Identification of Products from Oxidation of Uric Acid Induced by Hydroxyl Radicals

The aim of the present study was to separate and characterise products formed by oxidation of uric acid by hydroxyl radicals with a view to probing for these products in vivo in clinical contexts. Aerated solutions of 200 μM uric acid, or its oxidation products, allantoin or parabanic acid, were exposed to gamma radiolysis, (52.0 Gy/min), as a source of HO- radicals, at pH 3.4 and 7.4. Aliquots were taken every 5 minutes for 20 minutes and oxidation products were separated by HPLC and analysed with a diode array detector. Identities of oxidation products were confirmed on the basis of similarity of retention times and absorbance spectra and peak purity parameters of known standards. Hydroperoxides were measured by tri-iodide formation in the 20 minute sample. Exposure of uric acid to such HO fluxes produced a net loss of the parent compound with formation of a complex mixture of products with allantoin and parabanic acid being the predominant products at pH 3.4. The rate of uric acid degradation at physiological pH was slower and the distribution of oxidation products was different. A small but significant amount of uric acid hydroperoxide was detected at both pHs. A mechanism for uric acid oxidation under these conditions is presented.     

比色法测定Fenton反应产生的羟自由基及其应用

Fenton反应产生的羟自由基与二甲亚砜反应,生成甲基亚磺酸,再与坚牢蓝BB盐反应生成偶氮砜,比色法测定其含量可间接测定OH·的生成量. 通过对测定条件的研究,得到最佳实验方案. 抗氧化剂药物硫脲和抗坏血酸与羟自由基清除率具有明显的量效关系. 测定了核桃、黑芝麻等几种天然食物的水提取物清除羟自由基的功能. 此法可用于羟自由基清除剂的筛选.     

Role of Hydroxyl Radicals in Escherzchza Colz Killing Induced by Hydrogen Peroxide

Escherichia coli lethality by hydrogen peroxide is characterized by two modes of killing. In this paper we have found that hydroxyl radicals (OH -) generated by H₂O₂ and intracellular divalent iron are not involved in the induction of mode one lethality (i.e. cell killing produced by concentrations of H₂O₂ lower than 2.5 mM). In fact, the OH radical scavengers, thiourea, ethanol and dimethyl sulfoxide, and the iron chelator, desferrioxarnine, did not affect the survival of cells exposed to 2.5mM H₂O₂. In addition cell vulnerability to the same H₂O₂ concentration was independent on the intracellular iron content. In contrast, mode two lethality (i.e. cell killing generated by concentrations of H₂O₂ higher than 10mM) was markedly reduced by OH radical scavengers and desferrioxamine and was augmented by increasing the intracellular iron content.

It is concluded that OH. are required for mode two killing of E. coli by hydrogen peroxide.     

Alpha-Phenyl-N-Tert-Butylnitrone Attenuates Excitotoxicity in Rat Striatum by Preventing Hydroxyl Radical Accumulation

Various in vitro experiments have indicated that oxygen-derived free radicals may contribute to excitotoxic neuronal death. In the present study we induced excitotoxicity in rat striatum by perfusing glutamate at a high concentration through a microdialysis probe. We observed an increased formation of hydroxyl radicals (^˙OH) during the perfusion of the excitotoxin and an extensive striatal lesion 24 h after the insult. The spin trap, -phenyl-N-tert-butylnitrone (PBN), attenuated both hydroxyl radical levels and the volume of the lesion. This result suggests that the neuroprotection may be due to a free radical scavenging mechanism. It also implies that PBN may be used in pathological situations involving excitotoxicity such as stroke, brain trauma, and chronic neurologic diseases.     

Genetic vitamin E deficiency does not affect MPTP susceptibility in the mouse brain

Oxidative stress is involved in the degeneration of the nigrostriatal dopaminergic system in Parkinson's disease (PD). Vitamin E (alpha-tocopherol) is a potent antioxidant in the cell membrane that can trap free radicals and prohibit lipid peroxidation. The retention and secretion of vitamin E are regulated by alpha-tocopherol transfer protein (TTP) in the brain and liver. Dysfunction of TTP results in systemic deficiency of vitamin E in humans and mice, and increased oxidative stress in mouse brain. In this study, we investigated the effect of vitamin E deficiency in PD development by generating an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD using TTP knockout (TTP-/-) mice. Vitamin E concentration in the brains of TTP+/- mice was half that in TTP+/+ mice, and in TTP-/- mice, was undetectable. MPTP treatment tended to decrease striatal dopamine, but the effect was comparable and not significant in any of the three genotypes. Furthermore, the extent of loss of dopaminergic cell bodies in the substantia nigra did not differ among the groups. One the other hand, oral administration of vitamin E resulted in the partial protection of striatal dopaminergic terminals against MPTP toxicity. Our results suggest that vitamin E does not play a major protective role in MPTP-induced nigrostriatal dopaminergic neurodegeneration in the brain.     

Mazindol Attenuates the 3,4-Methylenedioxymethamphetamine-Induced Formation of Hydroxyl Radicals and Long-Term Depletion of Serotonin in the Striatum

The formation of hydroxyl radicals following the systemic administration of 3,4-methylenedioxymethamphetamine (MDMA) was studied in the striatum of the rat by quantifying the stable adducts of salicylic acid and D-phenylalanine, namely, 2,3-dihydroxybenzoic acid (2,3-DHBA) and p-tyrosine, respectively. The repeated administration of MDMA produced a sustained increase in the extracellular concentration of 2,3-DHBA and p-tyrosine, as well as dopamine. The MDMA-induced increase in the extracellular concentration of both dopamine and 2,3-DHBA was suppressed in rats treated with mazindol, a dopamine uptake inhibitor. Mazindol also attenuated the long-term depletion of serotonin (5-HT) in the striatum produced by MDMA without altering the acute hyperthermic response to MDMA. These results are supportive of the view that MDMA produces a dopamine-dependent increase in the formation of hydroxyl radicals in the striatum that may contribute to the mechanism whereby MDMA produces a long-term depletion of brain 5-HT content.     

A Kinetic and ESR Investigation of Iron(II) Oxalate Oxidation by Hydrogen Peroxide and Dioxygen as a Source of Hydroxyl Radicals

The reaction of Fe^II oxalate with hydrogen peroxide and dioxygen was studed for oxalate concentrations up to 20 mM and pH 2-5, under which conditions mono- and bis-oxalate comlexes (Fe^II(ox) and Fe^II(ox)₂^2-) and uncomplexed Fe²⁺ must be considered. The reaction of Fe^II oxalate with hydrogen peroxide (Fe²⁺ + H₂O₂ → Fe³⁺ + *OH + OH^-) was monitored in continuous flow by ESR with t-butanol as a radical trap. The reaction is much faster than for uncomplexed Fe²⁺ and a rate constant, k = 1 × 10⁴ M-¹ s^-1 is deduced for Fe^II(ox). The reaction of Fe^II oxalate with dioxygen is strongly pH dependent in a manner which indicates that the reactive species is Fe^II(ox)₂^2-, for which an apparent second order rate constant, k = 3.6 M^-1 s^-1, is deduced. Taken together, these results provide a mechanism for hydroxyl radical production in aqueous systems containing Fe^II complexed by oxalate. Further ESR studies with DMPO as spin trap reveal that reaction of Fe^II oxalate with hydrogen peroxide can also lead to formation of the carboxylate radical anion (CO₂^*-), an assignment confirmed by photolysis of Fe^III oxalate in the presence of DMPO.     

Inhibitors of Mitochondrial Respiration, Iron (II), and Hydroxyl Radical Evoke Release and Extracellular Hydrolysis of Glutathione in Rat Striatum and Substantia Nigra

In this investigation, microdialysis has been used to study the effects of 1-methyl-4-phenylpyridinium (MPP+), an inhibitor of mitochondrial complex I and alpha-ketoglutarate dehydrogenase and the active metabolite of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), on extracellular concentrations of glutathione (GSH) and cysteine (CySH) in the rat striatum and substantia nigra (SN). During perfusion of a neurotoxic concentration of MPP+ (2.5 mM) into the rat striatum or SN, extracellular concentrations of GSH and CySH remain at basal levels (both approximately 2 microM). However, when the perfusion is discontinued, a massive but transient release of GSH occurs, peaking at 5,000% of basal levels in the striatum and 2,000% of basal levels in the SN. The release of GSH is followed by a slightly delayed and smaller elevation of extracellular concentrations of CySH that can be blocked by the gamma-glutamyl transpeptidase (gamma-GT) inhibitor acivicin. Low-molecular-weight iron and extracellular hydroxyl radical (OH*) have been implicated as participants in the mechanism underlying the dopaminergic neurotoxicity of MPTP/MPP+. During perfusion of Fe2+ (OH*) into the rat striatum and SN, extracellular levels of GSH also remain at basal levels. When perfusions of Fe2+ are discontinued, a massive transient release of GSH occurs followed by a delayed, small, but progressive elevation of extracellular CySH level that again can be blocked by acivicin. Previous investigators have noted that extracellular concentrations of the excitatory/excitotoxic amino acid glutamate increase dramatically when perfusions of neurotoxic concentrations of MPP+ are discontinued. This observation and the fact that MPTP/MPP+ causes the loss of nigrostriatal GSH without corresponding increases of glutathione disulfide (GSSG) and the results of the present investigation suggest that the release and gamma-GT/dipeptidase-mediated hydrolysis of GSH to glutamate, glycine, and CySH may be important factors involved with the degeneration of dopamine neurons. It is interesting that a very early event in the pathogenesis of Parkinson's disease is a massive loss of GSH in the SN pars compacta that is not accompanied by corresponding increases of GSSG levels. Based on the results of this and prior investigations, a new hypothesis is proposed that might contribute to an understanding of the mechanisms that underlie the degeneration of dopamine neurons evoked by MPTP/MPP+, other agents that impair neuronal energy metabolism, and Parkinson's disease.     

EGb761 Pretreatment Reduces Monoamine Oxidase Activity in Mouse Corpus Striatum During 1-Methyl-4-Phenylpyridinium Neurotoxicity

EGb761 produces reversible inhibition of both monoamine oxidase (MAO) isoforms in the central nervous system. 1-Methyl-4-phenylpyridinium (MPP+) neurotoxicity is prevented by treatment with the MAO inhibitor pargyline. We investigated EGb761's effect on striatal MAO activity during MPP+ neurotoxicity. C-57 black mice were pretreated with EGb761 (10 mg/kg) daily for 17 days followed by administration of MPP+ (0.72 mg/kg). MPP+ enhanced striatal MAO (30%) activity at 6 h, and EGb761 prevented this effect. MAO-B activity in striatum was enhanced (70%) 6 h after MPP+ administration and was reduced to almost normal levels in EGb761 + MPP+ group compared to MPP+ group. Pretreatment with EGb761 partially prevented (32%) the striatal dopamine-depleting effect of MPP+ and prevented the reduction in striatal tyrosine hydroxylase activity (100%). Results suggest that EGb761 supplements may be effective in reducing MAO activity as well as enhancement in dopamine metabolism, thereby preventing MPP+-neurotoxicity.     

Superoxide Dismutase Enhanced the Formation of Hydroxyl Radicals in a Reaction Mixture Containing Xanthone under UVA Irradiation

To clarify the effect of superoxide dismutase (SOD) on the formation of hydroxyl radical in a standard reaction mixture containing 15 μM of xanthone, 0.1 M of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and 45 mM of phosphate buffer (pH 7.4) under UVA irradiation, electron paramagnetic resonance (EPR) measurements were performed. SOD enhanced the formation of hydroxyl radicals. The formation of hydroxyl radicals was inhibited on the addition of catalase. The rate of hydroxyl radical formation also slowed down under a reduced oxygen concentration, whereas it was stimulated by disodium ethylenediaminetetraacetate (EDTA) and diethyleneaminepentaacetic acid (DETAPAC). Above findings suggest that O₂, H₂O₂, and iron ions participate in the reaction. SOD possibly enhances the formation of the hydroxyl radical in reaction mixtures of photosensitizers that can produce O₂ ^?·.     

Superoxide-Dependent Formation of Hydroxyl Radicals from Ferric-Complexes and Hydrogen Peroxide: An Evaluation of Fourteen Iron Chelators

When a variety of ferric chelates are reacted with hydrogen peroxide in phosphate buffer deoxyribose is damaged and this damage is protected against by formate, thiourea and mannitol. Damage done by ferric complexes of citrate, EDTA, NTA, EGTA and HEDA is substantially inhibited by superoxide dismutase (SOD) whereas complexes of PLA. ADP and CDTA are moderately inhibited by SOD. The effects of SOD argue against hydrogen peroxide acting as a reductant in Fenton chemistry driven by ferric complexes and hydrogen peroxide. EDTA has proved to be a useful model for Fenton chemistry that is inhibited by SOD although, it is not unique in this respect.     

Interactions of the neurotoxin MPTP and its demethylated derivative (PTP) with monoamine oxidase-B

The kinetics of the interactions of MPTP and its N-des-methyl-derivative (PTP) have been studied. Both were mechanism-based inhibitors as well as substrates for the enzyme. Analysis of the reaction progress-curves for the formation of the corresponding dihydropyridine derivatives allowed the kinetic parameters for the process and the partition ratio, which corresponds to the number of mol. of product formed per mol. of enzyme inactivated, to be determined for both compounds. The conversion of MPTP to its corresponding pyridinium-ion derivative through the action of MAO-B is known to be essential for its neurotoxicity. PTP has been reported not to be neurotoxic, although it appears to be a relatively good substrate for MAO-B as well as acting a mechanism-based inhibitor. Studies of the changes in absorbance spectra during the MAO-B catalysed oxidation were consistent with the formation of the corresponding pyridinium-ion derivative (MPP+), which is known to be the effective neurotoxin, as the end-product when MPTP was oxidized. In contrast the oxidation of PTP appeared to stop at the dihydropyridine stage with no significant further oxidation to the corresponding pyridine-derivative.     

Oxidation of Dimethylsulphoxide to Formaldehyde by Oxyhaemoglobin and Oxyleghaemoglobin in the Presence of Hydrogen Peroxide is Not Mediated by “Free” Hydroxyl Radicals

In the presence of excess hydrogen peroxide. human oxyhaemoglobin and oxyleghaemoglobin from soybean root nodules cause oxidation of dimethylsulphoxide to formaldehyde. This reaction is inhibited by thiourea but not by phenylalanine. HEPES. mannitol or arginine. It is concluded that dimethylsulphoxide oxidation is not mediated by “free” hydroxyl radicals. consistent with previous conclusions that intact haemoglobin, leghaemoglobin or myoglobin molecules do not react with H₂O₂ to form hydroxyl radicals detectable outside the protein.     

1-甲基-4-苯基-吡啶盐致多巴胺能细胞系MES23.5的死亡机制

通过测定环境毒素1-甲基-4-苯基-吡啶盐(MPP )作用于多巴胺能细胞系MES23．5后细胞存活率的变化及细胞线粒体膜电位(△ψM)、活性氧(ROS)、羟自由基、超氧化物岐化酶(SOD)的变化,发现MPP^ 作用于多巴胺能细胞系MES23．5,可导致细胞存活率显著性减少,浓度达到200mol／L以上后,细胞存活率的下降呈时间与MPP^ 浓度依赖;以200μmol/L MPP^ 作用细胞6∽48h后,△ψM逐渐下降、ROS、羟自由基逐渐增加,48h后SOD开始显著性减少。结果表明早期线粒体能量代谢障碍和膜电位变化导致ROS(尤其是羟自由基)含量增加是MPP^ 导致多巴胺能细胞氧化应激的原因,而细胞内自由基的清除机制受损,则最终导致细胞变性死亡。     

Role of Oxygen Free Radicals in Respiratory Distress Induced by Arachidonic Acid In the Rat

The purpose of our present study is the possible implication of oxygen free radicals in the respiratory distress induced in rats by intravenous administration of arachidonic acid (20mg/kg). The respiratory frequency was measured and plasma TXB2 concentration was assayed by RIA from blood withdrawn I min after arachidonic acid administration. The substances studied were: SOD, catalase, manifold, DMSO, BHT, imidazole. All the drugs, except imidazole, significantly protect the rats from the respiratory distress induced by arachidonic acid. SOD, catalase, BHT and imidatole inhibit whereas mannitol and DMSO increase the plasma levels of TXB2. We suggest that oxygen free radicals generated in the respiratory burst induced by arachidonic acid are mainly responsible for the consequent respiratory distress.     

On the Specificity of Allopurinol and Oxypurinol as Inhibitors of Xanthine Oxidase. A Pulse Radiolysis Determination of Rate Constants for Reaction of Allopurinol and Oxypurinol with Hydroxyl Radicals

Allopurinol has been employed as a “specific” inhihitor of xanthine oxidase in studies of hypoxic/ reoxygenation injury. Pulse radiolysis was used to establish rate constants for the reactions of allopurinol and its major metabolite oxypurinol with hydroxyl radicals: values were (1.45 ± 0.241 × 10⁹ M^-1 s^-1 for allopurinol and (4.95 ± 0.84) × 10⁹ M^-1 s^-1 for oxypurinol. These rate constants show that, in view of the amounts of allopurinol that have been used in animal studies. hydroxyl radical scavenging by this molecule could contribute to its biological actions. especially if animals are pre-treated with allopurinol. so allowing oxypurinol to form. The ability of allopurinol to protect tissues not containing xanthine oxidase against reoxygenation injury may be related to radical scavenging by allopurinol and oxypurinol.     

On the Specificity of Allopurinol and Oxypurinol as Inhibitors of Xanthine Oxidase. A Pulse Radiolysis Determination of Rate Constants for Reaction of Allopurinol and Oxypurinol with Hydroxyl Radicals

Allopurinol has been employed as a “specific” inhihitor of xanthine oxidase in studies of hypoxic/ reoxygenation injury. Pulse radiolysis was used to establish rate constants for the reactions of allopurinol and its major metabolite oxypurinol with hydroxyl radicals: values were (1.45 ± 0.241 × 10⁹ M^-1 s^-1 for allopurinol and (4.95 ± 0.84) × 10⁹ M^-1 s^-1 for oxypurinol. These rate constants show that, in view of the amounts of allopurinol that have been used in animal studies. hydroxyl radical scavenging by this molecule could contribute to its biological actions. especially if animals are pre-treated with allopurinol. so allowing oxypurinol to form. The ability of allopurinol to protect tissues not containing xanthine oxidase against reoxygenation injury may be related to radical scavenging by allopurinol and oxypurinol.     

EPC-K1, a Hydroxyl Radical Scavenger,Prevents 6-Hydroxydopamine-Induced Dopamine Depletion in the Mouse Striatum by Up-Regulation of Catalase Activity

We examined the effect of pretreatment with EPC-K1, a potent hydroxyl radical scavenger, on 6-hydroxydopamine (6-OHDA)-induced reduction of dopamine (DA) and its metabolites in the mouse striatum. EPC-K1 was mixed with diet (0.2%, wt/wt) for 1 or 2 weeks, and then 6-OHDA (60 g in 2l of saline solution) was injected intracereberoventricularly. Mice continued to be fed EPC-K1-containing diet for another one week before they were sacrificed. The concentrations of DA and its metabolites in the striatum were measured by high performance liquid chromatography. 6-OHDA reduced the level of DA and its metabolites in the striatum. Pretreatment with EPC-K1 for 2 weeks, but not for 1 week, abrogated the neurotoxic effect of 6-OHDA on striatal concentrations of DA and its metabolites. Measurement of striatal concentrations of thiobarbituric acid reactive substances, glutathione, and malonaldehyde plus 4-hydroxynonenal, and the activities of superoxide dismutase and catalase in EPC-K1 treated mice showed an increase in catalase activity after 2 weeks of such treatment. No other changes in anti-oxidants levels were noted. Our results suggest that EPC-K1 counteracts the neurotoxicity of 6-OHDA by increasing catalase activities.     

In Vivo Electrochemical Studies of Dopamine Overflow and Clearance in the Striatum of Normal and MPTP-Treated Rhesus Monkeys

Abstract: Rapid chronoamperometric recordings, using Nafion-coated carbon-fiber electrodes (30–90 µm o.d.), were used to investigate overflow and uptake of dopamine (DA) in the striatum of normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated rhesus monkeys. The monkeys were anesthetized with isoflurane and placed in a stereotaxic apparatus. Magnetic resonance imaging-guided sterile stereotaxic procedures were used for implantations of the electrochemical electrodes coupled with single-barrel micropipettes that were used to apply potassium or DA locally. Potassium evoked a robust overflow of DA-like electrochemical signals into the brain extracellular space in the unlesioned or normal putamen and caudate nucleus of the rhesus monkeys. In contrast, potassium did not produce any detectable changes (> 97% depletion) of DA in the MPTP-lesioned striatum. In addition, the diffusion/clearance of locally applied DA was markedly altered in the lesioned caudate nucleus and putamen compared with unlesioned striatum. Cell counts of the number of residual tyrosine hydroxylase-positive neurons in MPTP-treated monkeys, in conjunction with whole-tissue levels of DA and its metabolites, showed that the MPTP lesions produced extensive damage of the nigrostriatal DA system. These data indicate that residual dopaminergic fibers remaining after MPTP lesions are dysfunctional and have a greatly diminished capacity for high-affinity DA uptake.     

Neuroprotective effect of Korea Red Ginseng extract on 1-methyl-4-phenylpyridinium-induced apoptosis in PC12 Cells

Background: Korean Red Ginseng (KRG) is a valuable herb in Asian countries that is used as a crude substance to inhibit inflammation and to enhance vitality, longevity and immunity. The protective effects of KRG against the toxicity of 1-methyl-4-phenylpyridinium (MPP+) were investigated in vitro in the present study.

Methods: PC12 cells were pretreated with the water extract of KRG for 24?h, then incubated with MPP+ for 24?h. The growth of the cells was assessed using a live cell viability assay, the ratio of apoptotic cells was measured using flow cytometry and morphology of the apoptotic cells was detected using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. The expressions of caspase-3 and -9 were measured by quantitative real-time PCR.

Results: Pretreatment of the KRG extract increased cell viability significantly when compared with that of only MPP+-treated cells (p?p?p?p?Conclusion: These results indicate that KRG treatment suppresses MPP+-induced apoptosis in PC12 cells by regulating caspase cascades, suggesting a possible role for KRG in the prevention and treatment of Parkinson’s disease.