Human neutrophils employ chlorine gas as an oxidant during phagocytosis.

Reactive oxidants generated by phagocytes are of central importance in host defenses, tumor surveillance, and inflammation. One important pathway involves the generation of potent halogenating agents by the myeloperoxidase-hydrogen peroxide-chloride system. The chlorinating intermediate in these reactions is generally believed to be HOCl or its conjugate base, ClO-. However, HOCl is also in equilibrium with Cl2, raising the possibility that Cl2 executes oxidation/ halogenation reactions that have previously been attributed to HOCl/ClO-. In this study gas chromatography-mass spectrometric analysis of head space gas revealed that the complete myeloperoxidase-hydrogen peroxide-chloride system generated Cl2. In vitro studies demonstrated that chlorination of the aromatic ring of free L-tyrosine was mediated by Cl2 and not by HOCl/ClO-. Thus, 3-chlorotyrosine serves as a specific marker for Cl2-dependent oxidation of free L-tyrosine. Phagocytosis of L-tyrosine encapsulated in immunoglobulin- and complement-coated sheep red blood cells resulted in the generation of 3-chlorotyrosine. Moreover, activation of human neutrophils adherent to a L-tyrosine coated glass surface also stimulated 3-chlorotyrosine formation. Thus, in two independent models of phagocytosis human neutrophils convert L-tyrosine to 3-chlorotyrosine, indicating that a Cl2-like oxidant is generated in the phagolysosome. In both models, synthesis of 3-chlorotyrosine was inhibited by heme poisons and the peroxide scavenger catalase, implicating the myeloperoxidase-hydrogen peroxide system in the reaction. Collectively, these results demonstrate that myeloperoxidase generates Cl2 and that human neutrophils use an oxidant with characteristics identical to those of Cl2 during phagocytosis. Moreover, our observations suggest that phagocytes exploit the chlorinating properties of Cl2 to execute oxidative and cytotoxic reactions at sites of inflammation and vascular disease.     

Oxidative decarboxylation of free and peptide-linked amino acids in phagocytizing guinea pig granulocytes.

The oxidative decarboxylation of amino acids by a system consisting of myeloperoxidase-hydrogen peroxide-chloride has been demonstrated previously by others and the process has been considered to be part of the microbicidal armamentarium of some phagocytic leukocytes. We were able to translate these earlier observations, made on model systems, to intact guinea pig granulocytes. We could demonstrate differences in the cellular handling of peptide-linked amino acids as particles, compared with free amino acids. Specific inhibitors were used to explore two routes of oxidative decarboxylation: (a) the myeloperoxidase-catalyzed direct decarboxylation-deamination reaction, and (b) oxidation of alpha-keto acids after transamination of amino acids. These inhibitors were cyanide, azide, and tapazole for the former pathway, and amino-oxyacetate for the latter. Amino-oxyacetate profoundly inhibited the decarboxylation of free 14C-amino acids (alanine and aspartate) in both resting and stimulated cells, but had only a minimal effect on 14CO2 production from ingested insoluble 14C-protein. On the other hand, the peroxidase inhibitors cyanide, azide, and tapazole dramatically inhibited the production of 14CO2 from ingested particulate 14C-protein, but had only small effects on the decarboxylation of free amino acid. Soluble, uniformly labeled 14C-protein was not significantly converted to 14CO2 even in the presence of phagocytizable polystyrene beads. These observation suggest that the amino acids taken up by phagocytosis (e.g., as denatured protein particles) are oxidatively decarboxylated and deaminated in the phagosomes by the myeloperoxidase-hydrogen peroxide-chloride system; soluble free amino acids that enter the cytoplasm by diffusion or transport are oxidatively decarboxylated after transamination by the normal cellular amino acid oxidative pathway.     

The cytotoxicity of clozapine metabolites: implications for predicting clozapine-induced agranulocytosis.

BACKGROUND: Therapy of schizophrenia with clozapine is associated with the unpredictable development of severe neutropenia and agranulocytosis in 1% to 2% of patients. The mechanism of this effect is unknown but may involve reactive products of clozapine generated by either hepatic metabolism or oxidation by the peroxidase-peroxide system of activated neutrophils. METHODS: Involvement of reactive metabolites was tested with in vitro cytotoxicity assays with use of peripheral blood mononuclear cells isolated from 3 groups of subjects: normal control subjects, patients with schizophrenia who tolerated clozapine therapy (control patients), and patients with schizophrenia in whom agranulocytosis developed while taking clozapine (patients with agranulocytosis). Cell viability was determined after incubations with clozapine and rat liver microsomes or clozapine and horseradish peroxidase-peroxide (HRP-H2O2). RESULTS: In microsomal incubations, clozapine significantly increased the cell death in all groups: control subjects (8.8%+/-1.6%), control patients (7.4%+/-0.4%), and patients with agranulocytosis (9.1%+/-1.5%). However, differences between mean values were not statistically significant. In similar incubations with HRP-H2O2, clozapine significantly increased toxicity (P < .05) in cells from patients with agranulocytosis (22%+/-4.6%) compared with those from normal control subjects (7.7%+/-4.1%) or control patients (6.5%+/-4.4%). CONCLUSIONS: These results suggest that both generating systems metabolized clozapine to toxic products. Some products may play a role in clozapine-induced agranulocytosis. Of diagnostic relevance is the observation the HRP-H2O2 produces significantly greater toxicity in cells from patients with agranulocytosis than in cells from control patients. Although the exact mechanism(s) of drug activation in vivo remains unclear, the bioactivation of clozapine by HRP-H2O2 may be a useful in vitro tool for predicting which patients are at risk for agranulocytosis before initiation of therapy.     

Sudden late onset of clozapine-induced agranulocytosis

OBJECTIVE: To report a patient who suddenly developed agranulocytosis after long-term clozapine therapy. CASE SUMMARY: A 41-year-old white man suddenly developed agranulocytosis after 89 months of nearly continuous clozapine therapy. During this time, which included the addition of risperidone to the treatment regimen, his white blood cell (WBC) and granulocyte counts remained stable. One week after having stable hematologic counts, the patient suddenly developed agranulocytosis. WBC and granulocyte counts returned to baseline shortly after discontinuation of all medications and administration of sargramostim. DISCUSSION: The main factor limiting the use of clozapine as a first-line agent in mentally ill patients is the risk of agranulocytosis. Although the greatest risk of developing this adverse reaction is during the initial 6-month exposure, clozapine-induced agranulocytosis continues to pose a risk after years of exposure. Current product labeling requires weekly WBC and granulocyte monitoring for the first 6 months of treatment with clozapine, which may be decreased to biweekly monitoring after 6 months. Based on the sudden and late onset of agranulocytosis in our patient, clinicians may consider opting for weekly monitoring of hematologic function for patients on long-term clozapine therapy. The likelihood that clozapine was the cause of the agranulocytosis was rated possible according to the Naranjo probability scale. CONCLUSIONS: Clinicians must remain vigilant to trends in WBC and granulocyte counts and may wish to consider weekly hematologic monitoring regardless of duration of clozapine therapy. Patient and treatment system compliance with the registries' protocol regarding WBC monitoring is instrumental in reducing morbidity and mortality rates associated with clozapine use.     

丁二酸洛沙平与氯氮平治疗精神分裂症对照研究

目的 评价国产丁二酸洛沙平对精神分裂症的临床疗效与安全性。方法 对122例住院精神分裂症患者随机分为两组,分别选用丁二酸洛沙平(治疗组)和氯氮平(对照组)进行8w治疗,采用阳性与阴性症状评定量表(PANSS)评定疗效,用副反应评定量表(TESS)评定副作用。结果 两组疗效无显著性差异(P>0.05)。洛沙平组副反应有锥体外系副反应,加用安坦后消失。氯氮平组副反应为白细胞减少及脑电图异常。结论 丁二酸洛沙平是一种有效、安全度大的抗精神病药物。     

氯氮平与其他抗精神病药物致粒细胞缺乏对照研究的Meta分析

为进一步认识氯氮平与其他抗精神病药物致粒细胞缺乏的差异,按标准对收集的、关于氯氮平与其他抗精神病药致粒细胞缺乏的对照研究论文结果作Meta分析。结果:(1)氯氮平与其他抗精神病药物导致粒细胞缺乏有非常显著性差异(X~2=11.20,df=1,P<0.01);(2)氯氮平引起粒细胞缺乏的危验性是其他抗精神病药物的8.8倍(OR=8.8);(3)病因学分数:EF_(暴露)因素=0.88,即氯氮平治疗组中出现粒缺者,88％是由氯氮平引起的,EF_(总体)=0.88,即在全部被治疗的精神分裂症出现粒缺者,88％由氯氮平引起。提示,氯氮平引起粒细胞缺乏效应显著高于其他抗精神病药物。     

Risk of clozapine-associated agranulocytosis and mandatory white blood cell monitoring

OBJECTIVE: To provide information for physicians and patients on which to base a decision as to whether to stop mandatory blood testing. DATA SOURCES: Articles on drug-induced blood dyscrasias were identified by searches of MEDLINE (1966-September 2005) and review of their bibliographies. Novartis was asked to provide additional data on clozapine, leukopenia, agranulocytosis, and suicidality. STUDY SELECTION AND DATA EXTRACTION: Data on the chance of clozapine-induced leukopenia and agranulocytosis were combined with data about possible fatality and compared with the risks associated with other medications and with life in general. DATA SYNTHESIS: The chance of clozapine-induced leukopenia or agranulocytosis decreases exponentially over time. In the US, the chance in the second 6 months of treatment is 0.70/1000 patient-years and, after the first year, 0.39/1000 patient-years. The case fatality rate of clozapine-induced agranulocytosis is estimated as 4.2-16%, depending on whether a granulocyte colony-stimulating factor is used. Nevertheless, treatment with clozapine reduces overall mortality, probably because it reduces suicidality. CONCLUSIONS: After at least 6 months' treatment with clozapine, the mortality involved in stopping white blood cell monitoring is about the same as the mortality associated with other medications, such as mianserin or phenylbutazone, and with life in general (traffic or occupational accident). If the patient has been well informed and wishes to stop the monitoring, it is a medically justifiable option to do so and is preferable to stopping treatment with clozapine since this drug reduces overall mortality.     

Clozapine overdose,a case report

Clozapine, an atypical antipsychotic agent with few of the extrapyramidal side effects of other antipsychotic agents, was approved for use in the United States in 1989. Because of drug-induced agranulocytosis, it is dispensed only under rigid guidelines. Overdose has been rare. Presented here is a case of clozapine overdose treated successfully with supportive care in which agranulocytosis did not develop.     

Binding of typical and atypical antipsychotic agents to transiently expressed 5-HT1C receptors.

We determined the affinities of clozapine and 21 other typical and atypical antipsychotic agents for the cloned 5-hydroxytryptamine-1C (5-HT1C) receptor. For these studies, 5-HT1C receptors were transiently expressed in COS-7 cells using the vector pSVK3-5HT1C. We discovered that clozapine and several other putative typical and atypical antipsychotic agents (loxapine greater than tiosperone greater than SCH23390 greater than fluperlapine greater than rilapine greater than chlorpromazine) had relatively high affinities (7-30 nM) for the cloned 5-HT1C receptor. Other antipsychotic agents (risperidone greater than tenilapine greater than mesoridazine greater than thioridazine greater than cis-fluphenthixol) had intermediate affinities (30-100 nM), whereas many other antipsychotics (fluphenazine greater than spiperone greater than amperozide greater than melperone greater than thiothixene greater than haloperidol, metoclopramide, pimozide, domperidone, sulpiride) had low affinities (greater than 500 nM) for the cloned 5-HT1C receptor. The results indicate that although several putative atypical antipsychotic agents have high affinities for the cloned rat 5-HT1C receptor, the spectrum of drug binding does not correlate with the atypical nature of these compounds.     

Metabolism of trimethoprim to a reactive iminoquinone methide by activated human neutrophils and hepatic microsomes.

The antibacterial agent, trimethoprim, is normally used synergistically with sulfonamides. Its use is associated with idiosyncratic reactions including liver toxicity and agranulocytosis. In this study, we demonstrated that trimethoprim was oxidized by activated human neutrophils, as well as a combination of myeloperoxidase/hydrogen peroxide/chloride or hypochlorous acid, to a reactive pyrimidine iminoquinone methide intermediate with a protonated molecular ion of m/z 289 as detected by mass spectrometry. In the presence of N-acetyl-L-cysteine (NAC), the pyrimidine iminoquinone methide could be trapped as three NAC adducts. The three NAC adducts were separable on HPLC, but showed the same protonated molecular ion of m/z 452. The proton NMR spectrum of the major adduct showed that the NAC group was at the 6 position of the pyrimidine ring. The mass spectra of the two minor NAC adducts indicated that they were the two diastereomers in which NAC was attached to the exo-cyclic prechiral carbon of the pyrimidine iminoquinone methide. Incubation of trimethoprim with isolated hepatic microsomes, both human and rat, in presence of NAC gave the same set of trimethoprim-NAC adducts. We propose that the formation of this pyrimidine iminoquinone methide by both hepatic microsomes and neutrophils may be responsible for trimethoprim-induced idiosyncratic hepatotoxicity and agranulocytosis.     

The antipsychotic clozapine is metabolized by the polymorphic human microsomal and recombinant cytochrome P450 2D6.

A large interindividual variability for clozapine bioavailability and plasma steady-state concentrations and clearance exists. The enzymatic system which is involved in clozapine metabolism has not been fully characterized, yet structurally related tricyclic drugs have been found to be metabolized by cytochrome P450 2D6 (CYP2D6), which is polymorphically expressed in humans. The involvement of CYP2D6 in clozapine and fluperlapine metabolism was studied with human liver microsomes and in recombinant RT2D6 7-8 (RT2D6) cells, which specifically express human CYP2D6. Clozapine and its structural analog fluperlapine both bind to the active site of CYP2D6, as demonstrated by the competitive inhibition of dextromethorphan metabolism at inhibitor concentrations up to 40 microM. The inhibition constants (Ki) for both clozapine and fluperlapine were about 4 microM in microsomes from human liver. Clozapine exhibited a higher inhibition constant of 18.7 microM in microsomes from RT2D6 cells, but the difference was not statistically significant (P less than .05). These concentrations are close to the plasma concentrations of 0.3 to 3 microM achieved during clozapine therapy. Both clozapine and fluperlapine are also metabolized by CYP2D6. RT2D6 cells produced a number of metabolites from clozapine, whereas only a single metabolite was obtained from fluperlapine. The clozapine metabolites were not identified; however, they were different from N-oxide and N-demethyl clozapine. The fluperlapine metabolite was found to be the 7-hydroxy fluperlapine, which is also a major metabolite in vivo. In conclusion, both drug-drug interactions on the binding site of CYP2D6 and polymorphic metabolism of clozapine by CYP2D6 could contribute to the observed variability in clozapine kinetics in humans.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxic activity in serum of patients with clozapine-induced agranulocytosis.

Studies were conducted on serum samples collected from 15 patients during the course of clozapine-induced agranulocytosis. During acute phases of agranulocytosis, serum was cytotoxic to peripheral polymorphonuclear neutrophils (PMNs), as indicated by complement-dependent suppression of postphagocytosis respiratory burst and by increased retention of trypan blue dye by test PMNs. Cytotoxicity was removed by adsorption with allogeneic PMNs, was attenuated by antibody to immunoglobulin M but not by antibody to immunoglobulin G antigen-binding fragment, was not dialyzable, and was partially removed by 2-mercaptoethanol and dialysis. Three patients in a longitudinal study all had perturbed postphagocytosis respiratory burst 20 days before agranulocytosis developed. In all patients cytotoxicity disappeared less than 40 days after treatment when the offending drug was discontinued. Trypan blue dye reactivity was similar when tested. At 20% of culture medium, all serum samples partially suppressed development of colony-forming units of granulocytes and macrophages (CFU-GM) in marrow cultures. At 40% of culture medium, agranulocytosis serum suppressed CFU-GM completely but did not inhibit development of colony-forming units of erythroblasts (CFU-E) or burst-forming units of erythroblasts (BFU-E). Addition of clozapine alone or to agranulocytosis serum neither enhanced nor suppressed toxicity to peripheral PMNs. Neither clozapine nor its toxic metabolic end-products directly produced equivalent cytotoxicity to cellular function or proliferation at 10(-5)mol/L. Serum from patients given clozapine who did not have agranulocytosis and samples from allogeneic normal subjects were not cytotoxic to test PMNs. Cytotoxicity was specific to granulocyte cell lines because development of CFU-GM was inhibited by agranulocytosis serum, whereas CFU-E and BFU-E were not similarly affected. Further studies are in progress to distinguish between immunogenic and non-immunogenic mechanisms.     

Spongy titanosilicate promotes the catalytic performance and reusability of WO3 in oxidative cleavage of methyl oleate

A tungsten containing catalyst catalyzed oxidative cleavage of methyl oleate (MO) by employing H₂O₂ as an oxidant and is known as an efficient approach for preparing high value-added chemicals, however, the tungsten leaching problem remains unresolved. In this work, a binary catalyst consisting of tungsten oxide (WO₃) and spongy titanosilicate (STS) zeolite is proposed for MO oxidative cleavage. The function of STS in this catalyst is investigated. On the one hand, STS converts MO to 9,10-epoxystearate (MES), which further forms nonyl aldehyde (NA) and methyl azelaaldehydate (MAA) with the catalysis of WO₃. In this way, MO oxidation and hydrolysis that generates unwanted diol product 9,10-dihydroxystearate (MDS) decreases obviously. On the other hand, STS decomposes peroxide and promotes the conversion of soluble peroxotungstate to insoluble polytungstate. Meanwhile, these tungsten species are allowed to precipitate on its surface instead of remaining in the liquid phase owing to its relative large specific area. Therefore, tungsten leaching can be reduced from 37.0% to 1.2%. Due to the cooperation of WO₃ and STS, 94.4% MO conversion and oxidative cleavage product selectivity of 63.1% are achieved, and the WO₃–STS binary catalyst maintains excellent catalytic performance for 8 recycling reactions.

Proposed “dissolve and precipitate” reaction mechanism of WO₃–STS catalyzed MO oxidative cleavage reaction.     

Primaquine-induced hemolytic anemia: formation of free radicals in rat erythrocytes exposed to 6-methoxy-8-hydroxylaminoquinoline

Primaquine is an important antimalarial drug that is often dose-limited in therapy by the onset of hemolytic anemia. We have shown recently that an N-hydroxy metabolite of primaquine, 6-methoxy-8-hydroxylaminoquinoline (MAQ-NOH), is a direct-acting hemolytic agent in rat red cells and that the hemolytic activity of this metabolite is associated with GSH oxidation and oxidative damage to both membrane lipids and skeletal proteins. To determine whether the formation of free radicals may be involved in this process, rat red cells (40% suspensions) were incubated with hemolytic concentrations of MAQ-NOH (150-750 microM) and examined by EPR spectroscopy using 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO) as a spin trap. Addition of MAQ-NOH to red cell suspensions containing 10 mM EMPO gave rise to an EPR spectrum with hyperfine constants consistent with those of an EMPO-hydroxyl radical adduct standard. Of interest, formation of EMPO-OH was constant for up to 20 min and dependent on the presence of erythrocytic GSH. Although no other radical adduct signals were detected in the cells by EPR, spectrophotometric analysis revealed the presence of ferrylhemoglobin, which indicates that hydrogen peroxide is generated under these experimental conditions. The data support the hypothesis that oxygen-derived and possibly other free radicals are involved in the mechanism underlying MAQ-NOH-induced hemolytic anemia.     

Clozapine: a novel antipsychotic.

Clozapine is the first truly new antipsychotic drug introduced in the last 40 years. Compared to traditional neuroleptic agents, clozapine appears to have a stronger effect on most schizophrenic symptoms. Thus, it seems to be more effective than other agents in severely ill, treatment-resistant patients. Clozapine rarely causes extrapyramidal symptoms such as pseudoparkinsonism or akathisia. To date, no confirmed cases of tardive dyskinesia have been attributed to the drug. Despite these advantages, the usefulness of clozapine is limited by its potentially life-threatening side effects, which include agranulocytosis and respiratory depression.     

Identification of 1,5-anhydroglucitol in thin-layer chromatograms

The chromatographical characteristics of 1,5-anhydroglucitol in thin-layer chromatograms can be studied using the customary carbohydrate solvents. Both hydrogen peroxide and sodium metaperiodate oxidation reactions were tested in order to find a specific colour reaction for the detection of the compound. 1,5-anhydroglucitol was readily converted by periodate into an intermediate product which produced an intense orange-red colour with diphenylamine aniline reagent. According to data obtained from periodate oxidation, IR spectroscopy and mass fragmentography, the intermediate product was a dialdehyde compound with a C6 structure, possibly formed through cleavage at C2-C3. The formation of a compound of this kind without chain cleavage in the periodate oxidation of C6 carbohydrates is uncommon. Periodate oxidation followed by diphenylamine-aniline reaction affords a sensitive and specific method for the detection of 1,5-anhydroglucitol.     

Macrophage scavenger receptor CD36 is the major receptor for LDL modified by monocyte-generated reactive nitrogen species

The oxidative conversion of LDL into an atherogenic form is considered a pivotal event in the development of cardiovascular disease. Recent studies have identified reactive nitrogen species generated by monocytes by way of the myeloperoxidase-hydrogen peroxide-nitrite (MPO-H(2)O(2)-NO(2)(-)) system as a novel mechanism for converting LDL into a high-uptake form (NO(2)-LDL) for macrophages. We now identify the scavenger receptor CD36 as the major receptor responsible for high-affinity and saturable cellular recognition of NO(2)-LDL by murine and human macrophages. Using cells stably transfected with CD36, CD36-specific blocking mAbs, and CD36-null macrophages, we demonstrated CD36-dependent binding, cholesterol loading, and macrophage foam cell formation after exposure to NO(2)-LDL. Modification of LDL by the MPO-H(2)O(2)-NO(2)(-) system in the presence of up to 80% lipoprotein-deficient serum (LPDS) still resulted in the conversion of the lipoprotein into a high-uptake form for macrophages, whereas addition of less than 5% LPDS totally blocked Cu(2+)-catalyzed LDL oxidation and conversion into a ligand for CD36. Competition studies demonstrated that lipid oxidation products derived from 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine can serve as essential moieties on NO(2)-LDL recognized by CD36. Collectively, these results suggest that MPO-dependent conversion of LDL into a ligand for CD36 is a likely pathway for generating foam cells in vivo. MPO secreted from activated phagocytes may also tag phospholipid-containing targets for removal by CD36-positive cells.     

Studies of the Metabolic Activity of Leukocytes from Patients with a Genetic Abnormality of Phagocytic Function

Polymorphonuclear leukocytes from patients with chronic granulomatous disease respond to the phagocytosis of latex particles with normal increments in glucose consumption, lactate production, Krebs' cycle activity, and lipid turnover.The leukocytes of these patients fail to show normal increments in respiration, direct oxidation of glucose, and hydrogen peroxide formation during particle uptake.It appears that the stimulation of respiration with the formation of hydrogen peroxide and stimulation of the direct oxidative pathway of glucose metabolism are closely linked to degranulation and intracellular killing of bacteria by polymorphonuclear leukocytes.     

Lowering effects of onion intake on oxidative stress biomarkers in streptozotocin-induced diabetic rats

The protective effect of onion against oxidative stress in streptozotosin-induced diabetic rats was investigated in comparison with that of quercetin aglycone. We measured oxidative stress biomarkers involving the susceptibility of the plasma against copper ion-induced lipid peroxidation, which was estimated by the amounts of thiobarbituric acid-reactive substances (TBARS) and cholesteryl ester hydroperoxides, and urine TBARS and 8-hydroxydeoxyguanosine contents. After the 12-week feeding period, plasma glucose levels and these biomarkers increased in diabetic rats compared to normal rats. In diabetic rats fed a 6.0% onion diet (quercetin equivalent: 0.023%), quercetin metabolites accumulated in the plasma at concentrations of approximately 35 microM. Onion intake decreased plasma glucose levels and lowered the oxidative stress biomarkers. On the other hand, quercetin metabolites in the plasma of rats fed a diet with 0.023% quercetin aglycone were found at lower concentrations (14.2 microM) than the rats fed the onion diet. Furthermore, oxidative stress biomarkers were higher in the quercetin diet group compared to the onion diet group. These results strongly suggest that onion intake suppresses diabetes-induced oxidative stress more effectively than the intake of the same amount of quercetin aglycone alone.