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.

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.

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.

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

Reaction time impairment in schizophrenia and affective illness: the role of attention.

A young but chronic group of schizophrenic and affective disorders patients was tested for simple reaction time (RT) and RT while engaged in a concurrent task. The affective disorders patients were subdivided by the presence of psychotic features. The results show that extreme slowing of RT is due to psychoticism and is not characteristic of nonpsychotic affective illness. Extreme intrasubject variability, however, was specific to schizophrenia, and may be a trait marker of the disorder.

Assays using horseradish peroxidase and phenolic substrates require superoxide dismutase for accurate determination of hydrogen peroxide production by neutrophils.

We used horseradish peroxidase and either scopoletin, homovanillic acid, or phenol red to measure hydrogen peroxide generated by human neutrophils. With these assays, superoxide dismutase significantly increased the amount of hydrogen peroxide detected. In contrast, it had no effect when the accumulation of hydrogen peroxide was measured with a hydrogen peroxide electrode. We propose that superoxide interferes with horseradish peroxidase-dependent assays so that hydrogen peroxide is underestimated. Thus, when using these assays, superoxide dismutase must be added to neutrophils to ensure that all the hydrogen peroxide they produce is detected.

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.

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.

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.

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.

Free radical inactivation of rabbit muscle creatinine kinase: catalysis by physiological and hydrolyzed ICRF-187 (ICRF-198) iron chelates.

Creatine kinase is a sulfhydryl containing enzyme that is particularly susceptible to oxidative inactivation. This enzyme is potentially vulnerable to inactivation under conditions when it would be used as a diagnostic marker of tissue damage such as during cardiac ischemia/reperfusion or other oxidative tissue injury. Oxidative stress in tissues can induce the release of iron from its storage proteins, making it an available catalyst for free radical reactions. Although creatinine kinase inactivation in a heart reperfusion model has been documented, the mechanism has not been fully described, particularly with regard to the role of iron. We have investigated the inactivation of rabbit muscle creatine kinase by hydrogen peroxide and by xanthine oxidase generated superoxide or Adriamycin radicals in the presence of iron catalysts. As shown previously, creatine kinase was inactivated by hydrogen peroxide. Ferrous iron enhanced the inactivation. In addition, micromolar levels of iron and iron chelates that were reduced and recycled by superoxide or Adriamycin radicals were effective catalysts of creatinine kinase inactivation. Of the physiological iron chelates studied, Fe(ATP) was an especially effective catalyst of inactivation by what appeared to be a site-localized reaction. Fe(ICRF-198), a non-physiological chelate of interest because of its putative role in alleviating Adriamycin-induced cardiotoxicity, also catalyzed the inactivation. Scavenger studies implicated hydroxyl radical as the oxidant involved in iron-dependent creatine kinase inactivation. Loss of protein thiols accompanied loss of creatine kinase activity. Reduced glutathione (GSH) provided marked protection from oxidative inactivation, suggesting that enzyme inactivation under physiological conditions would occur only after GSH depletion.

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.

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.

A monoclonal antibody recognizing the chlorohydrin derivatives of oleic acid for probing hypochlorous acid involvement in tissue injury.

A monoclonal antibody against hypochlorous acid-modified oleic acid has been raised to investigate involvement of HOCI in tissue injury. Mice were immunized with an isomeric mixture of chlorohydrin derivatives of oleic acid (18:0-chlorohydrin) conjugated to keyhole limpet haemocyanin (CH-KLH). The chlorohydrin was formed by the treatment of oleic acid with hypochlorous acid. Monoclonal antibodies were raised and the fusion was screened with 18:0-chlorohydrin-bovine serum albumin (CH-BSA) conjugate. A number of antibody-secreting clones were identified and the supernatants were characterized by binding studies and dose-response curves. In ELISA, mAb CH-1 had an equivalent titre when either the chlorohydrin or bromohydrin derivative of oleic acid, complexed to bovine serum albumin, was used as screening antigen. The mAb CH-1 recognition of CH-BSA was competed with chlorohydrin and bromohydrin conjugates of BSA and KLH. Similarly, free 18:0-chlorohydrin and the 18:0-chlorohydrin-phosphatidyl choline treated with hypochlorous acid competed with mAb CH-1 binding. The mAb CH-1 also recognised the chlorohydrin derivative of linoleic acid and chlorohydrin formed from palmitoyl, oleyl phosphatidyl choline but with a decreased avidity. Weak cross-reactivity was observed with hydroxy-linoleic acid and linoleic acid hdroperoxide, either as free fatty acid or in phosphatidyl choline. There was minimal competitive binding of mAb CH-1 to free oleic acid, 16:0/18:1 phosphatidylcholine, cholesterol, or cholesterol chlorohydrin. The mAb CH-1 described here may be a useful probe for assessing the involvement of hypochlorous acid in tissue injury.

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.

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.

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.

Automated cognitive assessment of elderly patients: a comparison of two types of response device.

Groups of 14 cognitively impaired elderly people were tested on two automated cognitive tests on several occasions. Patients registered their responses using either a touch-sensitive screen or a board with illuminated response buttons. The results indicate that the touch-sensitive screen is probably a more suitable response device than the button-board. Suggestions that it would prove disadvantageous to elderly patients were not supported, and its continued use is recommended.

Controlled study of self-exposure treatment for phobics: preliminary communication.

Patients with phobic disorder (mainly agoraphobics ) of minimum one year duration were treated by self-administered exposure in vivo treatment. Seventy-one patients were randomly assigned to one of three groups: (A) book-instructed, (B) computer-instructed, or (C) therapist-instructed. All three groups improved significantly to a similar extent on various phobic measures at the end of the treatment and maintained their treatment gains at 6-month follow up. Mean clinicians' time spent with each patient was 40 minutes, 4.2 hours and 3.2 hours in group A, B and C respectively. Similar small numbers of patients defaulted from each group.