RESUMO
PURPOSE: Changes in the circadian rhythm may contribute to the development of cancer and are correlated with the high risk of breast cancer (BC) in night workers. Melatonin is a hormone synthesized by the pineal gland at night in the absence of light. Levels of melatonin and the metabolite of oxidative metabolism AFMK (acetyl-N-formyl-5-methoxykynurenamine), are suggested as potential biomarkers of BC risk. The aims of this study were to evaluate levels of melatonin and AFMK in women recently diagnosed with BC, women under adjuvant chemotherapy, and night-shift nurses, and compare them with healthy women to evaluate the relation of these compounds with BC risk. METHODS: Blood samples were collected from 47 women with BC, 9 healthy women, 10 healthy night shift nurses, and 6 patients under adjuvant chemotherapy. Compound levels were measured by mass spectrometry. RESULTS AND CONCLUSIONS: Our results showed that women with BC had lower levels of melatonin compared to control group women, and even lower in night-shift nurses and in patients under adjuvant chemotherapy. There was no significant difference of AFMK levels between the groups. In addition to this, high levels of melatonin and AFMK were related to patients with metastasis, and high levels of AFMK were related to the presence of lymph node-positive, tumor > 20 mm and patients who sleep with light at night. Our results showed a reduction of melatonin levels in BC patients, suggesting a relation with the disease, and in addition, point to the importance of melatonin supplementation in women that work at night to reduce the BC risk.
Assuntos
Neoplasias da Mama/sangue , Cinuramina/análogos & derivados , Melatonina/sangue , Biomarcadores/sangue , Ritmo Circadiano/fisiologia , Feminino , Humanos , Cinuramina/sangue , Metástase Linfática , Pessoa de Meia-Idade , PrognósticoRESUMO
Melatonin is widely known for its antioxidant, immunomodulatory, and anti-inflammatory effects. Hypochlorous acid (HOCl) is one example of an endogenous oxidant that is promptly neutralized by melatonin. Melatonin also inhibits myeloperoxidase, the enzyme that catalyzes the oxidation of chloride to HOCl. Taurine is the most abundant free amino acid in leukocytes. In activated neutrophils, taurine is converted to taurine chloramine (Tau-NHCl) through a reaction with HOCl. In addition, the related compound taurine bromamine (Tau-NHBr) can be released by neutrophils and eosinophils. The aim of this study was to investigate the reactivity of Tau-NHCl and Tau-NHBr with melatonin. We found that melatonin can react with either Tau-NHCl or Tau-NHBr, leading to the production of 2-hydroxymelatonin and N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK). The reaction was pH-dependent, and it occurs more rapidly at a slightly acidic pH. Tau-NHBr was significantly more reactive than Tau-NHCl. Using Tau-NHBr as the oxidizing agent, 1 mm melatonin was oxidized in less than 1 min. The pH dependence of the reaction with Tau-NHCl and the increased reactivity of Tau-NHBr can be explained by a mechanism based on the initial attack of chloronium (Cl(+)) or bromonium (Br(+)) ions on melatonin. We also found that the addition of iodide to the reaction medium increased the yield of AFMK. These findings could contribute to the establishment of new functions for melatonin in inflammatory and parasitic diseases, where the role of this indoleamine has been extensively investigated.
Assuntos
Melatonina/química , Taurina/análogos & derivados , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Ácido Hipocloroso/química , Iodetos/química , Iodetos/metabolismo , Cinética , Cinuramina/análogos & derivados , Cinuramina/química , Cinuramina/metabolismo , Melatonina/análogos & derivados , Melatonina/metabolismo , Oxirredução , Taurina/química , Taurina/metabolismoRESUMO
In vertebrates, many studies verified different effects of melatonin in the antioxidant defense system (ADS). In crustaceans, few studies have been conducted to verify this possibility. We verified the melatonin effects in the crab Neohelice granulata using low (0.002 and 0.02 pmol/crab) and high (2.0 and 20.0 pmol/crab) melatonin dosages in short-term (0.5h) and long-term (9.5h) experiments. We analyzed the antioxidant capacity against peroxyl radicals (ACAP), reactive oxygen species (ROS) concentration, levels of by products of lipid peroxidation (LPO), oxygen consumption (VO(2)), the activity of glutamate cysteine ligase (gamma-GCL) and catalase (CAT) and glutathione content (GSH). Finally, the effects of exogenous melatonin were verified in terms of melatonin and N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) content in the muscles of N. granulata. In short-term experiment and low dosages, melatonin increased the VO(2), gamma-GCL activity and GSH content (p<0.05) and decreased melatonin content (p<0.05) without effects in ROS, ACAP and LPO (p>0.05). Possibly, melatonin is acting in the ADS increasing its efficiency and/or acting in mitochondrial activity and/or through signaling muscles to increase its consumption. AFMK was only detected in the eyestalk and cerebroid ganglia. In high dosages melatonin effects decreased, possibly by the desensitization of their receptors. In long-term experiment, melatonin decreased ACAP (p<0.05), and CAT activity (p<0.05) in low dosages. In high dosages melatonin reduced VO(2) (p<0.05) and increased ACAP (p<0.05), possibly stimulating others components of the ADS. In conclusion, melatonin in the locomotor muscles of N. granulata affects the antioxidant/pro-oxidant balance in a time and dosage dependent manner.
Assuntos
Braquiúros/efeitos dos fármacos , Catalase/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Melatonina/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/farmacologia , Gânglios dos Invertebrados/efeitos dos fármacos , Gânglios dos Invertebrados/metabolismo , Cinuramina/análogos & derivados , Cinuramina/metabolismo , Melatonina/farmacologia , Músculos/efeitos dos fármacos , Músculos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologiaRESUMO
N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) is the product of oxidative pyrrole ring cleavage of melatonin. AFMK and its deformylated derivative N(1)-acetyl-5-methoxykynuramine (AMK) are compounds for which there are increasing demands because of their antioxidant, immunomodulatory and anti-inflammatory properties. Here, we sought to determine the best reaction conditions for preparation of AFMK using chlorpromazine (CPZ) as a co-catalyst in the peroxidase-mediated oxidation of melatonin. The parameters studied were pH, identity and concentration of buffers, hydrogen peroxide (H(2)O(2)) and CPZ concentrations and the presence or absence of dissolved molecular oxygen in the reaction medium. The rate and efficiency of AFMK production were compared with a noncatalyzed method which uses a high concentration of H(2)O(2). We found that by using CPZ and bubbling molecular oxygen during the course of the reaction, the yield of AFMK was significantly increased (about 60%) and the reaction time decreased (about 30 min), as compared with the noncatalyzed reaction (yield 32% and reaction time 4 hr). Based on these data, we suggest that this could be a new, easily performed and efficient route for AFMK preparation. Additionally, we provide evidence that a radical chain reaction could be responsible for the formation of AFMK.
Assuntos
Cinuramina/análogos & derivados , Melatonina/química , Clorpromazina/química , Cromatografia Líquida de Alta Pressão , Peroxidase do Rábano Silvestre/metabolismo , Concentração de Íons de Hidrogênio , Cinuramina/síntese química , Concentração Osmolar , Oxirredução , Oxigênio/químicaRESUMO
Accumulating evidence points to relationships between increased production of reactive oxygen or decreased antioxidant protection in schizophrenic patients. Chlorpromazine (CPZ), which remains a benchmark treatment for people with schizophrenia, has been described as a pro-oxidant compound. Because the antioxidant compound melatonin exerts protective effects against CPZ-induced liver disease in rats, in this investigation, our main objective was to study the effect of CPZ as a co-catalyst of peroxidase-mediated oxidation of melatonin. We found that melatonin was an excellent reductor agent of preformed CPZ cation radical (CPZ(*+)). The addition of CPZ during the horseradish peroxidase (HRP)-catalyzed oxidation of melatonin provoked a significant increase in the rate of oxidation and production of N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK). Similar results were obtained using myeloperoxidase. The effect of CPZ on melatonin oxidation was rather higher at alkaline pH. At pH 9.0, the efficiency of oxidation of melatonin was 15 times higher and the production of AFMK was 30 times higher as compared with the assays in the absence of CPZ. We suggest that CPZ is able to exacerbate the rate of oxidation of melatonin by an electron transfer mechanism where CPZ(*+), generated during the peroxidase-catalyzed oxidation, is able to efficiently oxidize melatonin.
Assuntos
Antipsicóticos/farmacologia , Clorpromazina/farmacologia , Cinuramina/análogos & derivados , Melatonina/metabolismo , Oxidantes/farmacologia , Peroxidase/fisiologia , Animais , Antioxidantes/metabolismo , Humanos , Cinuramina/metabolismo , Oxirredução , Peroxidase/química , Ratos , Espectrofotometria UltravioletaRESUMO
The lipid peroxidation of triglycerides enriched in polyunsaturated fatty acids was investigated by photoemission techniques and the TBARS assay. Butylated hydroxytoluene, 5-OH-tryptophan and N-acetylserotonin inhibited light emission and TBARS formation in a concentration dependent manner. However, it was enhanced in the presence of melatonin and 5-methoxytryptamine and was dependent on its concentration. The total relative luminic units were found to be lower in those systems incubated in the presence of butylated hydroxytoluene, N-acetylserotonin or 5-OH-tryptophan; this decreased proportionally to the concentration of the compound tested. The order of inhibition was 5-OH-tryptophan>N-acetylserotonin>butylated hydroxytoluene with the following IC50 values: 0.65, 6.5 and 9.0 mM respectively. The free-radical scavenging activity of the indole derivatives was also analyzed by the DPPH method, and the results indicate that 5-OH-tryptophan, and N-acetylserotonin exhibited a dose-dependent free-radical scavenging ability at all of the tested concentrations. Thus, at 10 microM concentration a decrease of 84.71% and 73.50% of initial DPPH was observed, compared to 51.00% of BHT. Melatonin and 5-methoxytriptamine decreased the initial concentration of DPPH only 1.85% and 5.0%, respectively. The possible formation of N(1)-acetyl-N(2) formyl-5-methoxykynuramine (AFMK) during lipid peroxidation of triglycerides enriched in PUFAs with cumene hydroperoxide in the presence of melatonin was also analyzed.
Assuntos
Hidroxitolueno Butilado/química , Ácidos Graxos Ômega-3/química , Peroxidação de Lipídeos/efeitos dos fármacos , Melatonina/farmacologia , Triglicerídeos/química , 5-Metoxitriptamina/química , 5-Metoxitriptamina/farmacologia , Derivados de Benzeno/química , Compostos de Bifenilo , Hidroxitolueno Butilado/farmacologia , Sistema Livre de Células , Ácidos Docosa-Hexaenoicos/química , Sequestradores de Radicais Livres/química , Cinuramina/análogos & derivados , Cinuramina/química , Lipídeos/química , Medições Luminescentes , Melatonina/química , Oxirredução , Picratos/química , Espécies Reativas de Oxigênio/química , Serotonina/análogos & derivados , Serotonina/química , Serotonina/farmacologia , Substâncias Reativas com Ácido Tiobarbitúrico/química , Triptofano/química , Triptofano/farmacologiaRESUMO
We previously reported that intraerythrocytic malaria parasites have their development synchronized by melatonin and other products of tryptophan catabolism (i.e. serotonin, N-acetylserotonin and tryptamine). Here, we show that N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), a product of melatonin degradation, synchronizes Plasmodium chabaudi and Plasmodium falciparum. The synchronization is abrogated with a melatonin receptor antagonist, luzindole. We established quantitatively that a differential AFMK production occurred within the intraerythrocytic stages of rodent malaria parasite Plasmodium chabaudi (ring, trophozoite and schizont), when the infected erythrocytes were previously incubated with melatonin. Measurement of AFMK formation in P. chabaudi after incubation with melatonin at a concentration of 500 nmol/L revealed the following values for AFMK production: ring 0.1 +/- 0.1 nmol/L, trophozoite 22.9 +/- 0.5 nmol/L, schizont 29 +/- 5 nmol/L. Confocal and spectrofluorophotometer experiments with isolated parasites and infected-RBC, loaded with calcium indicator Fluo-4 showed that AFMK elicits an increase in the cytosol calcium concentration in these parasites. Our data suggest that AFMK could have an important role in modulating the cell cycle of malaria parasites mainly in the late stages (trophozoite and schizont).
Assuntos
Ciclo Celular/efeitos dos fármacos , Eritrócitos/metabolismo , Cinuramina/análogos & derivados , Plasmodium chabaudi/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Animais , Eritrócitos/parasitologia , Cinuramina/metabolismo , Cinuramina/farmacologia , Melatonina/metabolismo , Camundongos , Camundongos Endogâmicos BALB CRESUMO
There is a growing body of evidence that melatonin and its oxidation product, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), have anti-inflammatory properties. From a nutritional point of view, the discovery of melatonin in plant tissues emphasizes the importance of its relationship with plant peroxidases. Here we found that the pH of the reaction mixture has a profound influence in the reaction rate and products distribution when melatonin is oxidized by the plant enzyme horseradish peroxidase. At pH 5.5, 1 mm of melatonin was almost completely oxidized within 2 min, whereas only about 3% was consumed at pH 7.4. However, the relative yield of AFMK was higher in physiological pH. Radical-mediated oxidation products, including 2-hydroxymelatonin, a dimer of 2-hydroxymelatonin and O-demethylated dimer of melatonin account for the fast consumption of melatonin at pH 5.5. The higher production of AFMK at pH 7.4 was explained by the involvement of compound III of peroxidases as evidenced by spectral studies. On the other hand, the fast oxidative degradation at pH 5.5 was explained by the classic peroxidase cycle.
Assuntos
Peroxidase do Rábano Silvestre/metabolismo , Cinuramina/análogos & derivados , Melatonina/metabolismo , Concentração de Íons de Hidrogênio , Cinuramina/síntese química , OxirreduçãoRESUMO
Activated phagocytes oxidize the hormone melatonin to N1-acethyl-N2-formyl-5-methoxykynuramine (AFMK) in a superoxide anion- and myeloperoxidase-dependent reaction. We examined the effect of melatonin, AFMK and its deformylated-product N-acetyl-5-methoxykynuramine (AMK) on the phagocytosis, the microbicidal activity and the production of hypochlorous acid by neutrophils. Neither neutrophil and bacteria viability nor phagocytosis were affected by melatonin, AFMK or AMK. However these compounds affected the killing of Staphylococcus aureus. After 60 min of incubation, the percentage of viable bacteria inside the neutrophil increased to 76% in the presence of 1 mM of melatonin, 34% in the presence of AFMK and 73% in the presence of AMK. The sole inhibition of HOCl formation, expected in the presence of myeloperoxidase substrates, was not sufficient to explain the inhibition of the killing activity. Melatonin caused an almost complete inhibition of HOCl formation at concentrations of up to 0.05 mM. Although less effective, AMK also inhibited the formation of HOCl. However, AFMK had no effect on the production of HOCl. These findings corroborate the present view that the killing activity of neutrophils is a complex phenomenon, which involves more than just the production of reactive oxygen species. Furthermore, the action of melatonin and its oxidation products include additional activities beyond their antioxidant property. The impairment of the neutrophils' microbicidal activity caused by melatonin and its oxidation products may have important clinical implications, especially in those cases in which melatonin is pharmacologically administered in patients with infections.
Assuntos
Cinuramina/análogos & derivados , Cinuramina/farmacologia , Melatonina/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Células Cultivadas , Humanos , Ácido Hipocloroso/metabolismo , Cinuramina/química , Cinuramina/metabolismo , Ativação de Neutrófilo/imunologia , Neutrófilos/microbiologia , Neutrófilos/fisiologia , Oxirredução , Fagocitose , Staphylococcus aureus/imunologiaRESUMO
Many physiologic changes related to light-dark cycles and antioxidant effects have been related to melatonin (N-acetyl-5-methoxytryptamine) and its metabolites, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK). In this review, we discuss some methodologies, in particular, those employing high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) assays to quantitatively determine melatonin, AFMK, and AMK. These approaches offer a highly specific and an accurate quantification of melatonin and its metabolites. These characteristics are essential to point out correctly the biological effects of these compounds in physiological and pathological conditions.
Assuntos
Melatonina/análise , Melatonina/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Cinuramina/análogos & derivados , Cinuramina/metabolismo , Espectrometria de Massas , Melatonina/fisiologia , Padrões de ReferênciaRESUMO
Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics.
Assuntos
Melatonina/química , Peroxidase/química , Superóxidos/química , Animais , Antioxidantes/química , Catalase/química , Catálise , Bovinos , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Dimerização , Relação Dose-Resposta a Droga , Radicais Livres , Heme/química , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Radical Hidroxila , Ácido Hipocloroso/química , Ácido Hipocloroso/metabolismo , Cinuramina/análogos & derivados , Cinuramina/química , Fígado/enzimologia , Espectrometria de Massas , Modelos Químicos , Neutrófilos/química , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Oxigênio/química , Ozônio/química , Ligação Proteica , Taurina/análogos & derivados , Taurina/química , Fatores de Tempo , Xantina Oxidase/químicaRESUMO
We evaluated the presence of the melatonin metabolite N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), in cerebrospinal fluid (CSF) of patients with viral meningitis (n = 20) and control samples (n = 8) and correlate AFMK levels with inflammatory markers such as cellularity, protein, tumor necrosis factor (TNF)-alpha, interleukin (IL)-8 and IL-1beta levels. A portion of the CSF was extracted with dichloromethane (1:5) and analyzed by high-performance liquid chromatography (HPLC) under standardized conditions for AFMK. AFMK was detected in 16 of 20 CSF samples of patients with viral meningitis; the concentration of AFMK was found to be above the quantification limit (50 nmol/L) in six of these samples. AFMK was not detected in any of the eight control samples. The samples were classified into groups according to AFMK levels: undetectable (<10 nmol/L, group I), detectable but below the quantification limit (< 50 nmol/L, group II), and quantified (>50 nmol/L, group III). Group II presented the highest levels of proteins and IL-8, whereas group III showed the lowest levels of the inflammatory parameters. This study supports our hypothesis that inflammation favors the formation of AFMK and that this compound has immunomodulatory activity in vivo.
Assuntos
Adjuvantes Imunológicos/metabolismo , Cinuramina/análogos & derivados , Melatonina/metabolismo , Meningite/líquido cefalorraquidiano , Adjuvantes Imunológicos/fisiologia , Biomarcadores , Cromatografia Líquida de Alta Pressão , Humanos , Cinuramina/líquido cefalorraquidianoRESUMO
Porphyrias are defined as either inborn or acquired diseases related to enzymatic deficiencies in the heme biosynthetic pathway. Lead poisoning, hereditary tyrosinemia, and acute intermittent porphyria (AIP) are characterized by the absence of photosensitivity and the accumulation of 5-aminolevulinic acid (ALA) together with its increased urinary excretion. The main clinical manifestations of AIP are intermittent attacks of abdominal pain, neuromuscular weaknesses and neuropsychiatry alterations, and also an association with primary liver cancer, in which may be involved the oxidative potential of ALA which is able to cause DNA damage. The use of antioxidants in the treatment of ALA-induced oxidative stress is not well established. In the current work, we show the antioxidant efficacy of several compounds including melatonin, quercetin, resveratrol and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), a melatonin oxidation product, in terms of their ability to limit DNA damage induced by ALA/Fe2+ in an in vitro system. Damage was measured by plasmid DNA strand breaks and detection of 8-oxo, 7-8-dihydro,2'-deoxyguanosine (8-oxodGuo) by high-performance liquid chromatography coupled with electrochemical detection. All compounds tested showed a dose-dependent protective action against free radical damage. These results could be the first step toward studies of the possible use of these antioxidants in oxidative stress promoted by ALA or other pro-oxidants.
Assuntos
Ácido Aminolevulínico/farmacologia , Antioxidantes/farmacologia , DNA/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Ácido Aminolevulínico/metabolismo , Dano ao DNA , Relação Dose-Resposta a Droga , Eletroforese em Gel de Ágar , Cinuramina/análogos & derivados , Melatonina/farmacologia , Porfiria Aguda Intermitente/metabolismo , Quercetina/farmacologia , Resveratrol , Estilbenos/farmacologiaRESUMO
A growing body of evidence suggests that the pineal hormone, melatonin, has immunomodulatory properties, although very little is known about its effect on leukocytes. Therefore, we aimed to investigate the effect of melatonin and its oxidation product N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) on cytokine production by neutrophils and peripheral blood mononuclear cells (PBMCs). AFMK (0.001-1 mM) inhibits the lipopolysaccharide (LPS)-mediated production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) more efficiently in neutrophils than PBMCs. Moreover, the inhibitory activity of AFMK is stronger than that of melatonin. Interestingly, monocytes efficiently oxidize melatonin to AFMK. We conclude that neutrophils are one of the main targets for melatonin and that at least part of the effects described for melatonin on immune cells may be due to its oxidation product, AFMK. We also consider that the oxidation of melatonin may be an important event in the cross-talking between neutrophils and monocytes.
Assuntos
Citocinas/metabolismo , Cinuramina/análogos & derivados , Cinuramina/farmacologia , Melatonina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Análise de Variância , Células Cultivadas , Humanos , Cinuramina/metabolismo , Melatonina/metabolismo , Oxirredução/efeitos dos fármacosRESUMO
N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) and N(1)-acetyl-5-methoxykynuramine (AMK), two melatonin catabolites, have been described as potent antioxidants. We aimed to follow the kinetics of AFMK and AMK formation when melatonin is oxidized by phorbol myristate acetate (PMA) and lipopolysaccharide (LPS)-activated leukocytes. An HPLC-based method was used for AFMK and AMK determination in neutrophil and peripheral blood mononuclear cell cultures supernatants. Samples were separated isocratically on a C18 reverse-phase column using acetonitrile/H(2)O (25:75) as the mobile phase. AFMK was detected by fluorescence (excitation 340 nm and emission 460 nm) and AMK by UV-VIS absorbance (254 nm). Activation of neutrophils and mononuclear cells with PMA produces larger amounts of AFMK than activation with LPS, probably due to the lower levels of reactive oxygen species formation and myeloperoxidase (MPO) degranulation that occurs when cells are stimulated with LPS. The concentration of AMK found in the supernatant was about 5-10% (from 18-hr cultures) compared with AFMK. This result may reflect its reactivity. Indeed AMK, but not AFMK, is easily oxidized by activated neutrophils in a MPO and hydrogen peroxide-dependent reaction. In conclusion, we defined a simple procedure for the determination of AFMK and AMK in biological samples and demonstrated the capacity of leukocytes to oxidize melatonin and AMK.
Assuntos
Cinuramina/análogos & derivados , Cinuramina/metabolismo , Leucócitos/metabolismo , Melatonina/metabolismo , Catalase/metabolismo , Catalase/farmacologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Humanos , Peróxido de Hidrogênio/farmacologia , Cinética , Cinuramina/análise , Leucócitos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Melatonina/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Oxirredução , Peroxidase/efeitos dos fármacos , Peroxidase/metabolismo , Espectrofotometria Ultravioleta/métodos , Espectrofotometria Ultravioleta/normas , Superóxido Dismutase/metabolismo , Superóxido Dismutase/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Melatonin (N-acetyl-5-methoxytryptamine) is implicated in physiologic changes related to light-dark cycles and has been recently found to display antioxidant properties. It is known that the reaction of melatonin with certain reactive oxygen and nitrogen species, such as hydrogen peroxide and singlet oxygen, produces N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK). We report herein on the development of a new liquid chromatography/tandem mass spectrometry (LC/ESI/MS-MS) assay to quantitatively determine melatonin and AFMK. The stable isotopic internal standard of melatonin-D3 was synthesized by the reaction of 5-methoxytryptamine with deuterated acetyl chloride (CD3COCl). Labeled AFMK (AFMK-D3) was obtained after photooxidation of melatonin-D3. The predominant ion [M + H]+ in the full scan mass spectra of melatonin, melatonin-D3, AFMK and AFMK-D3 were located, respectively, at m/z = 233, 236, 265 and 268. The collision-induced dissociation of the molecules revealed a predominant fragment at m/z = 174 for melatonin and melatonin-D3 (loss of the N-acetyl group), and at m/z = 178 for AFMK and AFMK-D3 (loss of both the N-acetyl and the N-formyl groups). The m/z transitions from 233 to 174 (melatonin), from 236 to 174 (melatonin-D3), from 265 to 178 (AFMK), and from 268 to 178 (AFMK-D3) were therefore chosen for the multiple reaction monitoring detection experiments, ensuring a high specificity and an accurate quantification of melatonin and AFMK in human plasma.
Assuntos
Cromatografia Líquida/métodos , Cinuramina/análogos & derivados , Cinuramina/sangue , Cinuramina/síntese química , Melatonina/sangue , Melatonina/síntese química , Espectrometria de Massas por Ionização por Electrospray/métodos , Bioquímica/métodos , Calibragem , Cromatografia Líquida/normas , Humanos , Espectrometria de Massas por Ionização por Electrospray/normasRESUMO
It has been shown that melatonin exhibits antioxidant properties. Chemical structures of some of the products formed by the interaction of melatonin with reactive oxygen and nitrogen species have been elucidated. Despite some evidence that the reaction of melatonin with singlet molecular oxygen (O2(1deltag)) produces N1-acetyl-N2-formyl-5-methoxykynurenine (AFMK), it has not been fully documented. In this investigation, melatonin was oxidized by photosensitization with methylene blue or by a clean chemical source of O2(1deltag), the thermodecomposition of N,N'-di(2,3-dihydroxypropyl)-1,4-naphtalenedipropanamide (DHPNO2). The resulting product was characterized by high performance liquid chromatography, coupled to electrospray ionization mass spectrometry and also by 1H, 13C and dept135 nuclear magnetic resonance spectroscopy. An isotopically labeled DHPN18O2 was also prepared and used as a chemical source of labeled 18[O2(1deltag)] to unequivocally characterize the end product. The results uncovered by this work confirm the hypothesis that oxidation of melatonin by O2(1deltag) produces AFMK.
Assuntos
Cinuramina/análogos & derivados , Cinuramina/metabolismo , Melatonina/metabolismo , Oxirredução , Oxigênio Singlete/metabolismo , Cromatografia Líquida de Alta Pressão , Cinética , Cinuramina/síntese química , Espectrometria de MassasRESUMO
Mononuclear phagocytes appear to synthesize kynurenine-like products from the oxidation of biologically active indole compounds including melatonin, catalyzed by interferon (IFN)-gamma-inducible enzyme indoleamine 2,3-dioxygenase (IDO). Concanavalin A (Con A) is a plant lectin that induces interferon-gamma (IFN-gamma) production by T cells. In this study we investigated whether Con A-primed peritoneal macrophages are able to oxidize melatonin to N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK). The AFMK production was accompanied by chemiluminescence. It was found that Con A-primed but not resident macrophages produce AFMK. Surprisingly, Con A-primed macrophages from IFN-gamma-deficient mice were as effective as macrophages from IFN-gamma-sufficient mice in oxidizing melatonin. Moreover, addition of an inhibitor of IDO (1-methyltryptophan) did not affect melatonin oxidation. Con A-primed but not resident macrophages have a significant content of myeloperoxidase (MPO) and inhibition of MPO by azide completely blocked chemiluminescence and AFMK production. Thus, our findings provide evidence that melatonin oxidation by macrophages may occur through a mechanism dependent of MPO and independent of IFN-gamma and IDO activity.
Assuntos
Interferon gama/metabolismo , Cinuramina/análogos & derivados , Macrófagos/metabolismo , Melatonina/metabolismo , Oxirredução , Triptofano/análogos & derivados , Animais , Azidas/metabolismo , Peróxido de Hidrogênio/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase , Interferon gama/genética , Cinuramina/metabolismo , Camundongos , Camundongos Knockout , Peroxidase/antagonistas & inibidores , Peroxidase/metabolismo , Acetato de Tetradecanoilforbol/metabolismo , Triptofano/metabolismo , Triptofano Oxigenase/antagonistas & inibidores , Triptofano Oxigenase/metabolismoRESUMO
We recently described that horseradish peroxidase (HRP) and myeloperoxidase (MPO) catalyze the oxidation of melatonin, forming the respective indole ring-opening product N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) (Biochem. Biophys. Res. Commun. 279, 657-662, 2001). Although the classic peroxidatic enzyme cycle is expected to participate in the oxidation of melatonin, the requirement of a low HRP:H(2)O(2) ratio suggested that other enzyme paths might also be operative. Here we followed the formation of AFMK under two experimental conditions: predominance of HRP compounds I and II or presence of compound III. Although the consumption of substrate is comparable under both conditions, AFMK is formed in significant amounts only when compound III predominates during the reaction. Using tryptophan as substrate, N- formyl-kynurenine is formed in the presence of compound III. Both, melatonin and tryptophan efficiently prevents the formation of p-670, the inactive form of HRP. Since superoxide dismutase (SOD) inhibits the production of AFMK, we proposed that compound III acts as a source of O(-*)(2) or participates directly in the reaction, as in the case of enzyme indoleamine 2,3-dioxygenase.