Reaction Of Melatonin Research Articles

P.527 Comparison between paediatric and adult suspected neurological and psychiatric adverse drug reactions of melatonin reported to the European medicines agency

P.527 Comparison between paediatric and adult suspected neurological and psychiatric adverse drug reactions of melatonin reported to the European medicines agency

Dynamics of oxidative stress and urinary excretion of melatonin and its metabolites during acute ischemic stroke

Oxidative stress is a leading cause of neuronal damage in ischemic stroke. Melatonin may play a role in the antioxidant response. Melatonin and its metabolites may be involved in the modulation of oxidative stress in human acute stroke. No data are available in humans to establish this relationship. In this context, on the first and the fifth days post-stroke, we assessed serum total antioxidant capacity (TAC) and urine levels of melatonin, 6-sulfatoxymelatonin (aMT6S), and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), the last compound being produced in the brain after reaction of melatonin with reactive oxygen species. Compared to controls’ values, TAC and levels of melatonin and aMT6S were reduced, without difference between the first and the fifth days post-stroke, whereas AFMK levels remained in the normal range at both time points. Melatonin catabolism might be speeded up in acute ischemic stroke in order to increase the antioxidant response.

Involvement of electron transfer in the radical-scavenging reaction of melatonin in a protic medium

Involvement of electron transfer in the radical-scavenging reaction of melatonin in a protic medium

Melatonin as a natural ally against oxidative stress: a physicochemical examination

Oxidative stress has been proven to be related to the onset of a large number of health disorders. This chemical stress is triggered by an excess of free radicals, which are generated in cells because of a wide variety of exogenous and endogenous processes. Therefore, finding strategies for efficiently detoxifying free radicals has become a subject of a great interest, from both an academic and practical points of view. Melatonin is a ubiquitous and versatile molecule that exhibits most of the desirable characteristics of a good antioxidant. The amount of data gathered so far regarding the protective action of melatonin against oxidative stress is overwhelming. However, rather little is known concerning the chemical mechanisms involved in this activity. This review summarizes the current progress in understanding the physicochemical insights related to the free radical-scavenging activity of melatonin. Thus far, there is a general agreement that electron transfer and hydrogen transfer are the main mechanisms involved in the reactions of melatonin with free radicals. However, the relative importance of other mechanisms is also analyzed. The chemical nature of the reacting free radical also has an influence on the relative importance of the different mechanisms of these reactions. Therefore, this point has also been discussed in detail in the current review. Based on the available data, it is concluded that melatonin efficiently protects against oxidative stress by a variety of mechanisms. Moreover, it is proposed that even though it has been referred to as the chemical expression of darkness, perhaps it could also be referred to as the chemical light of health.

On the direct scavenging activity of melatonin towards hydroxyl and a series of peroxyl radicals

The reactions of melatonin (MLT) with hydroxyl and several peroxyl radicals have been studied using the Density Functional Theory, specifically the M05-2X functional. Five mechanisms of reaction have been considered: radical adduct formation (RAF), Hydrogen atom transfer (HAT), single electron transfer (SET), sequential electron proton transfer (SEPT) and proton coupled electron transfer (PCET). It has been found that MLT reacts with OH radicals in a diffusion-limited way, regardless of the polarity of the environment, which indicates that MLT is an excellent OH radical scavenger. The calculated values of the overall rate coefficient of MLT + ˙OH reaction in benzene and water solutions are 2.23 × 10(10) and 1.85 × 10(10) M(-1) s(-1), respectively. MLT is also predicted to be a very good ˙OOCCl(3) scavenger but rather ineffective for scavenging less reactive peroxyl radicals, such as alkenyl peroxyl radicals and ˙OOH. Therefore it is concluded that the protective effect of MLT against lipid peroxidation does not take place by directly trapping peroxyl radicals, but rather by scavenging more reactive species, such as ˙OH, which can initiate the degradation process. Branching ratios for the different channels of reaction are reported for the first time. In aqueous solutions SEPT was found to be the main mechanism for the MLT + ˙OH reaction, accounting for about 44.1% of the overall reactivity of MLT towards this radical. The good agreement between the calculated and the available experimental data, on the studied processes, supports the reliability of the results presented in this work.

Antioxidative effect of melatonin on DNA and erythrocytes against free-radical-induced oxidation

The aim of this work is to investigate the antioxidative effect of melatonin ( N-acetyl-5-methoxytryptamine) on the oxidation of DNA and human erythrocytes induced by 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH). First, the 50% inhibition concentration (IC 50) of melatonin is measured by reacting with two radical species, i.e., 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS +) and 2,2′-diphenyl-1-picrylhydrazyl (DPPH). The IC 50 of melatonin are 75 μM and 300 μM when melatonin reacts with ABTS + and DPPH, respectively. Especially, the reactions of melatonin with ABTS + and DPPH are the direct evidence for melatonin to trap radicals. Then, melatonin is applied to protect DNA and human erythrocytes against oxidative damage and hemolysis induced by 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH). The presence of melatonin prolongs the occurrence of the oxidative damage of DNA and hemolysis of erythrocytes, generating an inhibition period ( t inh). The proportional relationship between t inh and the concentration of melatonin ([MLT]) is treated by the chemical kinetic equation, t inh = ( n/ R i)[MLT], in which n means the number of peroxyl radical trapped by an antioxidant, and R i stands for the initiation rate of the radical reaction. It is found that every molecule of melatonin can trap almost two radicals in protecting DNA and erythrocytes. Furthermore, quantum calculation proves that the indole-type radical derived from melatonin is much stable than amide-type radical. Finally, melatonin is able to accelerate hemolysis of erythrocytes induced by hemin, indicating that melatonin leads to the collapse of the erythrocyte membrane in the presence of hemin. This may provide detailed information for the usage of melatonin and helpful reference for the design of indole-related drugs.

First insights into regiospecific transnitrosation reactions between tryptophan derivatives: melatonin as an effective target

Melatonin, a derivative of the essential amino acid tryptophan, has been portrayed as a hormone, a tissue factor, an autocoid, a paracoid, and a vitamin with antioxidative capabilities. In the present study a novel reaction which cannot be attributed to any of these suggested features, i.e. the transfer of the nitroso-function from N-nitrosotryptophan derivatives to melatonin, is unequivocally demonstrated. In the lipophilic buffer dimethylsulfoxide reaction of N-acetyl-N-nitrosotryptophan (NANT) with melatonin was very slow (k = 1.5 x 10(-6)/m/s), but reversible as shown by 15N-NMR spectrometry. These measurements demonstrated also that the thermodynamical equilibrium lies on the side of N-nitrosomelatonin (NOMela). Quantum-chemical calculations performed with the third-generation density functional B97-2 additionally predicted that this is also the case in an aqueous environment. In fact, reaction of melatonin with either NANT or N-nitrosotryptophan located at the endothelin-1 fragment 16-21 yielded NOMela with a rate constant of 1.7 +/- 0.5/m/s as shown by capillary zone electrophoresis. Interestingly, the known reactive nitrogen oxide species scavenger, piperazine, did not inhibit the NANT-dependent nitrosation of melatonin, thus very strongly indicating a direct transnitrosation reaction. All of these capabilities are known from the reaction of S-nitrosothiols with thiolate anions and are believed to be highly important in the transport and targeting of nitric oxide.

Synthesis of internal labeled standards of melatonin and its metabolite N1-acetyl-N2-formyl-5-methoxykynuramine for their quantification using an on-line liquid chromatography-electrospray tandem mass spectrometry system.

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.

Oxidation of melatonin by singlet molecular oxygen (O2(1deltag)) produces N1-acetyl-N2-formyl-5-methoxykynurenine.

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.

Further insights into the reaction of melatonin with hydroxyl radical

Recent interest has focused on the use of exogenous melatonin as an antioxidant, particularly to scavenge the highly cytotoxic hydroxyl radical (HO ) which may be generated in many pathological conditions. However, in vitro and in vivo studies aimed at assessing the antioxidant properties of melatonin have produced conflicting results. While it is known that HO reacts with melatonin at a diffusion limited rate, very little is known about the products of this reaction. In this investigation it is shown that incubation of melatonin with a Fenton-type HO -generating system at pH 7.4 forms a complex mixture of primary products. These include 2-hydroxymelatonin, which was isolated as its more stable oxindole tautomer, 4- and 6-hydroxymelatonin, N-acetyl- N 2-formyl-5-methoxykynurenine and 7,7 ′-bi-(5-methoxy- N-acetyltryptamine-4-one). Reaction pathways that might lead to these products are described. The differing biological effects of these products, while currently incompletely understood, might account for the controversy concerning the antioxidant properties of melatonin.

Reaction of melatonin with hemoglobin-derived oxoferryl radicals and inhibition of the hydroperoxide-induced hemoglobin denaturation in red blood cells.

Melatonin has been shown to act as a radical scavenger in various chemical and biological model systems in vitro. Kinetic evidence is now provided showing that melatonin inhibits the irreversible degradation of hemoglobin (Hb), when incubated with red blood cells exposed to the oxidant activity of cumene hydroperoxide (cumOOH). A decrease of heme loss and accumulation of soluble methemoglobin (met-Hb) are explained in terms of the interaction of the indoleamine with perferryl Hb (Hb[Fe(IV)=O]), a highly reactive Hb-derived radical species responsible for the irreversible Hb degradation. A kinetic study, in pure chemical solution, showed that melatonin can effectively reduce the oxoferryl heme group of perferryl-Hb, thus forming met-Hb. The reducing activity of melatonin is of the same order as that of Trolox, the water-soluble vitamin E analog. This novel radical-scavenging activity of melatonin may contribute to the previously observed protective effects of melatonin in ischemia-reperfusion injury.

Oxidation of melatonin by oxoferryl hemoglobin: A mechanistic study

Reaction of melatonin with the hypervalent iron centre of oxoferryl hemoglobin, produced in aqueous solution from methemoglobin and H2O2, has been investigated at 37°C and pH 7.4, by absorption spectroscopy. The reaction results in reduction of the oxoferryl moiety with formation of a heme-ferric containing hemoprotein. Stopped-flow spectrophotometric measurements provide evidence that the reduction of oxoferryl-Hb by melatonin is first-order in oxoferryl-Hb and first-order in melatonin. The bimolecular reaction constant at pH 7.4 and 37°C is 112 ± 1.0 M-1 s-1.Two major oxidation products from melatonin have been found by gas chromatography-mass spectroscopy: the cyclic compound 1,2,3,3a,8,8a-hexahydro-1-acetyl-5-methoxy-3a-hydroxypyrrolo[2,3-b]indole (cyclic 3-hydroxy-melatonin), and N-acetyl-N′-formyl 5-methoxykynuramine (AFMK). The percentage yield of the two major products appears dependent on the ratio [oxoferryl-Hb]: [melatonin]—the higher the ratio the higher the yield of AFMK. The observed stoichiometry oxoferryl-Hbreduced:melatoninconsumed is 2, when the ratio [oxoferryl-Hb]:[melatonin] is 1:1, but appears >2 at higher molar ratios. The reduction of the hypervalent iron of the oxoferryl moiety may be consistent with an oxidation of melatonin by two one-electron steps.

New Colorimetric Methods for Microdetermination of Melatonin in Pure and in Dosage Forms

ABSTRACT Two simple, rapid, sensitive and accurate methods (A and B) for the microdetermination of melatonin in pure form and in pharmaceutical formulations are developed. Method A is based on the formation of tris(o-phenanthroline)iron(II) complex (ferroin) upon reaction of melatonin with an iron(III)-o-phenanthroline mixture in sodium acetate-acetic acid buffer media. The ferroin complex is colorimetrically measured at λmax 510 nm against reagent blank. Method B is based on the reduction of Fe (III) by the drug which forms a colored complex (λmax 522 nm) with 2,2-bipyridyl. Optimizations of the experimental conditions are described. Beer's law is obeyed in the concentration range 0.4 – 6.4 μg ml− for A and 0.4 – 7.4 μg ml− for B, respectively. The developed methods are applied successfully for the determination of melatonin in pure form and in melatonin tablets without any interference from common excipients.

The Reaction of Melatonin with Peroxynitrite: Formation of Melatonin Radical Cation and Absence of Stable Nitrated Products

Peroxynitrite is capable of hydroxylating and nitrating aromatic species. However, nitromelatonin is not found as a final product when melatonin was allowed to react with peroxynitrite either in the presence or absence of added bicarbonate. In the absence of bicarbonate, the two major products formed are 6-hydroxymelatonin and 5-methoxy-2-hydro-pyrroloindole, and the latter is the only major product with excess bicarbonate. A transient purple intermediate with a maximum absorbance at about 520 nm is observed upon mixing solutions containing peroxynitrite and melatonin. These observations indicate that the melatoninyl radical cation is formed in the peroxynitrite/melatonin reaction, providing a direct evidence for the one-electron oxidation ability of peroxynitrite. The melatoninyl radical cation also is observed with excess bicarbonate.

Reactions of melatonin and related indoles with free radicals: a computational study.

Melatonin is being increasingly promoted as a therapeutic agent for the treatment of jet lag and insomnia and has been recently suggested to act as an efficient free-radical scavenger. In the present work, its mechanisms of action for scavenging hydroxyl radicals have been investigated using semiempirical AM1 and density functional theory (DFT) computational tools. Two different reactions were proposed as follows: one involving the abstraction of an indolic hydrogen to yield a neutral radical and another involving the addition of the hydroxyl radical to the indolic moiety. Our results show that, from a thermodynamical standpoint, melatonin may directly scavenge hydroxyl radicals both in vacuum and in aqueous solution. The structural requirements for free-radical-trapping ability have been examined comparing melatonin with related indoles. Computational data suggest that 5-methoxy and N-acetyl groups of melatonin do not significantly affect its thermodynamical capacity of free-radical trapping. The present results support experimental data on the potential of melatonin as a physiological or pharmacological antioxidant agent.

Reactions of melatonin with radicals in deoxygenated aqueous solution

Reactions of melatonin (N-acetyl-5-methoxytryptamine) with radiolytically generated radicals were studied. Reaction of melatonin with OH radicals is diffusion-controlled (k=1.2·1010 dm3 mol−1·s−1), the main (but not the only one) intermediate being the indolyl-type radical, while the rate constant for the reaction with hydrated electrons isk=4.3·108 dm3·mol−1·s−1. Melatonin is capable of scavenging tert-butanol radicals, while its reactivity towards polymer radicals of poly(acrylic acid) and poly(vinyl pyrrolidone) is very low.

Colorimetric and Spectrofluorimetric Methods for the Determination of Melatonin in Tablets and Serum

Two sensitive and accurate colorimetric and spectrofluonmetric methods, are presented for the determination of melatonin in tablets and serum. The first method utilizes the reactions of p-dimethylaminobenzaldehyde in hydrochloric acid (van Urk reagent)-ferric chloride in sulphuric acid (Salkowski reagent) mixture. The blue color of the resulting reaction product is measured at 630 nm. The second method is based on the reaction of melatonin with o-phthalaldehyde in acid medium which yields highly fluorescent condensation product that is measured at 465 nm as emission wavelength, using excitation wavelength at 355 nm. No interference was observed from tableting additives, and the applicability of the methods was examined by analysing tablets containing melatonin (single and combined with pyridoxine). Mean percentage recoveries from tablets were found to be 99.9+0.31 for single and 100.5+0.15 for combined tablets using colorimetric method, while by applying spectrofluorimetric method the recoveries were found to be 100.610.41 for single and 100.2+0.39 for combined tablets. Furthermore, the proposed methods were extended to the in-vitro determination of melatonin in serum. The detection limits are 0.27 ug ml−1 for colorimetric method and 0.00035 ug ml−1 for spectrofluorimetric method.

Reaction of Melatonin and Related Indoles With Hydroxyl Radicals: EPR and Spin Trapping Investigations

It has been suggested that the indole hormone melatonin ( N-acetyl-5-methoxytryptamine, MLT) is an important natural antioxidant and free radical scavenger [ J. Pineal Res., 14: 51; 1993]. In the present work we determined the rate constants, k r, for scavenging OH radicals by melatonin, 5-methoxytryptamine (5-MeO-T), 5-hydroxytryptamine (serotonin, 5-OH-T), 6-chloromelatonin (6-Cl-MLT), 6-hydroxymelatonin (6-OH-MLT), and kynurenine (KN) in aqueous solutions. Hydroxyl radicals were generated using a Fenton reaction in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which competed with the indoles for the radicals. It was found that MLT reacts with OH with k r = 2.7 × 10 10 M −1 s −1. Other indoles and KN reacted with OH radicals with similarly high rates (k r > 10 10 M −1 s −1). In contrast to nonhydroxylated indoles (MLT, 6-Cl-MLT, and 5-MeO-T), hydroxylated indoles (5-OH-T and 6-OH-MLT) may function both as OH promoters and OH scavengers. The melatonin precursor serotonin promoted the generation of OH radicals in the presence of ferric iron and H 2O 2, and the melatonin metabolite 6-hydroxymelatonin generated large quantities of OH radicals in aerated solutions containing Fe 3+ ion, even in the absence of externally added hydrogen peroxide. These reactions may be relevant to the biological action of these physiologically important indolic compounds.

Reaction of Melatonin With Lipoperoxyl Radicals in Phospholipid Bilayers

Melatonin, at 5 to 500 μM was incorporated in unilamellar soybean phosphatidylcholine (PC) liposomes, the peroxidation of which was induced by 2,2′-azobis(2-amidinopropane-hydrochloride) (AAPH), and measured as production of conjugated diene lipid hydroperoxides. Concentration as low as 5 and 10 μM were poorly effective in reducing lipid peroxidation. Melatonin at 30 to 500 μmM caused short inhibition periods, increasing with, but not linearly related to concentration, with a concurrent net decrease of the propagation rate. The time course of melatonin oxidation, measured as loss of fluorescence, was studied during the AAPH-stimulated peroxidation of soybean PC liposomes, or when melatonin was incorporated in nonperoxidable unilamellar dimirystoyl phosphatidylcholine (DMC) liposomes. Consumption kinetics of 30 μM melatonin were linear with time in DMC liposomes and disappearance of melatonin occurred at a rate of 0.058 M −8s −1. On the other hand, the consumption of melatonin during the oxidation of soybean PC liposomes, was not linear with time. The rate of disappearance was calculated as 0.19 M −8s −1 at the beginning of the propagation phase, then it slowed down to reach the same rate observed in DMC liposomes. This evidence suggests a reaction with lipid-derived peroxyl radicals, possibly in addition to reaction with peroxyl radicals derived from AAPH. Scavenging of lipoperoxyl radicals by melatonin was also evident in experiments where melatonin was incorporated in multilamellar soybean PC liposomes and peroxidation was initiated by 2,2′-azobis(2,4-dimethyl-valeronitrile). The antioxidant activity of melatonin in soybean PC liposomes is much lower than that of α-tocopherol, under comparable assay conditions. However, a combination of melatonin and α-tocopherol, at 5 μM, resulted in a synergistic antioxidant effect. Time course of α-tocopherol consumption, monitored in the absence and in the presence of melatonin, showed a significant decrease of the consumption rate when compounds were combined, indicating some protection by melatonin. Regeneration mechanisms were not evident and depletion of α-tocopherol was coincident with the inhibition time.

A nonisotopic assay for acetylserotonin methyltransferase

A nonisotopic assay for acetylserotonin methyltransferase (ASMT) was devised. Melatonin, the product of the enzyme reaction, is measured fluorometrically after its reaction with o-phthaldialdehyde (OPT). The reaction of melatonin with OPT is carried out in 1 n HCl to suppress the reaction of N-acetylserotonin, the substrate of ASMT, with OPT. The mixture is gassed with nitrogen just before incubation at 60°C for 60 min in order to secure the linear relationship between the concentration of melatonin and the fluorescence intensity. This method is much simpler than the isotopic assay and also has as much high sensitivity. Moreover, in this assay the enzyme can be well saturated with S-adenosylmethionine, whereas in the isotopic assay it cannot.