The effect of stress on the antioxidative potential of serum: implications for Alzheimer's disease.

There is growing consensus in the literature that oxidation status is increased in Alzheimer's disease (AD), and that antioxidant supplementation as prevention or treatment strategy should be investigated further. In the present study the total antioxidant status (TAS) was found to be highly significantly lower in 22 AD patients (p < 0.0001) than in 22 age- and gender matched non-demented controls. The TAS was also lower than controls in 22 patients with vascular dementia, but not significantly. The increased oxidation status in AD was verified using the benzoate hydroxylation method. The origin of the enhanced oxidation status in AD has not been elucidated. To determine whether a causal effect between stress and oxidative status of serum can be demonstrated, a rat model was used with two different kinds of stressors, swim stress (exercise) and restraint stress (non-exercise stress). Following swim stress the maximum oxidative effect was observed at one hour post stress (p < 0.001). At 24 h the oxidative status had recovered significantly to below control values. Restraint stress, however, showed progressively increased oxidation which attained significance after 24 h (p < 0.005). It is postulated that stress may contribute to the higher oxidation status in AD patients.

Population screening for isoniazid acetylator phenotype.

PURPOSE: To establish a useful method for acetylator phenotypification and therapeutic drug monitoring of patients receiving isoniazid. METHODS: Sixty patients with uncomplicated pulmonary tuberculosis were given a 5-mg/kg oral dose of isoniazid each. Plasma concentrations of isoniazid and its metabolite, acetyl-isoniazid, were determined by HPLC analyses at various post-dose times. From the isoniazid concentration and the concentration ratio of acetyl-isoniazid and isoniazid (metabolic ratio), phenotypification methods were assessed. RESULTS: The metabolic ratios at 3 h post-dose revealed a trimodal distribution; a fast, intermediate and slow acetylator phenotype group. The 2-h and 6-h data showed different bimodal combinations of these phenotype groups. The metabolic ratio phenotypification method could be simplified by using the HPLC data directly without converting it to absolute concentrations. CONCLUSIONS: A single-sample test based upon the plasma isoniazid concentration, combined with the metabolic ratio of acetyl-isoniazid and isoniazid, appears to be a reliable parameter for phenotype discrimination and for bioavailability testing.

Trimodality of isoniazid elimination: phenotype and genotype in patients with tuberculosis.

The study was undertaken to show that polymorphic isoniazid elimination in humans is trimodal; that the acetylator genotype and eliminator phenotype of the individual patient are concordant; and that the differences in the pharmacokinetic parameters of fast, intermediate, and slow eliminator subgroups are statistically significant. Sixty adult patients of both sexes and of mixed race with tuberculosis participated in the trial. The apparent elimination rate constant (k, h(-1)) and the area under the isoniazid concentration-time curve (AUC, mg/L/h), over the interval 2 to 6 h after oral isoniazid were determined in all patients; NAT2 allele composition was determined in 47 patients. Serum INH concentrations were determined by HPLC and genotypes by PCR/restriction enzyme analysis. Three eliminator phenotypes could be distinguished, and concordance between the phenotype and the genotype of the individual could be demonstrated. The isoniazid concentration-time profiles of the three eliminator subgroups were significantly different (p < 0.05). The NAT2*12A allele, which codes for fast acetylation, has a high frequency in the population studied, the intermediate acetylator genotype is constituted of codominant fast and slow alleles, and the distribution of phenotypes/genotypes in the population is consistent with Hardy-Weinberg predictions. The therapeutic implications of polymorphic isoniazid metabolism are discussed.

Free radical scavenging effects of melatonin and serotonin: possible mechanism.

Melatonin has been reported to be a potent free radical scavenger, but the mechanism by which it protects membranes from lipid peroxidation is poorly understood. The present study addresses this problem by comparing the free radical scavenging properties of melatonin and serotonin, two indoles with similar structure, but differing solubilities. Both serotonin and melatonin significantly prevented lipid peroxidation of platelet membranes. Additionally, melatonin significantly decreased the microviscosity (increased the fluidity) of platelet membranes, while serotonin had the opposite effect. These data led us to postulate that serotonin exerts its free radical scavenging action in the aqueous phase, or at the water-membrane interface, while melatonin positions itself within the lipid bilayer where it protects membrane phospholipids against free radical attack.

Transferrin C2 and Alzheimer's disease: another piece of the puzzle found?

A significant increase in the occurrence of the transferrin C2 genetic subtype has been found in patients with Alzheimer's disease. This variant has previously been linked to diseases thought to be associated with free radical damage. We hypothesize that Alzheimer's disease is caused by free radical damage to membranes of endocytic vesicles due to defective binding of iron and aluminium by Tf C2. The aluminium binds to the membranes, creating pores, while the iron reacts with H2O2 and superoxide radicals produced by activated microglia (brain phagocytes), to produce hydroxyl radicals (oxidative toxins), which attack the fatty acids in the membranes through these pores. In order to treat the disease successfully, it would be necessary to alleviate the multiple deficiencies caused by these toxins by constantly providing the cells with antioxidants and other essential nutrients. In addition, a drug that would stimulate the regrowth of neurons is needed.

Apparent hydroxyl radical generation without transition metal catalysis and tyrosine nitration during oxidation of the anti-tubercular drug, isonicotinic acid hydrazide.

Aromatic hydroxylation and formation of thiobarbituric acid-reactive substances occurred in a mixture of isonicotinic acid hydrazide (isoniazid) and catalase. Since these reactions were stimulated by phytic acid (a potent metal chelator), rather than inhibited, transition metal-catalysed hydroxyl radical generation was not implicated. Hydroxylation also occurred with isoniazid and phytic acid in the absence of catalase, albeit to a lesser extent. The independent effects of catalase and phytic acid are related to their abilities to catalyse isoniazid oxidation. In the presence of tyrosine, both the isoniazid/phytic acid system and authentic peroxynitrite generated dityrosine. Authentic peroxynitrite, as well as a phytic acid-mediated isoniazid oxidation product, have absorbance maxima at 302 nm. The yield of this isoniazid-derived product increased with pH and in the presence of a superoxide-generating system. A good correlation existed between absorbance at 302 nm and aromatic hydroxylation. Acid-induced decomposition of the 302 nm absorbance in the presence of superoxide dismutase led to the formation of a product absorbing in the same region as peroxynitrite-modified superoxide dismutase (350 nm at acid pH). Catalase catalysed peroxynitrite-mediated, as well as isoniazid/phytic acid-mediated tyrosine nitration, which was accompanied by Compound II formation (ferryl-catalase) in both cases. We postulate that peroxynitrite or a similar species is formed during isoniazid oxidation.

Lipid peroxidation and platelet membrane fluidity--implications for Alzheimer's disease?

In humans, the fluidity of cell membranes generally decreases with age. Unexpectedly, several laboratories have found increased fluidity of platelet membranes (mainly endoplasmic reticulum) in patients with Alzheimer's disease (AD) compared with controls. In the present study, free radical induced lipid peroxidation was found to increase the fluidity of platelet membranes. Hydroxyl radicals were generated in the presence of Fe2+ and EDTA at low concentrations of ascorbate. It is hypothesised that platelet membranes are unable to restore their microviscosity by incorporating cholesterol. There may be a link between the result obtained in this study, the recently discovered decreased cholesterol content of affected AD neuronal membranes, and the increased frequency of epsilon 4 apolipoprotein E (a cholesterol carrier) found in AD patients.

Different oxidative pathways of isonicotinic acid hydrazide and its meta-isomer, nicotinic acid hydrazide.

1. Superoxide was generated during the auto-oxidation of the antituberculous drug, isonicotinic acid hydrazide (INH), but not with its meta-isomer, nicotinic acid hydrazide (NH). During Fe(3+)-stimulated oxidation of INH and NH, aromatic hydroxylation occurred which was inhibited by the chelating agent, phytic acid. 2. A mixture of myeloperoxidase (MPO) and a hydrazide induced formation of compound III (oxyperoxidase) and aromatic hydroxylation which was stimulated by phytic acid. INH was considerably more potent than NH. 3. Co-oxidation of a hydrazide and thyroxine (T4) in the MPO system resulted in the formation of a pink-coloured product (maximum absorbance at 504 nm) which was more stable with NH than with INH. 4. The hydrazides and Cl- acted synergistically on MPO haem modification when co-oxidised in the MPO-H2O2 system. INH was more destructive than NH. 5. The different oxidative pathways of the hydrazides are consistent with the fact that an acyl intermediate of INH, unlike that of NH, is resonance stabilized.

Aromatic hydroxylation during the myeloperoxidase-oxidase oxidation of hydrazines.

Benzoic acid was found to be hydroxylated by a mixture of myeloperoxidase (MPO) and the mycobactericidal drug, isoniazid. Aromatic hydroxylation and formation of compound III (oxyperoxidase) were coincident during the MPO-oxidase oxidation of isoniazid which proceeded without augmentation from the reagent hydrogen peroxide. An intermediate of isoniazid reduced ferric MPO to ferrous MPO which associated with dioxygen to form compound III. Aromatic hydroxylation also occurred in a mixture of isoniazid (or phenylhydrazine) and a ferric salt. Hydroxylations in both the enzymatic and nonenzymatic reaction systems were inhibited by the iron chelator, desferal, as well as by the specific hydroxyl radical scavenger, mannitol. To distinguish between the hydroxylating intermediates in the different reaction systems, the unique properties of the natural antioxidant, phytic acid, were exploited. Phytic acid inhibited aromatic hydroxylation in the Fe(3+)-INH system, which is in accordance with its known properties as a powerful inhibitor of iron-driven reactions (.OH formation). By contrast, phytic acid stimulated hydroxylation in the enzymatic system which was accompanied by a concomitant stimulation in the rate of compound III formation. These events were, however, not directly related to each other. Phytic acid had a direct effect on the redox transformation of isoniazid by stimulating superoxide generation during auto-oxidation of the drug. In addition, phytic acid also facilitated compound III decay in the absence of isoniazid, suggesting that it may also regulate the oxygen affinity of MPO, similar to its effect on the oxygenation of haemoglobin. The data on aromatic hydroxylation in the MPO-isoniazid system do not support a role for .OH in the reaction and may fit the model for the P450 mixed oxidase system.

Thyroxine-supported oxidation in the myeloperoxidase system.

Thyroxine and other iodothyronines (concentrations in the nanomolar range) stimulated the oxidation of NADH in the myeloperoxidase-H2O2-Cl- system. In the absence of chloride, thyroxine had only a marginal effect. This suggests that thyroxine increased the generation of chlorinating oxidants. A peroxidase-catalysed oxidation product of thyroxine, 3,5-diiodotyrosine, was inactive. Pre-incubation of thyroxine in the myeloperoxidase system showed that thyroxine was oxidized to a product capable of stimulating NADH oxidation. Reduction and alkylation of myeloperoxidase under nondenaturing conditions also increased the oxidative activity of the enzyme. It is postulated that both iodoacetamide and a thyroxine-derived oxidation product (presumably a quinone) alkylate sulphydryl groups near the active centre of myeloperoxidase making it more accessible for its substrate.

Anti-oxidant properties of H2-receptor antagonists. Effects on myeloperoxidase-catalysed reactions and hydroxyl radical generation in a ferrous-hydrogen peroxide system.

Ulcerogenesis of the gastroduodenal mucosa is caused by the digestive action of gastric juice and initially involves an inflammatory reaction with infiltration of phagocytes. The anti-inflammatory activity of many drugs have been attributed to the inhibition of the leukocyte enzyme, myeloperoxidase (MPO). In this study, the H2-antagonists in clinical use were found to be potent inhibitors of MPO-catalysed reactions (IC50 < 3 microM) under conditions resembling those in experiments with intact neutrophils. Since peak plasma concentrations of cimetidine, ranitidine and nizatidine are well within the micromolar range, after oral therapeutic dosing, our results may be of clinical relevance. The inhibitory actions of cimetidine and nizatidine were largely due to scavenging of hypochlorous acid (HOCl), a powerful chlorinating oxidant produced in the MPO-H2O2-Cl- system. In contrast to famotidine, ranitidine was also a potent scavenger of HOCl, while both drugs inhibited MPO reversibly by converting it to compound II, which is inactive in the oxidation of Cl-. The HOCl scavenging potencies of ranitidine and nizatidine were about three times higher than that of the anti-rheumatic drug, penicillamine, which had a potency similar to that of cimetidine. The rapid HOCl scavenging ability of penicillamine is thought to contribute to its anti-inflammatory effects. Using riboflavin as a probe, the H2-antagonists were found to be inhibitors of hydroxyl radical (.OH) generated in a Fe(2+)-H2O2 reaction mixture. Spectral analyses of the interaction of iron ions with the drugs and studies with chelators, suggest that the drugs were efficient chelators of Fe2+, in addition to their .OH scavenging abilities. Since the gastrointestinal tract can contain potentially reactive iron, the simultaneous presence of H2-antagonists may help to suppress iron-driven steps in tissue damage.

Interaction of the di-catechols rooperol and nordihydroguaiaretic acid with oxidative systems in the human blood. A structure-activity relationship.

The effects of the di-catechols rooperol [(E)-1,5-bis(3',4'- dihydroxyphenyl)pent-4-en-1-yne; P2] and nordihydroguaiaretic acid (NDGA) on oxidative systems in the human blood were investigated. P2 and NDGA gave comparable results in the inhibition of leukotriene synthesis in the polymorphonuclear leukocyte and prostaglandin synthesis in platelet microsomes. The oxidation states of myeloperoxidase in the presence of H2O2 were also similarly affected by both P2 and NDGA. In these systems, the 4'4'-beta-D-diglucopyranoside of rooperol, hypoxoside [(E)-1,5-bis(4' beta-D-glucopyranosyloxy-3'- hydroxyphenyl) pent-4-en-1-yne; P2A] had no effect. The only system which showed significant differences in the effects of the catechols was the red blood cell. NDGA in the presence of H2O2 had a pronounced haemolytic effect, which did not correlate with its ability to induce methaemoglobin formation, while P2 had a much lower haemolytic effect, but stimulated the oxidation of haemoglobin to a greater extent than NDGA. NDGA is more hydrophobic than P2 which would result in a greater membrane effect. The pent-4-en-1-yne chain which links the catechol moieties in P2 can take part in the resonance structures of the semiquinone free radical, thus assisting in its stabilization and leading to increased methaemoglobin formation. This stabilization is also demonstrated by the fact that P2A affected the oxidation of haemoglobin to nearly the same extent as NDGA.

Interaction of methyl-xanthines with myeloperoxidase. An anti-inflammatory mechanism.

1. Inhibition of myeloperoxidase (MPO)-catalyzed reactions by methyl-substituted xanthines has been investigated. 2. Except for theobromine and caffeine, all xanthines tested were potent inhibitors of the MPO-H2O2-Cl- system. 3. In contrast to methyl substitution in the 1 or 8 position of xanthine, substitution in the 3 or 7 position had a marked effect on the inhibition of MPO catalysis. 4. Two different inhibitory mechanisms were induced; scavenging of hypochlorous acid (HOCl) generated by the MPO system and accumulation of Compound II (ferryl MPO) which is inactive as a catalyst of Cl- oxidation.

Mechanisms by which clofazimine and dapsone inhibit the myeloperoxidase system. A possible correlation with their anti-inflammatory properties.

The mechanisms by which two anti-leprotic drugs (clofazimine and dapsone), both with anti-inflammatory properties, inhibit myeloperoxidase (MPO)-catalysed reactions, were investigated. The disappearance of NADH fluorescence was used as an assay for its oxidation. Chloride stimulated the oxidation of NADH in the MPO-H2O2 system in a concentration-dependent manner (50-fold at 150 mM NaCl). Under these conditions Cl- is oxidized and the oxidant formed, presumably hypochlorous acid (HOCl), oxidizes NADH. Observations demonstrating the effect of the drugs on the MPO system, are: (1) Inhibition of Cl(-)-stimulated oxidation of NADH. (2) Inhibition of polypeptide modification in a model protein, thyroglobulin (TG). (3) Protection of MPO against loss of catalytic activity caused by chlorinating oxidants generated by the system. (4) Inhibition of haemoglobin oxidation. Only dapsone was active here. HPLC analyses suggested that the drugs were not significantly metabolized in the MPO-H2O2 system in the absence of Cl-. Bleaching of clofazimine was stimulated by Cl- in the MPO system, suggesting the involvement of HOCl. Clofazimine was found to be a more potent scavenger of HOCl than dapsone when the inhibition of NADH oxidation by reagent HOCl was used as an assay. This finding is also supported by HPLC analyses which indicated a greater sensitivity of HOCl for clofazimine than for dapsone. Relatively low concentrations of dapsone inhibited the oxidation of oxygenated haemoglobin (HbO2), suggesting that the drug was not metabolized to its N-hydroxylated derivative which is thought to be responsible for methaemoglobin (metHb) formation in vivo. It is proposed that the inhibitory mechanism of action of clofazimine is to scavenge chlorinating oxidants generated by the MPO-Cl(-)-H2O2 system, while dapsone converts MPO into its inactive compound II (ferryl) form. The different inhibitory mechanisms of clofazimine and dapsone towards the MPO system may contribute to the anti-inflammatory actions of the drugs.

Cytotoxicity of myeloperoxidase-activated catechols: oxidative injury to the red blood cell.

The effects of two catechols (1,2-benzenediol and nordihydroguaiaretic acid) on the myeloperoxidase-Cl(-)-H2O2 antimicrobial/cytotoxic system of the human neutrophil were investigated. To determine the cytotoxicity of myeloperoxidase-generated oxygen metabolites (mainly chlorinated oxidants such as hypochlorite) and catechol oxidation products, the well characterized erythrocyte was used as a target. At relatively low concentrations (less than 10 microM), the catechols acted as redox catalysts by stimulating the generation of chlorinated oxidants. This is visualized as a promotion of haemolysis which reached a maximum and then decreased again with increasing concentrations of the catechol. In this respect, the dicatechol, nordihydroguaiaretic acid, was more potent. At higher concentrations, the catechols competed more effectively with Cl- as electron donors and the generation of chlorinated oxidants decreased with a consequent decrease in haemolysis. Above 200 microM nordihydroguaiaretic acid, complete haemolysis occurred which might be due to high membrane concentrations of the catechol due to its high lipid solubility. In contrast, high 1,2-benzenediol concentrations did not induce haemolysis. The catechols stimulated methaemoglobin formation in a concentration-dependent fashion with 1,2-benzenediol more potent than nordihydroguaiaretic acid. There was some correlation between membrane microviscosity and haemolysis which in turn did not correlate with haemoglobin oxidation. No direct correlation existed between intracellular methaemoglobin formation and the precipitation of haemoglobin oxidation products on the membrane. Disulphide crosslinks were not involved in the covalent polymerization of haemoglobin subunits.

Activation of chlorpromazine by the myeloperoxidase system of the human neutrophil.

The univalent oxidation of chlorpromazine (CPZ) by the myeloperoxidase (MPO-H2O2) system led to the formation of a cation free radical (CPZ+) which was observed optically at 527 nm. CPZ protected MPO against loss of catalytic activity when co-oxidized in a MPO-Cl(-)-H2O2 system. Due to the stability of CPZ+ either further oxidation, or reduction back to the mother compound, become important mechanisms for disappearance of the free radical. Thus, the rate of formation and decay of CPZ+ were higher in the presence of Cl- than in its absence, since the radical can also be oxidized further by hypochlorous acid (HOCl), which is formed in the MPO-Cl(-)-H2O2 system. Decay of CPZ+ can also be due to electron acceptance from ascorbic acid or oxygenated haemoglobin (HbO2), resulting in regeneration of CPZ. When CPZ+ was generated in the MPO-H2O2 system, addition of HbO2 resulted in a sudden decrease in CPZ+ absorbance at 527 nm and a concomitant formation of metHb. When HbO2 was not added, the decay of CPZ+ was much slower. CPZ (in the absence of the MPO system) also stimulated the oxidation of HbO2 in the presence of 20 microM H2O2, but this reaction was considerably slower than when CPZ+ (generated by the MPO system) was allowed to react directly with HbO2. These results suggest that HbO2 was oxidized by CPZ+. To study the effect of CPZ intermediates, thyroglobulin (TG) was used as a model polypeptide. Chlorinated oxidants formed in the MPO system (in the absence of CPZ) induced TG peptide bond splitting. In contrast, CPZ metabolites generated by the MPO system (in the absence of Cl-) induced polymerization of TG, as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

Stimulation of the chlorinating activity of human myeloperoxidase by thyroid hormones and analogues.

Thyroxine and triiodothyronine concentrations of 50 nM or lower can stimulate the chlorinating activity of the myeloperoxidase-H2O2-Cl- antimicrobial system in vitro. The initial rates of the chlorinating reaction with monochlorodimedone were similar for both thyroid hormones. Maximum stimulation occurred around pH 6 and a linear relationship exists between stimulation and thyroxine concentrations up to at least 1 microM. Of the various thyroxine analogues tested, stimulation was in the order: T4 (or T3) greater than triiodothyropropionic acid greater than 3,5-T2. Diiodotyrosine did not have any significant stimulatory effect. The oxidised product of the phenolic ring of T4, presumably a hydroxyquinone, may act as an additional electron carrier and thereby facilitates redox reactions.

Isoniazid-mediated irreversible inhibition of the myeloperoxidase antimicrobial system of the human neutrophil and the effect of thyronines.

During aerobic myeloperoxidase-catalysed oxidation of isoniazid at pH 7.8, compound III was generated. Oxidation of isoniazid or hydrazine sulphate at pH values of 6.5 or 7.8 in a myeloperoxidase-H2O2 system caused considerable haem loss, which was associated with compound III formation. Haem loss and also compound III formation could be inhibited when 8 microM thyroxine was included in the reaction mixtures. During the reaction with isoniazid, an intense pink-coloured pigment with maximum absorbance at 500 nm was formed which could be bleached with ascorbate or hypochlorous acid. The pigment was more stable at pH 7.8 than at pH 6.5. A similar pink colour was generated when a mixture of isoniazid and thyroxine in alkaline solution was irradiated with light of wavelength greater than 300 nm. A possible product of thyroxine oxidation, 3,5-diiodotyrosine, could not protect the enzyme against isoniazid-mediated haem loss and no colour formation was observed. Haem loss was most extensive when isoniazid was oxidised in a myeloperoxidase system at pH 7.8 in the presence of 0.1 M NaCl. Thyroxine (8 microM), however, could still inhibit haem loss under these conditions. A good correlation was found between haem loss and irreversible loss of peroxidase activity.

The inhibitory effect of acetaminophen on the myeloperoxidase-induced antimicrobial system of the polymorphonuclear leukocyte.

Acetaminophen binds via its acetamido side chain to purified myeloperoxidase in a pH-dependent manner and maximum binding occurred around pH 6. The H2O2-dependent myeloperoxidase-catalysed polymerization products of acetaminophen had excitation maxima at 304 nm and 334 nm in acid and alkaline solutions, respectively, and an intense blue fluorescence maximum at 426 nm. Acetaminophen can compete effectively with Cl- as myeloperoxidase substrate and thus HOCl formation is suppressed while HOCl, nevertheless present, can be scavenged by the drug. In this way the microbicidal action of the myeloperoxidase-H2O2-Cl- system can be seriously limited in the presence of high concentrations of acetaminophen. To study the effect of acetaminophen on peptide bond splitting in the myeloperoxidase antimicrobial system, thyroglobulin was used as a model peptide. Peptide bond splitting was inhibited at acetaminophen concentrations below the accepted toxic range for plasma values.

The effect of thyroxine and related compounds on the aerobic myeloperoxidase--catalysed oxidation of NADH.

Thyroxine concentrations as low as 1 microM significantly stimulate compound III formation during aerobic oxidation of NADH by highly purified myeloperoxidase. This increased compound III formation is paralleled by an increased oxidation of NADH. Stimulation of various thyronine and tyrosine analogues was in the order T4 greater than T3 greater than 3,5-T2 (or triiodothyropropionic acid). Thyronine and diiodotyrosine had no significant effect. From the potencies of the various thyronines to stimulate compound III formation, the following structural features seem necessary: (1) Substitution of thyronine with four iodine atoms. (2) An amino group on the alanine side chain. (3) Both aromatic rings of thyronine.