Search

Over the last few years, an increasing interest in the scientific research of natural antioxidant molecules from plants capable to attenuate, or even prevent, stress oxidative-related disorders, such as diabetes, inflammatory, and neurological pathologies has been observed. Serotonin and melatonin are two of these molecules, being widely found in Mediterranean foodstuffs, fruits, vegetables, and medicinal herbs. The consumption of foods containing both antioxidants can raise their physiologic concentrations in blood and, consequently, enhance antioxidant defenses, improve mood, and treat sleep disorders, depression, and anxiety. Currently, there exist several analytical methods suitable for the determination of melatonin and serotonin in fruits and edible plants. Among them, the most used include chromatographic techniques and immunological assays. This work aims to present a comprehensive overview of the biosynthesis of these indolamines in nature, the most common techniques used to detect and quantify both compounds in fruits, vegetables, medicinal herbs, and other food sources, as well as to address their health benefits, particularly their antioxidant, neuroprotective, and sleep-promoting effects.

Seven varieties of cherry from the “Valle del Jerte” in the Spanish Region of Extremadura were analyzed quantitatively and qualitatively for their content of the amino acid tryptophan. Tryptophan is an essential amino acid in humans, being the precursor of the indolamines serotonin and melatonin which are biogenic amines with great importance in human sleep function. It is also of great importance in the synthesis of indole alkaloids in fruit. The objective of the present work was to determine the levels of tryptophan in “Valle del Jerte” (Extremadura, Spain) cherry varieties by high-performance liquid chromatography with fluorescence detection. Tryptophan was found in all seven varieties tested, the mean concentration being 54.48 ± 17.14 g/ml. There were major differences between the varieties. The highest levels corresponded to the variety Navalinda and the lowest to Pico colorado. The tryptophan content of all the cherry varieties tested, in particular Navalinda, will have importance as the precursor for the biosynthesis of the biogenic amines.

In this study, we examined the relative efficacies of alpha-tocopherol, N-acetyl-serotonin, and melatonin in reducing ascorbate-Fe(2+) lipid peroxidation (LPO) of rat testicular microsomes and mitochondria. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids (PUFAs) C20:4 n6 and C22:5 n6. The LPO of testicular microsomes or mitochondria produced a significant decrease of C20:4 n6 and C22:5 n6. Both long-chain PUFAs were protected when the antioxidants were incorporated either in microsomes or mitochondria. By comparison of the IC50 values obtained between alpha-tocopherol and both indolamines, it was observed that alpha-tocopherol was the most efficient antioxidant against the LPO induced by ascorbate-Fe(2+) under experimental conditions in vitro, IC50 values from the inhibition of alpha-tocopherol on the chemiluminescence were higher in microsomes (0.14 mM) than in mitochondria (0.08 mM). The protective effect observed by alpha-tocopherol in rat testis mitochondria was higher compared with microsomes, associated with the higher amount of [C20:4 n6] + [C22:5 n6] in microsomes than that in mitochondria. Melatonin and N-acetyl-serotonin were more effective in inhibiting the LPO in mitochondria than that in microsomes. Thus, a concentration of 1 mM of both indolamines was sufficient to inhibit in approximately 70% of the light emission in mitochondria, whereas a greater dosage of 10 times (10 mM) was necessary to produce the same effect in microsomes. It is proposed that the vulnerability to LPO of rat testicular microsomes and mitochondria in the presence of both indolamines is different because of the different proportion of PUFAs in these organelles.

Quantitative analysis of signaling molecules from single cells and cellular materials requires careful validation of the analytical methods. Strategies have been investigated that enable single neurons and neuronal tissues to be stored before being assayed for many low-weight, biologically active molecules, such as serotonin, dopamine, and citrulline. Both metacerebral cell and pedal ganglia homogenates isolated from Pleiuohbrain-Chae californica have been studied by capillary electrophoresis with two complimentary laser-induced fluorescence detection methods. For homogenized ganglia samples, several cellular analytes (such as arginine and citrulline) are unaffected by standing at room temperature for days. Many other analytes in the biological matrix, including the catecholamines and indolamines, degrade by 20% within 10 h at room temperature. Rapidly freezing samples or preserving them with ascorbic acid preserves more than 80% of the dopamine and about 70% of the serotonin even after five days. In addition, serotonin and dopamine remain completely stable for at least five days by combining the ascorbic acid preservation and freezing at -20 degrees C. The timing of preservation is critical in maintaining the original composition of the biological samples. Using our optimum storage protocol of freezing the sample within 2 h after isolation, we can store frozen homogenate ganglia samples for more than four weeks before assay while still obtaining losses less than 10% of the original serotonin and dopamine. The nanoliter-volume single cell samples, however, must be analyzed within 4 h to obtain losses of less than 10% for serotonin related metabolites.

Copper toxicity has been implicated in various neurodegenerative disorders such as Wilson's disease and Alzheimer's disease. Free copper in the brain is toxic and leads to neuronal and cellular damage, through free radical generation. Melatonin has been investigated as a possible copper ion chelator. Melatonin could prevent copper-induced neuronal and cellular damage through binding with copper and preventing copper-induced free radical generation. The effect of copper on pineal indolamine synthesis has not been studied extensively. In the present study, copper (2 mg/kg) and melatonin (12 mg/kg) were administered daily to Wistar rats for a 2-week and 6-week period. Pineal organ culture was utilized to monitor pineal indolamine synthesis. The pineals from the 2-week copper/melatonin-treated group showed a statistically significant decrease in 5-methoxytryptophol synthesis (p < 0.01), compared to the pineals from the copper-treated group. Conversly, in the 6-week experiment, 5-methoxytryptophol synthesis was increased in both the copper- and copper/melatonin-treated groups. There was a statistically significant decrease in the N-acetyl serotonin level in the pineals from the 6-week copper-treated animals, as compared to the control- and copper/melatonin-treated group (p < 0.01). These results imply that copper reduces N-acetyltransferase activity, which results in a decrease in N-acetyl serotonin synthesis. Melatonin when coadministered with copper appears to prevent the N-acetyltransferase inhibition by copper. Copper exerts contradictory effects on 5-methoxytryptophol synthesis. Further investigations need to be carried out to examine the effects of copper on the pineal enzymes.

The content of melatonin and its precursors in the pineal gland of healthy rats and rats with hereditary retinal degeneration is studied on days 20 and 40 of postnatal development. The measurements are performed between 02:30 and 03:00, when the content of melatonin is maximal. In December, the content of melatonin and N-acetylserotonin in 20-day-old rats with hereditary retinal degeneration is 1.9- and 3.2-fold higher than in healthy controls. The content of norepinephrine in the pineal gland of these rats is increased 1.8-fold. On postnatal day 40, no differences in the pineal content of indolamines are noted. An increase in the content of melatonin in 20-day-old rats with hereditary retinal degeneration in comparison with healthy controls is also observed in May. A decrease in the maximum nocturnal level of melatonin in May in comparison with December is shown both in rats with hereditary retinal degeneration (by 46%) and healthy controls (by 41%) only on postnatal day 40.

The pineal complex of the three-spined stickleback (Gasterosteus aculeatus L.) was investigated by light and electron microscopy, as well as fluorescence histochemistry for demonstration of catecholamines and indolamines. The pineal complex of the stickleback consists of a pineal organ and a small parapineal organ situated on the left side of the pineal stalk. The pineal organ, including the entire stalk, is comprised mainly of ependymal-type interstitial cells and photoreceptor cells with well-developed outer segments. Both unmyelinated and myelinated nerve fibres are present in the pineal organ. Nerve tracts from the stalk enter the habenular and posterior commissures. A small bundle of nerve fibres connects the parapineal organ and the left habenular body. The presence of indolamines (5-HTP, 5-HT) was demonstrated in cell bodies of both the pineal body and the pineal stalk, and catecholaminergic nerve fibres surround the pineal complex.

The distribution of serotonin-immunoreactive cell bodies and fibers in the ventral nerve cord of the earthworm has been investigated from whole-mount preparations and serial sections. Serotonin-immunoreactive neurons are organized in seven soma groups per ganglion; these are defined by cell number, soma shape, diameter and fiber projections. Positional variations of this pattern have been studied quantitatively. The number of labeled perikarya is constant in midbody ganglia, but increases markedly rostral to the posterior margin of the clitellum. Variability of position and cell number differs between the cell groups. Stained nerve fibers could only be partially traced; their distribution is described in relation to defined neuronal fiber bundles and segmental nerves. The distribution and morphology of serotonin-immunoreactive cells are compared with previous observations, based on pattern analysis of primary catecholamines and indolamines with the use of a formaldehyde-induced fluorescence technique. The possible role of serotonin-immunoreactive nervous elements in different nerve cord compartments is discussed with respect to physiological effects of serotonin in earthworms and other invertebrates.

Kynuramines occur endogenously in brain and peripheral tissues as metabolites of indoleamino acids and indolamines but little is known regarding their possible physiological and/or pharmacological activity. The present study has investigated the effects of kynuramine and 5-hydroxykynuramine on the cardiovascular system of pithed rats and attempted to correlate effects seen on adrenergic and serotonergic receptors with ligand binding experiments done in vitro using rat brain membranes. Kynuramine was found to release cardiac catecholamines and to act as a weak partial agonist on vascular alpha-adrenoceptors. Hydroxylation in the 5-position (5-hydroxykynuramine) did not alter cardiac potency but increased pressor activity 100-fold. Vasopressor responses to 5-hydroxykynuramine were mediated via a dual agonistic action on both postsynaptic alpha 2-adrenoceptors and vascular serotonin2 receptors. These findings were supported by ligand binding studies, in which both kynuramine and 5-hydroxykynuramine showed affinity for cortical [3H]spiroperidol and [3H]clonidine binding sites. Overall, the results show that kynuramines can exert peripheral (and possibly central) actions which may prove to be of physiological and/or pharmacological significance.

N-acetylated and tertiary indolamines, some of which are possible neurotransmitter candidates in the CNS, cannot be visualized with the standard Falck-Hillarp histofluorescence method and very little is known about their cellular localization. The present investigation demonstrates that glyoxylic acid (GA), formaldehyde (FA) in combination with aluminum ions (the ALFA method) and trifluoroacetic acid anhydride (TFAA) are capable of forming fluorescent compounds from N-acetylated (e.g. melatonin and N-acetyl-5-hydroxytryptamine) and tertiary (e.g. bufotenin) indolamines in histochemical protein models. With GA and FA-aluminum more vigorous reaction conditions were required for demonstration of these compounds compared to those needed for optimal visualization of primary catecholand indolamines (prolonged reaction time and higher concentration of GA and FA and aluminum ions). The fluorophore formation from N-acetylated and tertiary indolamines, which represents a new reaction principle in amine fluorescence histochemistry, is proposed to proceed as follows. In the first step, the indole reacts in 2-position with the reagent. The intermediate formed is dehydrated in the second step, yielding a strongly fluorescent 2-methylene derivative, which either per se or as the corresponding autoxidized dimer constitutes the main fluorophore. TFAA and related anhydrides represent new and potent reagents for histochemical visualization of N-acetylated indolamines such as melatonin. In contrast to the GA and ALFA reactions the optimal formation of fluorphores with TFAA required only mild reaction conditions (2–10 min at 0–20° C). The main fluorophore formed from melatonin has been identified and the reaction with TFAA is proposed to proceed as follows. An unstable intermediate, the isoimidinium carboxylate, is formed in the first step and this compound is then cyclized to form the fluorophore, 6-methoxy-1-methyl-3,4-dihydro-β-carboline. The GA and ALFA methods are already widely used for visualization of catecholamine systems. The fluorescence microscopical and microspectrofluorometric analysis did not, however, veveal any specific structures containing N-acetylated or tertiary indolamines in the rat CNS. The TFAA reaction was highly specific for N-acetylated indolamines when applied to protein models. However, in tissue a disturbing background fluorescence appeared, which under all reaction conditions tested, developed concomitantly with the specific fluorescence from melatonin. The problem with this background reaction has to be solved before the TFAA reaction can be applied for demonstration of N-acetylated indolamines in tissue.

Les sites d'incorporation du 5-HTP-3H (5-hydroxytryptophane-3H) et de retention de ses derives ont ete recherches dans l'organe pineal de Lacerta vivipara (J.). Les animaux recoivent au cours de l'apres-midi (aux environs de 15h, le 5/VII, T°=21 a 28°C) une injection intraperitoneale de 5-HTP-3H. Ils sont sacrifies apres 2, 10, 15, 21, 30 minutes, 1, 2, 3, 4, 5, 10, 15 heures et 1, 2, 3, 4, 5, 6, 7, jours. De l'etude qualitative et quantitative, il ressort que des reactions radioautographiques specifiques apparaissent dans les photorecepteurs rudimentaires secretoires (=PRS) entre 2 et 10 minutes. La concentration de radioactivite atteint un maximum au niveau des PRS, apres 5 heures. Puis l'intensite du marquage diminue pour disparaitre entre 2 et 3 jours. La radioactivite des PRS est toujours concentree dans les regions de grains denses de secretion proteique (500 a 3400 A) d'origine golgienne. Les autres compartiments cellulaires sont nettement moins radioactifs. Les autres cellules (interstitielles de type ependymaire; nerveuses sensorielles et probablement noradrenergiques) de l'organe pineal et les regions voisines du cerveau ne retiennent pas significativement les composes radioactifs. Ces resultats et ceux anterieurs, cytophysiologiques et biochimiques, montrent le role important des PRS dans la biosynthese et le stockage de la serotonine. D'autres conclusions concernant la biosynthese et le metabolisme des indolamines ne seront exposees que dans une etude ulterieure ou l'incorporation du 5-HTP-3H est envisagee dans les conditions experimentales. Le turnover des indolamines semble plus lent chez Lacerta que chez les Mammiferes.

Catecholamines and indolamines react with formaldehyde to form fluorescent condensation products. This reaction forms the basis for the histochemical method of Falck and Hillarp which permits the demonstration of monoamines at the cellular level. The formation of the fluorophores is believed to require the presence of catalyzing proteins, peptides or amino acids. A similar reaction, however, can be induced also on silica gel thin-layers and this has been utilized for micro-analysis of tissue amines. In the present study formaldehyde-induced fluorophores of noradrenaline and 5-hydroxytryptamine in dry protein films and on silica gel thin-layers were analysed by microspectrofluorometry. The results indicated that the fluorophores formed in protein films and in silica gel were identical. It is suggested that the reaction on thin-layers can be used as a convenient model system permitting a detailed analysis of the various steps in the histochemical technique for the demonstration of catecholamines and 5-hydroxytryptamine.