University-affiliated private patient centers. PATIENT(S): Included were 700 women with a history of two or more consecutive pregnancy losses, 688 women with a history of infertility who were undergoing ART, and 200 healthy, reproductive-aged female controls. INTERVENTION(S): Blood was collected before ART cycles, frozen, and assayed. MAIN OUTCOME MEASURE(S): Standardized ELISAs were used to measure antithyroid antibodies and TSH levels. Statistical analysis was performed with use of the two-tailed Fisher's exact test. RESULT(S): Antithyroid antibodies were identified in 29 of 200 (14.5%) of controls and 158 of 700 (22.5%) of women with recurrent pregnancy loss and 132 of 688 (19.2%) of women undergoing ART. Less than 20% of the women with antithyroid antibodies were clinically hypothyroid. CONCLUSION(S): Antithyroid antibodies are identified more frequently in women with recurrent pregnancy loss than in controls but not in women undergoing ART. These autoantibodies may be markers of autoimmune activation and have been associated with an increased risk of pregnancy loss and postpartum thyroid disease.

Differential reactivities of hypochlorous and hypobromous acids with purified Escherichia coli phospholipid: formation of haloamines and halohydrins.

Hypochlorous (HOCl) and hypobromous (HOBr) acids are strong oxidants derived from myeloperoxidase and eosinophil peroxidase, the major antimicrobial enzymes of neutrophils and eosinophils, respectively. These oxidants are highly reactive with a wide range of biomolecules. At physiological pH, both HOCl and HOBr react readily with amines to form haloamines and with the unsaturated bonds of fatty acids to form halohydrins. We have investigated which of these reactions occur with phosphatidylethanolamine (PE), the predominant phospholipid of Escherichia coli. The formation of haloamines was determined by TLC and colorimetrically and the formation of halohydrins was determined by TLC and GC-MS. With HOCl, chloramines were much the preferred product and chlorohydrins were formed in substantial amounts only when HOCl was in excess of the amount required to convert the amine to the dichloramine. With HOBr at all concentrations, bromamines and bromohydrins were formed concurrently, indicating a greater relative reactivity with unsaturated fatty acids than with HOCl. The bromamine derivatives of PE, and other primary amines, were found to be more reactive than the equivalent chloramines, and were able to brominate the unsaturated bonds of fatty acids. Bromohydrins (formed directly or through the action of bromamines) may, therefore, be suitable biomarkers for the production of HOBr in vivo.

Comparison of human red cell lysis by hypochlorous and hypobromous acids: insights into the mechanism of lysis.

Human red blood cells are lysed by the neutrophil-derived oxidant hypochlorous acid (HOCl), although the mechanism of lysis is unknown. Hypobromous acid (HOBr), a similarly reactive oxidant, lysed red cells approx. 10-fold faster than HOCl. Therefore we compared the effects of these oxidants on thiols, membrane lipids and proteins to determine which reactions are associated with lysis. There was no difference in the loss of reduced glutathione or membrane thiols with either oxidant, but HOBr reacted more readily with membrane lipids and proteins. Bromohydrin derivatives of phospholipids and cholesterol were seen at approx. one-tenth the level of oxidant than chlorohydrins were. However, these products were detected only with high concentrations of HOCl or HOBr, which caused instant haemolysis. Membrane protein modification occurred at much lower doses of oxidant and was more closely correlated with lysis. SDS/PAGE analysis showed that band 3, the anion transport protein, was lost at the lowest dose of HOBr and at the higher concentrations of HOCl. Labelling the red cells with eosin 5-maleimide, a fluorescent label for band 3, suggested possible clustering of this protein in oxidant-exposed cells. There was also irreversible cross-linking of all the major membrane proteins; this reaction occurred more readily with HOBr. The results indicate that membrane protein modification is the reaction responsible for HOCl-mediated lysis. These effects, and particularly cross-link formation, might result in clustering of band 3 and other membrane and cytoskeletal proteins to form haemolytic pores.

Oxidation of neutrophil glutathione and protein thiols by myeloperoxidase-derived hypochlorous acid.

Neutrophils, when stimulated, generate reactive oxygen species including myeloperoxidase-derived HOCl. There is an associated decrease in reduced glutathione (GSH) concentration. We have shown that neutrophil GSH levels decrease on exposure to reagent HOCl, whereas the equivalent concentration of H2O2 had no effect. GSH loss occurred without cell lysis, was not reversible, and was accompanied by the loss of an equivalent proportion of the total protein thiols. No glutathione disulphide was formed. Studies with 35S-labelled cells indicated that much of the GSH lost was accounted for by mixed disulphides with protein and a product that co-migrated on HPLC with a novel compound formed in the reaction of HOCl and pure GSH. The properties of this compound are consistent with an intramolecular sulphonamide. Neutrophils stimulated with PMA lost 30-40% of their GSH and a similar proportion of protein thiols. Little glutathione disulphide was formed and the products were the same as seen with HOCl-treated cells. From the results and studies with inhibitors and scavengers, we conclude that HOCl was responsible for the GSH loss. Propargylglycine and buthionine sulphoximine, inhibitors of glutathione synthesis, enhanced GSH loss, but their effects were due to the production of long-lived chloramines that oxidized GSH with greater efficiency than HOCl, rather than to the inhibition of GSH synthesis. The lack of thiol selectivity by HOCl and irreversibility of oxidation means that GSH will provide limited antioxidant protection for thiol enzymes in stimulated neutrophils.

Nuclear magnetic resonance characterization of 6 alpha-chloro-5 beta-cholestane-3 beta,5-diol formed from the reaction of hypochlorous acid with cholesterol.

Hypochlorous acid generated by neutrophil myeloperoxidase has been shown to convert cholesterol into three different chlorohydrin isomers which previously had not been fully characterized. We have reacted hypochlorous acid with cholesterol/1,2-dipalmitoyl phosphatidylcholine liposomes to give these three major products and established that they are 6 beta-chloro-5 alpha-cholestane-3 beta,5-diol (chlorohydrin 1), 5 alpha-chloro-6 beta-cholestane-3,6-diol (chlorohydrin 2) and 6 alpha-chloro-5 beta-cholestane-3 beta,5-diol (chlorohydrin 3). These products were separated by thin-layer chromatography and fully characterized by 1H, 13C, attached proton test, doublequantum correlation spectroscopy, total correlation spectroscopy, heteronuclear multiple bond correlation and heteronuclear multiple quantum coherence nuclear magnetic resonance spectroscopy.

Modification of red cell membrane lipids by hypochlorous acid and haemolysis by preformed lipid chlorohydrins.

Hypochlorous acid (HOCl), a strong oxidant generated by the myeloperoxidase system of neutrophils and monocytes, has been implicated in inflammatory tissue damage by these cells. Reaction of HOCl with the double bonds of unsaturated lipids produces alpha, beta-chlorohydrin isomers. We have exposed red cell membranes to HOCl and used thin layer chromatography (TLC) of the extracted lipids and enzyme-linked immunosorbent assay (ELISA), using an antichlorohydrin monoclonal antibody, to show that fatty acyl chlorohydrins are formed. The ELISA was approximately 25 fold more sensitive than TLC, and chlorohydrins were detected when membranes from 10(6) cells were treated with > or = 0.16 nmoles HOCl. Lipid chlorohydrins are more polar and bulky than their parent lipids and as such could affect membrane stability and function. To determine the effect of incorporation of lipid chlorohydrins into cell membranes, preformed fatty acid and cholesterol chlorohydrins were incubated with red cells. Lysis was measured as release of haemoglobin and incorporation of lipids was determined by 14C scintillation counting. Addition of HOCl-treated oleic acid to red cells resulted in rapid lysis of a fraction of the cells in a concentration dependent manner. HOCl-treated cholesterol also caused a small amount of cell lysis that was predominantly due to chlorohydrin 3, one of the three major cholesterol chlorohydrin products. Chlorohydrin 3, which has a decreased planarity and polarity, was also primarily responsible for altering the critical micelle concentration of HOCl-treated cholesterol-containing liposomes.