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Melatonin - Science topic

A biogenic amine that is found in animals and plants. In mammals, melatonin is produced by the pineal gland. Its secretion increases in darkness and decreases during exposure to light. Melatonin is implicated in the regulation of sleep, mood, and reproduction. Melatonin is also an effective antioxidant.
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Melatonin and serotonin are easily lost because of their low content and light-sensitive. The extraction steps should be quick. Is there a simple way to extract them from hundreds of plants or any proper extraction buffer recommended? Thanks!
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Please if get answer reply i still have the same question
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Hi everyone,
can anyone tell me which is the best way to quantify Melatonin? I've read articles that refer hplc-dad and also hplc-fluorescence. I'm interested in analyze melatonin in raw milk, making the extraction following ISO14156.
Do you have any sugestions ?
Thank you for your help!
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Hendrik-Jan Brouwer thank you for your answer. It was very helpful.
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Does anyone have any research on - if artificial ingestion (pills/drops) of Melatonin can lead to addiction or lighter dependence? Due to or if, the brain's own production (epiphysis) goes down with extra intake. I.e., In Sweden, melatonin tablets are considered a prescription drug, while other countries classify it as a supplement.
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A recent review of melatonin and sleep (PMID: 28460563) concluded that “Exogenous melatonin supplementation is well tolerated and has no obvious short- or long-term adverse effects.” Another recent article suggests, based on animal studies, that melatonin supplements “may be beneficial in the management of behavioural manifestations of drug addiction” (PMID: 29988891). Melatonin’s precursor tryptophan, like melatonin, is treated as a drug in some countries and sold over the counter in others. These differences are possible related to cultural factors that influence how a government balances the right of people to take whatever they want against the role of governments in protecting the population from possible harms associated with a compound.
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People please help me to find out
1.Melatonin production by yeast
2.Down stream process
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Agreed with Chinaza Godswill Awuchi.
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Hello everbody. I just started my first scientif research and i'm having a little bit of trouble converting some units. I'm actually studing the possibily of building a biosensor to melatonine. I'm doing a review of what we already have in the literature about methods of detection, and I wanted to put every detection limit in pg/ml. For example, I found one that has a LD of " 1 fmol per well". I did some conversions (another question: I know the volume of a well is usely 350 uL, but there was this affirmation "the final volume of incubation was 126 uL", so I consider the volume of 126 uL. Am I right?), and found out that the limit were 6,67 pmol/L. Now I'm very confused of what I should do, because I don't have a cleary mass of melatonin (the objeticv of the research was to found that out in humam serum), just that they used 20 ng of melatonin-p-carboxybenzyl-AP conjugate, and I'm not so sure about what to do with this information. I hope this is not a very silly question. Thanks for the attention!
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Melatonin (pg/ml) x 4.30 = Melatonin (pmol/l)
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I tried to do a Slot Blot and was not successful and melatonin is only .23 Kda so it would probably run off a western blot. Has anyone had success with other methods?
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Have you considered doing a DOT blot instead?
Rather than run the sample on a gel and transfer, you just prep some membrane and sport the sample onto it. Once dried, block and run as usual.
It's normal to do serial dilutions of the sample and spot across the membrane.
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The protocol used in managing COVID-19 for Mr. president Trump containing:
Regenerons poly-clonal antibody cocktail
Zinc
Vit. D
Fomatidine
Melatonin
Aspirin
what are the differences and advantages by utilizing this protocol?
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President Trump was declared to have COVID-19 symptoms that could be graded to moderate disease. However, he was treated like a patient with severe or critical disease (Remdesivir, Dexamethasone, and Immunoglobulins)
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Hello,
Recently, I am doing my research on melatonin receptor two (MT2) using bovine granulosa cells. My result show that down-regulation of MT2 resulted in less apoptotic rate in transfected bovine granulosa cells while the control has high apoptotic rate. My result seems strange because melatonin and its membrane receptors have long been known as ati-apoptotic factor. And the apoptotic factors Bax and Bcl2 were up and down-regulated, respectively in MT2 silenced cells. Can anyone comment on this result?
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Hi Tesfaye Worku , I accidentally found your question here. I'm curious about the exact mechanisms of MT1 and MT2 on granulosa cells, too. Could you share with me your finding on MT2?
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I understand that light is one of the strongest external cues to affect our clock system. I am curious about other possible factors that are less discussed. I am finding a great amount of literature of EMFs on insects such as fruit flies, but when it comes to human studies the results are always contradictory.
I would be open to any suggestions on researchers and the latest studies in the field.
Thank you,
Korey
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There ar edifferneces between species in the effects on electromagnetic fields on the secretion of melatonin. Our team showed that in rats at relatively high levels of exposure melatonin secretion declined whereas no effect could be shown in humans, whatever the duration of exposure, short or long..
See the several papers by B. Selmaoui and Y. Touitou on PubMed on the topic
All best
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I tried with DI water but it cannot fully dissolve melatonin.Some people suggested to use little ethanol. But i have a confusion that if i use ethanol then it may have interference duringelectrochemical detection.I need some good suggestions.
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Thank you very much@Firas A. Al-Lolage
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Some healthcare providers are reluctant to use melatonin because potential suppression of endogenous production and/or the theoretical potential for pineal gland atrophy.
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Thanks for your response.
Jason
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What is the value of in vitro maturation and embryonic development of oocytes?What is the end result we want?
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I am starting a project with melatonin supplementation and would like to make a stock solution for dilution when giving the melatonin to rats; I suppose if I dilute the melatonin in alcohol and store in -20 degrees celsius, it would work, but I am not sure. Couldn´t find any research paper or technical publication on that... Does anyone have an idea?
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Thank you
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I'm thinking about using night vision goggles for a series of experiments I plan to perform in the absolute darkness. I understand that some light is emitted from the goggles when used in complete darkness and wondered whether anyone could advise how bright this light is in general? I'm wondering whether the light is going to be disruptive to melatonin levels.
Thanks in advance!
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Dear Susannah,
I am not sure the relation between night vision optic and circadian expeeiments.It will be possible
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This is for a research protocol in spider monkeys' chronobiology. So far the best saliva extracting device has been an aspirator that allows collecting about 1 ml in 2-8 min. The issue is that most monkeys are unwilling to cooperate if they don't receive a food reward while aspirating the saliva, thus contaminating the sample with the treats used to lure them.
The three analytes will be quantified by ELISA, nevertheless, if someone knew of a similar assay less prone to disturbance by the use of salivary stimulants it would be of great assistance as well. Thanks to anyone that can help me in this matter.
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I,m working on beneficial role of melatonin on vegetable crops. How I can analyze the level of endogenous melatonin in plants?
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I am interested in analyzing melatonin from human saliva using ELISA kit. There are quite some manufacturers (IBL-international, Amco, salimetrics etc.) that produce such kits but I already heard some negative feedbacks, like a lack of fluctuation in salivary melatonin from ELISA comparing with radioimmunoassay. It would be very interesting to know if anyone has tried any and could give a feedback on which you found useful? Thank you!
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I am with MP Biomedicals and we offer a Melatonin ELISA kit for use with saliva, if you are interested in the details or to request an evaluation kit to test, please let me know. Best of luck with your studies!
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Melatonin is said to be inolved in the circadian rythms as it plays a role in the sleep-wake process. How exactly does melatonin play this role?
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Please read our recent review in Endocrine Reviews. There is two sessions discussing the important role played by melatonin in the regulation of circadian and seasonal rhythms, including mechanisms. If you are not able to get the pdf in the journal site, please, ask me for it: cipolla@icb.usp.br
Melatonin as a Hormone: New Physiological and Clinical Insights
Jose ́ Cipolla-Neto and Fernanda Gaspar do Amaral
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Hello,
I currently have a product that has a small amount of melatonin dissolved in it (aqueous solution), and I need a sufficient amount to get a reading on an IR spec. I was thinking of doing an extraction in a sep funnel using DMSO-does anyone have any resources, links. or experiences with this? Thank you!
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Hi! Melatonine is indicated in Europe for sleep disorders in patients > 65 years old .
The secretion of melatonin is regulated by light. Apparently, the following mechanism is observed: absence of light => increase of NE => action on beta1 and alpa-adrenergic receptors of the pineal gland = > activation of N-acetyltransferase => synthesis and release of melatonin (Touitou Y., 2005).
My question is this: When we give the patient melatonin tablets (or a melatonin agonist as the agomelatine), should we be concerned about a potential inhibitory feedback on endogenous melatonin secretion (as is the case for corticosteroids and inhibition of the hyphalamic-pituitary axis): Do we have info about the regulation of this key enzyme, the N-acetyltransferase, by melatonine ? Do cells of pineal gland express melatonin receptors ?
Thank you in advance for your reply :) !
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No, there is no classical negative feedback at all. However, exogenous melatonin might advance or delay the endogenous pineal melatonin production accordingly to the intake time. See work by Alfred Lewy defining dim light melatonin onset and melatonin phase response curve. Alternatively, please, read our review recently published in Endocrine Review.
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Dear researchers, I want to find some genes related to SOD, POD, CAT, APX, MDA, and melatonin biosynthesis gene under heavy metal stress in cotton or other crops?
Kindly upload the related papers.
Thanks
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thank you very much prof. Jai Ghosh
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To determinefree Melatonin in serum or plasma samples is not so easy as it always found to be bounded to large proteins. Countless methods of the pretreatment of serum/plasma samples are described. But those methods use methanol or dichloromethane to extract melatonin and those are tedious and time consuming. I came to know that some commercial kits use digestive enzyme preferably pepsin for extraction. Does any one know what is the concentration of pepsin to be used or any other chemicals can be used?. I kindly request you to share your ideas or experience. Thank you.
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Yes, this operation is possible through the definition of derivated spectra of high orders
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I am looking for melatonin solvent, particularly chloroform,.I found DMF and DMSO concentration but I did not see for chloroform.
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please determine experimentally, no data available
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I am working on cotton crop. I want to know that which level of Melatonin can improve cotton seedling growth under cadmium stress
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Dear Aziz Khan,
I think singlet oxygen (1O2)‐producing mutant, upon light exposure may be suggestive to melatonin induction by chloroplastidic against
heavy metal stress tolerance in cotton seedlings.
This publication may describe more about this phenomenon:
Good luck.
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Just curious about any potent natural fungal or bacterial sources of melatonin. Thanks!
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Dear Nancy,
One paper which may be of your interest could be the following one by Rodriguez-Naranjo et al. (see citation below)
J. Pineal Res. 2012; 53:219–224
and perhaps this one: https://doi.org/10.1111/jpi.12005
It would be of great interest to investigate and unravel gene cluster(s) behind such production.
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Hi, I've been trying to find an antibody against melatonin that workd for immunohistochemistry (i.e., frozen or FA fixed sections of tissues). So far we have found very few commercially available and none that is able to detect free melatonin in our insect brain sections. ¿Does anyone know of a commercially available and not discontinued anti-melatonin? Alternatively, ¿does anyone have an anti-melatonin that works and is willing to send a small aliquote (5µl)?
Thanks in advance,
J.
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I agree with Jan. We have not been able to detect/quantify melatonin from Apis mellifera brain samples using our MS method. Either melatonin is not present at all or it is present below our detection limit of 7pg.
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Dear Colleague (s)
I am having little confusion regarding the best solvent to dissolve melatonin. Among ethanol, isopropanol and water, which will be best keeping in mind the duration of its half-life? I am finding it confusing in Sigma-Aldrich data sheet (Catalogue number # M5250).
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Melatonin isn't very soluble in water. I would dissolve it in DMSO, where it's soluble in the 10s of mM range, and then add the DMSO/melatonin mixture to your medium to get the appropriate melatonin concentration.
But if ethanol. iso and water are your only options, then go with ethanol. Just make sure you use a vehicle contrpl.
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Hi fellows,
Does any body knows an alternative procedure to pinealectomy to inhibit or attenuate the melatonin synthesis in vertebrate models?
Thanks a lot!
g.
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Hi Marius.
Thanks for your answer.
I was thinking in a non light dependent procedure, a pharmacological may be.
g.
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Melatonin secretion follows circardian rhythm.. what is the best way to estimate its levels in body? Serum sample, urine or saliva.... whats the most feasible method to follow for a stu in pediatric patients?
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Go to:
Abstract
Background and Purpose
The circadian rhythm of melatonin in saliva or plasma, or of the melatonin metabolite 6-sulfatoxymelatonin (a6MTs) in urine, is a defining feature of suprachiasmatic nucleus (SCN) function, the body’s endogenous oscillatory pacemaker. The primary objective of this review is to ascertain the clinical benefits and limitations of current methodologies employed for detection and quantification of melatonin in biological fluids and tissues.
Data Identification
A search of the English-language literature (Medline) and a systematic review of published articles were carried out.
Study Selection
Articles that specified both the methodology for quantifying melatonin and indicated the clinical purpose were chosen for inclusion in the review.
Data Extraction
The authors critically evaluated the methodological issues associated with various tools and techniques (e.g. standards, protocols, and procedures).
Results of Data Synthesis
Melatonin measurements are useful for evaluating problems related to the onset or offset of sleep and for assessing phase delays or advances of rhythms in entrained individuals. They have also become an important tool for psychiatric diagnosis, their use being recommended for phase typing in patients suffering from sleep and mood disorders. Additionally, there has been a continuous interest in the use of melatonin as a marker for neoplasms of the pineal region. Melatonin decreases such as found with aging are or post pinealectomy can cause alterations in the sleep/wake cycle. The development of sensitive and selective methods for the precise detection of melatonin in tissues and fluids has increasingly been shown to have direct relevance for clinical decision making.
Conclusions
Due to melatonin’s low concentration, as well as the coexistence of numerous other compounds in the blood, the routine determination of melatonin has been an analytical challenge. The available evidence indicates however that these challenges can be overcome and consequently that evaluation of melatonin's presence and activity can be an accessible and useful tool for clinical diagnosis.
Keywords: Melatonin, Circadian rhythms, Radioimmunoassay, Enzyme-linked immunoassay, High-performance liquid chromatography, Mass spectrometry, Capillary electrophoresisGo to:
Introduction
Melatonin (N-acetyl-5-methoxytryptamine) is a compound secreted mainly by the pineal gland, but synthesized also in many other tissues and cells, including the retina [1–3], human and murine bone marrow cells [4], platelets [5], the gastrointestinal tract [6], skin [7], or lymphocytes [8]. Because of the multiplicity of sites of formation and the presence of melatonin receptors in diverse locations, melatonin appears to be a versatile physiological signal that has been found involved in the control of numerous physiologic processes [9,10]. In mammals, photoperiodic information is relayed through the secretion of melatonin by the pineal gland, which then acts on the brain, and the neuroendocrine system to produce adaptive changes in endocrinology, anatomy, and physiology, thus affecting sleep, reproduction, molting, immune responses, energy balance, and behavior, among others [11–14]. Moreover, melatonin exhibits direct and indirect antioxidant properties, and there is strong evidence that this compound can counteract the deleterious effects of reactive oxygen and nitrogen species in different systems [15–19].
Although numerous physiological functions have been attributed to melatonin, the mechanisms involved in such functions are frequently unclear, especially when parallel signaling pathways that are initiated via the membrane receptors MT1 or MT2 are considered, or when other melatonin binding sites are involved [20]. Therefore, further investigation at cellular and molecular levels is needed to elucidate how this compound really acts as a relevant physiological regulatory signal.
Although in vitro studies can furnish important information on the effects of melatonin in cell culture or perfused tissues, studies of the fluctuations of melatonin concentration in body fluids and tissues generally have the most relevance for understanding its function in organisms. Thus, the development of sensitive and selective methods for the precise detection of these compounds in tissues and fluids of animals has become necessary. However, due to its low concentration and the coexistence of many other endogenous compounds in blood, the determination of melatonin has been an analytical challenge. This review discusses current methodologies employed for detection and quantification of melatonin in biological fluids and tissues.
Melatonin fluctuations in organisms
In vertebrates, the chronobiologically relevant fraction of melatonin is mainly produced and released into the circulation by either the pineal gland, especially in mammals, or pineal gland plus retina, e.g., in some birds and amphibians [21, 22]. The pineal glands of non-mammalian species are directly photosensitive while those of mammals are controlled by neuronal phototransduction pathways originating in the retina and then processed by the hypothalamic circadian pacemaker, the suprachiasmatic nucleus (SCN) [21, 23, 24]. In various birds, light influences circadian oscillators present in the pineal gland and acts additionally on neuronal pathways of retinal or hypothalamic origin modulating the gland [25–27]. The contribution of extrapineal/extraretinal melatonin to blood plasma concentrations of the hormone are either very low or, in the case of gastrointestinal origin, only episodic and without profound chronobiological significance [6].
In humans, nocturnally peaking high-amplitude oscillations of melatonin in plasma are paralleled by corresponding variations in saliva [28]. Although plasma levels are generally about ten times higher than those found in saliva, determinations of salivary melatonin can be advantageous, especially when it is preferred to avoid invasive procedures. The primary melatonin metabolite in the urine, 6-sulfatoxymelatonin, also oscillates consistently with melatonin concentration in urine, plasma, and saliva [28]. Melatonin levels in human plasma usually begin to increase between 18:00 and 20:00 h, and peak between midnight and 05:00 h [28–32], being followed by a rapid decrease. The duration of the nocturnal melatonin peak has been shown to be the crucial signal for encoding season [33]. However changes in the duration also occur in nonseasonal animals. For example, in the rat the duration of the melatonin increase is closely tied to the photoperiod [34]. Seasonal changes in the nocturnal peak of melatonin have also been widely reported with the amount of change varying between species. The nocturnal peak of pineal melatonin secretion in the Siberian hamster during the winter is two times greater than during the summer [35], while that of the European hamster shows a 10-fold increase [36]. Seasonal variations in melatonin duration and levels are also found in humans, but less prominently so. Again, the duration and amplitude of the melatonin peak is negatively correlated with the length of the photoperiod [37, 38].
Another important aspect of melatonin fluctuation in humans concerns variable environmental light intensities. It has been proposed that melatonin secretion during dark phases is greatly influenced by dim light, whereas very bright light can “mask” melatonin production [39]. Dim light can be particularly important in the circadian entrainment of rhythms. Moreover, the suppression of melatonin formation and release by nocturnal light represents a well-known phenomenon, of particular importance due to its health consequences in shift workers [40]. The photic shutoff mechanisms depend on the respective organism and may be based either on dephosphorylation of a key enzyme in melatonin biosynthesis, arylalkylamine N-acetyltransferase (AANAT), thus preventing interaction with a 14-3-3 protein, and followed by rapid proteasomal degradation, and/or on downregulation of AANAT expression [41, 42]. Consequently, melatonin’s actions are not only influenced by the phase of the light–dark cycle, but can be strongly affected by variations in light and dim light intensities. Moreover, both normal melatonin patterns and the influence of light can vary considerably between individuals, either in terms of personal characteristics [43–46] or as a consequence of aging or a chronic disease [47–52]. Studies in siblings have indicated that some of this variation has a genetic origin [53].
The close linkage between secretion levels of melatonin and the great variations in the pacemaker processes controlling circadian and annual rhythms suggests that a causal relationship may exist in these processes. In etiological terms, changes in melatonin have been repeatedly suspected to have involvement in numerous diseases, in the susceptibility to inflammatory processes or in genetic predispositions. The health-related roles of melatonin seem to reflect a mixture of hormonal, immunomodulatory, neuromodulatory, and various types of antioxidant actions, and its efficacy in safeguarding these underlying processes is observed at very low concentrations [20]. Therefore, it seems that fluctuations in melatonin duration and levels which may appear, at first glance, to be of only minor significance, may cause, in the long run, important pathophysiological changes. Because melatonin levels are relatively low even at nighttime highly specific and sensitive methods for melatonin measurements in biological samples are essential.
Relevance of melatonin assay in pediatric neurosurgery
Since the description of validated assays for melatonin in plasma, there has been a continuous interest in the use of melatonin as a marker for neoplasms of the pineal region [54–58]. The elevated diurnal melatonin levels reported in some initial studies [54, 56] were not verified in later descriptions of relatively larger series of pineal region neoplasms [58]. For example, in two series of tumors that included four [59] and five pineal parenchymal neoplasms [57] only one of the patients displayed high diurnal levels of melatonin.
The best evidence on the validity of plasma melatonin assay for diagnosis of neoplasms of the pineal region was given by a recent study on 29 tumors of the pineal region, including germ cell tumors, pineal parenchymal tumors, meningiomas, and glial tumors [58]. Presurgically, the daily rhythm of plasma melatonin was suppressed in undifferentiated or invasive tumors. Postsurgically, the absence of a melatonin rhythm could be correlated with a damage of the pineal gland by surgery.
Melatonin deficiency could be the cause of a post-pinealectomy syndrome that includes alteration of sleep/wake cycle [60] and recurrent headache and depression [61]. Somnolence after pinealectomy is amenable of exogenous melatonin treatment [62] as is the daily somnolence seen among craniopharyngioma survivors, the most common extraneural tumors of the CNS in children [63, 64]. In craniopharyngiomas the disrupted sleep pattern presumably results from dysfunction of the hypothalamic circadian pacemaker located in the SCN which suppresses the circadian rhythmicity of melatonin [65].
Determination of melatonin in biological samples
Considering its low levels an important issue of melatonin measurements is its adequate extraction from biological samples. In serum samples, melatonin can be extracted by simple liquid/liquid procedures, such as the addition of dichloromethane (1:1, v/v). Samples are then vigorously mixed and centrifuged to obtain aqueous and organic phases. With this procedure, melatonin is retained in the dichloromethane phases that are collected and dried under nitrogen atmosphere to concentrate melatonin. This yields a satisfactory recovery rate (generally more than 70%), and can be also applied to buffer-homogenized tissues. However, a low precision and accuracy with single liquid–liquid extractions of melatonin for high performance liquid chromatography (HPLC) coupled to fluorescence detector have been reported [66]. For multiple analyses of melatonin and its precursors or metabolites, more profound liquid–liquid extractions have been described using a combination of different solvents [67].
In older investigations, chloroform (trichloromethane) was mostly used for melatonin extraction and is still in use today. Although this method is effective, dichloromethane is preferred for reasons of lower toxicity. Generally, chlorinated methane should be of highest purity and protection from light and redox-active compounds is of utmost importance for avoiding formation of reactive intermediates which can destroy melatonin.
Laganà et al. [31] described an extraction procedure for serum samples through an LC-18 cartridge plus a Carbograph cartridge with a recovery ranging from 86.3 to 91.7% for 10 to 200 pg melatonin/ml. Briefly, 2 ml of serum sample is passed through an LC-18 cartridge, which is then washed with 2 ml of water and 2 ml of water–methanol (90:10, v/v). Thereafter, melatonin can be eluted from the column with pure methanol, dried and resuspended in an appropriate solution for analysis [68] or can be further purified by eluting with 2 ml of water–methanol (40:60, v/v) and loading onto a Carbograph cartridge [31]. The cartridge is then washed with 10 ml of methanol and 3 ml of methanol–dichloromethane (80:20, v/v), and melatonin is finally eluted with 1.5 ml of methanol–dichloromethane (10:90, v/v). The eluate is evaporated to dryness under N2 atmosphere and resuspended in 100 μl of water–methanol (75:25, v/v) for analyses.
Sample preparation will also depend on the method used for analysis, since the presence of other compounds in the sample can interfere with the melatonin signal. The extent of melatonin prepurification from biological samples can, in some cases, be fundamental for the sensitivity of the method used. The procedure described above allows melatonin detection with high sensitivity and without interference from other components in the sample [31]. It has been shown that homogenization in 10 vol of ice-cold 0.1 M perchloric acid can also represent an accurate means for melatonin determination in tissues by HPLC coupled to electrochemical or fluorescence detection [69]. In this case, the homogenate is centrifuged at 10,000 × g for 20 min at 4°C and the resulting supernatant can be directly injected into the HPLC system. It has also been suggested that 90 μl of the supernatant fraction be mixed with 10 μl of 1 M sodium phosphate, pH 4.3, for better resolution of peaks.
Depending on the method used, further treatment of melatonin extracts may be needed. Gas chromatography-mass spectrometry (GC-MS) detection of melatonin requires sample derivatization for melatonin volatilization by, for example, the use of pentafluoropropionic anhydride or heptafluorobutyrylimidazole [70, 71]. In another approach, human plasma samples have been directly injected into and evaluated in an HPLC system with fluorescence detection without prior extraction or purification, achieving a detection limit of 1 ng per ml of human plasma (4 pmol/ml) [72]. Also, it has been reported that derivatization of melatonin with sodium carbonate and hydrogen peroxide increases sensitivity almost 10-fold for measurement in HPLC systems coupled to fluorescence detectors [73].
Rolčik et al. [74] described a highly specific method for melatonin isolation and purification from complex biological matrices by immunoaffinity chromatography [74]. Polyclonal antibodies highly specific against melatonin were raised by Mannich synthesis [75] and used for preparation of immunoaffinity gel, with a 95% recovery rate for melatonin extraction. In these samples, melatonin concentration was determined by HPLC-mass spectrometry (HPLC-MS) with a detection limit of 10 fmol.
Regarding sample preparation for analysis by MS, the use of adequate isotopically labeled internal standards represents an important issue; this step improves quantification of the hormone and underestimation of actual levels of melatonin due to losses which might have occurred in the samples during extraction.
Finally, the correct handling and maintenance of samples is also important. Samples of melatonin should be kept constantly on ice and protected from light radiation, in order to avoid degradation. Despite its relative stability, melatonin oxidation can occur over time, including reactions with singlet oxygen. The probability of this occurrence varies, and is dependent on oxygen availability and light incidence. For sample freezing, it is recommended that samples be dried and preferentially kept under vacuum or nitrogen atmosphere.
Immunoassay
For the monitoring of melatonin in biological fluids, use of immunological methods is the most widespread method. Several commercial kits based on these methods are available for melatonin determination. Some of these methods are highly sensitive and simple to use (lower limit of detection: 0.5 pg/ml) but may suffer from a potential risk of cross-reactivity to structurally similar compounds if melatonin is not extracted [76, 77].
The most crucial aspect of immunoassays is the preparation of the antiserum. Because melatonin is too small to be capable of producing antisera on its own it must be coupled to an antigenic protein. In such a conjugate the small molecular weight substance is called a hapten. The resulting antiserum binds both the protein and the hapten plus a portion of the adjacent protein. The hapten has few antigenic determinants relative to the protein. Specificity studies of antisera produced by steroid–protein conjugates have shown that antisera are not able to discriminate structural differences in the hapten that are immediately at or close to the site of coupling [78].
The choice of the hapten and conjugation reaction should therefore be determined by the type of discrimination that is required. Indolealkylamines have in common a ring nitrogen (position 1) and an adjacent carbon (position 2). Thus for melatonin, coupling via the position 1 or position 2 should allow resulting antisera to discriminate different indoles that are commonly found in tissues.
Studies of antisera resulting from Mannich coupling of melatonin to bovine serum albumin (BSA) have revealed that this approach leads to a highly specific melatonin antiserum as shown by cross-reactivity studies in radioimmunoassay (RIA) [76, 79–81]. To determine the locus of attachment of melatonin to protein, model reactions have been conducted and the resulting products analyzed by nuclear magnetic resonance and infrared spectroscopy [78]. The results of the study indicated that coupling was likely at position 2. Further studies were done of cross-reactivity of intermediate reaction products revealing that the highest cross-reactivity occurred with C-2 substituted melatonin derivatives. Thus it was concluded that the methylene bridge conjugating melatonin to BSA occurred at the number 2 position of the indole molecule. This approach has been used widely for melatonin immunoassays. Recently, two different groups have used this approach for generating monoclonal antisera against melatonin [81, 82].
Coupling at the ring nitrogen using 1-(p-carboxybenzyl)-melatonin [83] coupled to BSA as antigen [75] results in antisera that bind melatonin specifically. Melatonin-1-propionic acid coupled to BSA also stimulates production of highly specific antisera. A similar approach by coupling 1-(4-carboybutyl)-melatonin to protein resulted in a highly specific RIA [84]. The melatonin derivative, 3-(3-(2-acetamidoethyl)-5-methoxy indol-l-yl) propionic acid coupled to bovine gamma globulin produces a specific antiserum that has been used widely in RIA [85, 86]. Yet another derivative N-[3-(2-aminoethyl)-5-methoxy indole] hemisuccinamide has been used to generate antiserum as the basis for a specific RIA [87]. Thus melatonin coupled at the N position gives rise to antisera that are highly specific for melatonin as compared to other indoles.
Coupling at the side chain has also successfully produced useable melatonin antiserum. The methods used include N-acetyl-5-methoxytryptophan coupled using carbodiimide [88, 89], succinyl-5-methoxy-tryptamine coupled to protein [90] and indomethacin coupled to protein [91]. Melatonin coupled via a diazo linkage has also been reported to produce a reasonably specific antiserum, however the sensitivity of the resulting assay was found to be low [92, 93].
Coupling of N-acetylserotonin using formaldehyde generates antiserum that binds melatonin and N-acetylserotonin equally; cross-reactivity studies and model reactions have shown that coupling occurs at the 4th position of the molecule [94, 95]. The resulting antiserum has been used as the basis of an RIA that required prior extraction and column chromatography to eliminate the cross-reacting indole [95].
The chief metabolite of melatonin in urine, 6-sulfatoxymelatonin (6- hydroxymelatonin sulfate) has also been measured by immunologic means. The antiserum typically used for this assay is generated by use of the Mannich reaction and is highly specific [94, 96]. Antisera produced using these approaches have been used extensively not only for RIA, but also for immunohistochemistry and for enzyme-linked immunoassays (ELISA).
Radioimmunoassay
The principle of RIA method for melatonin measurement is that a known amount of radioactive melatonin (2-I125-iodomelatonin or 3H-melatonin) is mixed with a fixed amount of antibody raised against melatonin. Increasing concentrations of unlabeled melatonin are added to the mixture, which will compete with labeled melatonin causing its displacement from the antibody. Free labeled melatonin is then separated from remaining antibody-bound radioactive melatonin and radioactivity is measured. As the concentration of unlabeled melatonin increases in the mixture, competition for the antibodies also increases and bound labeled melatonin decreases. A calibration curve constructed from known amounts of labeled and unlabeled melatonin allows the determination of unknown melatonin concentrations in biological samples.
Fraser et al. [97] described a protocol for melatonin measurement by RIA in plasma that has been adopted by several researchers, some with slight modifications. Briefly, 200 μl of 1000-fold diluted antibody is added to 500 μl of solutions containing different amount of melatonin standard (2.5 to 250.0 pg). The solution is vortexed and kept at room temperature for 30 min. 3H-melatonin is added to the tubes (100 μl, 4,000 cpm), mixed, and kept at 4°C for 18 h. Then, 0.5 ml of Dextran-coated charcoal solution (0.1 g of dextran 75 plus 10 g of charcoal per 500 ml of buffer) is added and the solution is centrifuged for 15 min at 1,500 × g and 4°C, in order to separate the antibody-bound melatonin from the free fraction. The supernatant fraction is finally decanted into 10 ml of scintillation fluid and radioactivity is counted on a beta scintillator counter [97].
Several variations in RIA methods have been described, by using different antibodies (as noted above), by changing 3H-melatonin to 2-I125-iodomelatonin, or by altering the separation procedure. In general, because of its higher specific activity 2-I125-iodomelatonin allows a lower detection limit thus allowing the use of a smaller amount of sample. The concentration of melatonin during daylight can be as low as 0.2 to 0.3 fM [74]. This could be especially important if measurements are not preceded by melatonin purification. However, 125I is more prone to nonspecific binding so that some determinations can be faulty.
Sieghart et al. [68] reported that prior melatonin purification from plasma using reversed-phase column chromatography greatly reduces the problems of cross-reactivity. Moreover, Rolčik et al. [74] used immunoaffinity chromatography employing specific antisera to process samples prior to HPLC-MS analysis. Nonetheless, it should be recognized that even a weak cross-reactivity can be a problem if the cross-reacting molecule is present in large quantities. Thus independent validation of the procedure is essential when a different matrix is assayed.
One example of such a different matrix is saliva for which several RIAs have been described [98–101]. To obtain saliva, different methods have been used, from chewing gum, chewing on cotton swabs, or using commercial apparatus. Again, extraction is usually essential especially since levels in saliva are about 40% of those in plasma. Saliva is particularly useful if repeated sampling is required: for example to characterize the full 24 h rhythm of melatonin or to determine the dim light melatonin onset (DLMO), a measure that has been shown to be very useful in studies on circadian rhythmicity in sleep disorders [30, 102, 103].
Several variants of the time-consuming charcoal separation procedure have been developed and successfully applied. In the so-called scintillation proximity assay, the melatonin antibody is bound to a secondary antibody (e.g., antisheep) attached to scintillator-containing microbeads (“fluomicrospheres”) [104]. This relatively convenient procedure depends, however, usually on the commercial availability of suitable fluomicrospheres, since preparation and standardization of such beads is too time-consuming for the average laboratory. In the proximity assay, bound radioactivity is detected directly by the scintillator system of the microspheres. For physical and geometrical reasons, such a system has to have a lower scintillation efficiency than a homogeneous scintillation cocktail. However, this procedure has other advantages. Apart from being more rapid, the system is less affected by nonspecific binding (values close to background) such as occur in the charcoal procedure, has a better reproducibility and shows a much lower assay drift upon repetitive measurements (proximity assay: about 10% change within 84 h; charcoal method: about 25% over the same period) [104]. Other variants include separation using a double antibody procedure [77] and ammonium sulfate precipitation [34].
Considerable interest has also been shown in the major urinary metabolite of melatonin, 6-sulfatoxymelatonin [96]. The 24-h pattern of excretion of the metabolite accurately reflects the pattern of melatonin in blood [105, 106]. RIAs for this substance are available and have been useful in assessing pineal function in various conditions [49, 50, 107–110].
Enzyme-linked immunoassay
A variety of ELISAs for melatonin have also been reported that employ antisera identical to those used in the RIA described above. One such immunoassay employed melatonin–hemisuccinate–human serum albumin absorbed on polystyrene spheres, with the melatonin competing for a fixed amount of peroxidase labeled IgG antibody to melatonin [111]. This method had a detection limit of 22 fmol per tube and therefore required extraction. A competitive solid phase ELISA for human and rat serum and rat pineal gland has been described and validated using microtiter plates that has a much lower detection limit (1.0 fmol per well) as well as precision comparable with other methods and that can be applied without extraction to rat serum [112]. An improved version of this assay with a shorter incubation time was subsequently reported [113]. A comparative study of an RIA and a commercial ELISA reported that the ELISA required a purification step to be valid when applied to human serum, a step that was not part of the procedure recommended by the manufacturer [114]. With the extraction step, the assay had distinct advantages, Enzyme assays have major advantages in that the enzyme conjugate is stable, is more convenient than 3H or 125I and present no problem with disposal of radioactive waste. Furthermore if microtiter plates are used centrifugation is not necessary. Although not an enzyme immunoassay, it is of interest that a time resolved fluoroimmunassay has also been described [115]. An enzyme immunoassay for 6-sulfatoxymelatonin has been reported [116], and commercial kits are available.
HPLC coupled to electrochemical and fluorescence detection
In many studies, RIA methodology has been replaced by HPLC with electrochemical and fluorescence detection for melatonin evaluation, due to its great sensitivity and specificity [117]. However, this procedure is more adequate for melatonin alone, and not for mixtures of several indoles, such as serotonin and tryptamine among others, that can cause disruptions in the assay. For example, serotonin/melatonin ratio is higher than 100 in rat pineal. This high ratio can cause disturbances in chromatographic separations that can make melatonin detection difficult, and thus requires a good procedure for melatonin extraction. However, the avoidance of partial coelution with other indoles is mostly a matter of the art of chromatography. In our work we have been able to detect melatonin with great accuracy in blood plasma after simple dichlorometane extraction as described above, and using an HPLC system connected to electrochemical detection. Figure 1 shows a chromatogram of melatonin detection in human plasma by this procedure. Good peak separation was achieved by using a LC-18 column and 50 mM sodium acetate—100 mM acetic acid (pH 4.3), 0.1 mM Na2-EDTA, and acetonitrile (75:25, v/v) as mobile phase pumped isocratically at 1 ml/min.
📷Fig. 1
Chromatogram of melatonin standard (2 pmol; a) and melatonin extracted from 2 ml of a plasma sample (b), by HPLC with coulometric electrochemical detector
Harumi et al. [69] also successfully determined melatonin by HPLC with electrochemical detection, with very clear peak separation for different indoleamines among melatonin. However, the sensitivity of this procedure depends on the model of electrochemical cell. Amperometric-based electrochemical cells are generally less sensitive than coulometric cells, so that the adequate potential should be previously optimized by the construction of hydrodynamic voltammograms. With our coulometric electrochemical system, the best melatonin signal is obtained at 600 mV. Sensitivity can be also greater with coulometric electrochemical detectors such as the ESA coulochem III model (ESA, Bedford, MA, USA), which uses porous electrochemical cells that allow greater accuracy in melatonin peak resolution. Harumi et al. reported the use of a higher potential, 900 mV, for good melatonin signal with their graphite carbon working electrode, and even so they detected melatonin at very low levels [69]. Rizzo et al. [66] also used 900 mV for melatonin detection with an amperometric electrochemical detector.
With respect of fluorescence detection, some highly sensitive methodologies have been reported for melatonin detection at the femtomole level [118–120]. Melatonin can be separated on a C18 column by using 75 mM sodium acetate pH 5.0 and acetonitrile (72:28, v/v) as the mobile phase pumped isocratically at 1.0 ml/min, and directly detected by setting up the fluorescence detector at an excitation wavelength of 275 nm and an emission wavelength of 345 nm [66]. Nevertheless, in some cases in which melatonin concentration is very low, derivatization is recommended to enhance the melatonin signal [118]. An oxidation procedure that can enhance melatonin fluorescence by 6.8 times (allowing its determination at attomole levels) has been described using biological samples [73]. Melatonin was oxidized to a new fluorescent compound with sodium carbonate and hydrogen peroxide. However, precautions should be taken when using this kind of approach, because other components in the biological sample may lead to the generation of fluorophores, which in turn could interfere with the determination of the correct level, thus preventing method specificity [73].
In any case, care with sample preparation can improve the melatonin signal. Prepurification of melatonin as described before will decrease chromatogram noise and avoid the coelution of melatonin with other compounds that can interfere with melatonin peaks. Generally, the use of fluorescence techniques are affected not only by co-elution with other fluorescent compounds in the sample, but also by the presence of quenchers. This should not be underrated since the majority of aromates absorb around the excitation maximum of melatonin. Therefore, samples should be tested in advance for quenching by adding known amounts of melatonin.
Mass spectrometry
The GC-MS technique is very sensitive and offers more specificity than HPLC with electrochemical or fluorescence detectors; however, a difficulty with this technique is the need of derivatization, and thus it has been gradually substituted by liquid chromatography-mass spectrometry procedures. Thus, alternative HPLC-MS methods appropriate for use in biological issues have been developed [119, 121–123]. However, this approach is limited by the need of adequate internal standards. Yang et al. described their methodology which used acetyltryptamine as the internal standard [119]; however, several factors make this approach less than ideal. It is appropriate to use a labeled internal standard whose structure is the same of the analyte except for the mass difference. The addition of an isotopically labeled internal standard prior to the analysis improves the method’s confidence level.
Another analytical method has been developed which uses column-switching semi-microcolumn liquid chromatography/mass spectrometry and selected ion monitoring (SIM) for detecting endogenous melatonin in human saliva. In the relevant study melatonin was monitored based on its fragment ion at m/z 174 by in-source dissociation and using deuterated melatonin as the internal standard, and a detection limit of 10 fmol was obtained [74]. The main limitation of this methodology is the use of the SIM mode to detect the ions generated in the probe, which does not imply an absolute specificity. Yet, Eriksson et al. [123] reported a method for the determination of melatonin in human saliva by HPLC-MS/MS, using 7-D-melatonin as internal standard. The limit of detection was 1.05 pg/ml and the limit of quantification was 3.0 pg/ml. One of us has reported the development of a new HPLC-MS/MS assay with electrospray ionization (ESI) to quantitatively determine melatonin and also its degradation product N1-acetyl-N2-formyl-5-methoxykynuramine with high sensitivity and specificity [122]. A stable isotopic internal standard melatonin-D3 (deuterated melatonin) was easily synthesized by the reaction of 5-methoxytryptamine with deuterated acetyl chloride (CD3COCl) (Fig. 2).
📷Fig. 2
Synthesis of labeled D3-melatonin for its use as internal standard during melatonin measurement in plasma samples
The predominant ion [M+H]+ in the full scan mass spectra of melatonin, and melatonin-D3 were located (Fig. 3a–b). The fragments generated in collision-induced dissociation chamber revealed a predominant fragment at m/z = 174 for melatonin and melatonin-D3 (loss of the N-acetyl group) (Fig. 3c–d). The m/ztransitions from 233 to 174 (melatonin) and from 236 to 174 (melatonin-D3) were therefore chosen for the Multiple Reaction Monitoring (MRM) detection experiments, which ensured a higher specificity and an accurate quantification of melatonin in human plasma (Fig. 3e). A chromatogram of melatonin and deuterated-melatonin is depicted in Fig. 4.
📷Fig. 3
Full mass scan spectra of melatonin (a) and deuterated melatonin (b) in the ESI+ mode, and their respective daughters (c and d) after fragmentation in the collision cell of the mass spectrometer. e A chromatogram of melatonin from a human plasma sample (MEL) and deuterated melatonin (MELD3) as internal standard by HPLC-MS/MS
Other techniques
Some laboratories have taken and developed capillary electrophoresis (CE) for the separation and determination of melatonin in blood plasma [124–126] and in pineal gland [127, 128]. Detection of analyte was performed with a UV and fluorescence [124–126, 129] or electrochemical detector [127, 128]. The detection limit of melatonin with CE is comparable with the data obtained by HPLC methods reported previously. Recently, for the separation of melatonin from related compounds, CE with micellar electrokinetic chromatography was applied [125, 126, 128, 129]. This technique permitted the effective separation of melatonin and its precursors or metabolites. Sodium dodecyl sulfate is used to produce a pseudostationary phase.
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Conclusions
The most common methods for determination of melatonin in blood or saliva are RIAs and ELISAs, and several commercial kits are now available for these assays. They are convenient to use, especially the enzyme based assays, but of the possibility of cross-reactions, and nonspecific effects must be considered. This is important because of the very low levels of melatonin that are to be measured. These potential problems can be reduced by determining when extraction is necessary and by comparing the results obtained with other established methods. It is expected that these methods will continue to improve and that enzyme assays will continue to gain ground for routine measurements of melatonin in blood and saliva.
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Acknowledgments
Part of this work (Paolo Di Mascio) was supported by the Brazilian research funding institutions FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), CNPq (Conselho Nacional para o Desenvolvimento Científico e Tecnológico), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), L'ORÉAL-UNESCO for Women in Science (S.M. Fellow) and The John Simon Memorial Guggenheim Foundation (P.D.M. Fellow), and INCT de Processos Redox em Biomedicina – Redoxoma.
D.P.C. is a Research Career Awardee from the Argentine National Research Council (CONICET), Argentina.
Conflicts of interest S.R. Pandi-Perumal is a stockholder and the President and Chief Executive Officer of Somnogen Inc., a New York Corporation. He declared no competing interests that might be perceived to influence the content of this article.
Disclosures All remaining authors declare that they have no proprietary, financial, professional, nor any other personal interest of any nature or kind in any product or services and/or company that could be construed or considered to be a potential conflict of interest that might have influenced the views expressed in this manuscript.
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Somnolence associated with melatonin deficiency after pinealectomy. Lancet. 1996;347:323. [PubMed]63. Muller HL, Handwerker G, Wollny B, Faldum A, Sorensen N. Melatonin secretion and increased daytime sleepiness in childhood craniopharyngioma patients. J Clin Endocrinol Metab. 2002;87:3993–3996. [PubMed]64. Muller HL, Handwerker G, Gebhardt U, Faldum A, Emser A, Kolb R, Sorensen N. Melatonin treatment in obese patients with childhood craniopharyngioma and increased daytime sleepiness. Cancer Causes Control. 2006;17:583–589. [PubMed]65. Lipton J, Megerian JT, Kothare SV, Cho YJ, Shanahan T, Chart H, Ferber R, Adler-Golden L, Cohen LE, Czeisler CA, Pomeroy SL. Melatonin deficiency and disrupted circadian rhythms in pediatric survivors of craniopharyngioma. Neurology. 2009;73:323–325. [PMC free article] [PubMed]66. Rizzo V, Porta C, Moroni M, Scoglio E, Moratti R. Determination of free and total (free plus protein-bound) melatonin in plasma and cerebrospinal fluid by high-performance liquid chromatography with fluorescence detection. J Chromatogr B Anal Technol Biomed Life Sci. 2002;774:17–24. [PubMed]67. Harumi T, Matsushima S. Separation and assay methods for melatonin and its precursors. J Chromatogr B Biomed Sci Appl. 2000;747:95–110. [PubMed]68. Sieghart W, Ronca E, Drexler G, Karall S. Improved radioimmunoassay of melatonin in serum. Clin Chem. 1987;33:604–605. [PubMed]69. Harumi T, Akutsu H, Matsushima S. Simultaneous determination of serotonin, N-acetylserotonin and melatonin in the pineal gland of the juvenile golden hamster by high-performance liquid chromatography with electrochemical detection. J Chromatogr B Biomed Appl. 1996;675:152–156. [PubMed]70. Degen PH, DoAmaral JR, Barchas JD. A gas-liquid chromatographic assay of melatonin and indoleamines using heptafluorobutyryl derivatives. Anal Biochem. 1972;45:634–644. [PubMed]71. Covaci A, Doneanu C, Aboul-Enein HY, Schepens P. Determination of melatonin in pharmaceutical formulations and human plasma by gas chromatography-electron impact mass spectrometry. Biomed Chromatogr. 1999;13:431–436. [PubMed]72. Bechgaard E, Lindhardt K, Martinsen L. High-performance liquid chromatographic analysis of melatonin in human plasma and rabbit serum with on-line column enrichment. J Chromatogr B Biomed Sci Appl. 1998;712:177–181. [PubMed]73. Tomita T, Hamase K, Hayashi H, Fukuda H, Hirano J, Zaitsu K. Determination of endogenous melatonin in the individual pineal glands of inbred mice using precolumn oxidation reversed-phase micro-high-performance liquid chromatography. Anal Biochem. 2003;316:154–161. [PubMed]74. Rolčik J, Lenobel R, Siglerova V, Strnad M. Isolation of melatonin by immunoaffinity chromatography. J Chromatogr B Anal Technol Biomed Life Sci. 2002;775:9–15. [PubMed]75. Grota LJ, Snieckus V, Silva SO, Tsui HW, Holloway WR, Lewy AJ, Brown GM. Radioimmunoassay of melatonin in rat serum. Prog Neuropsychopharmacol. 1981;5:523–526. [PubMed]76. Lemaitre BJ, Hartmann L. Preparation of anti-melatonin antibodies and antigenic properties of the molecule. J Immunol Methods. 1980;32:339–347. [PubMed]77. Di WL, Kadva A, Djahanbakhch O, Silman R. Radioimmunoassay of bound and free melatonin in plasma. Clin Chem. 1998;44:304–310. [PubMed]78. Grota LJ, Snieckus V, Silva SO, Brown GM. Antibodies to indolealkylamines II: site of conjugation of melatonin to protein using formaldehyde. Can J Biochem Cell Biol. 1983;61:1096–1101. [PubMed]79. Pang SF, Brown GM, Grota LJ, Chambers JW, Rodman RL. Determination of N-acetylserotonin and melatonin activities in the pineal gland, retina, harderian gland, brain and serum of rats and chickens. Neuroendocrinology. 1977;23:1–13. [PubMed]80. Bubenik GA, Brown GM, Grota LJ. Immunohistochemical localization of melatonin in the rat Harderian gland. J Histochem Cytochem. 1976;24:1173–1177. [PubMed]81. Yang T, Wang J, Qu L, Zhong P, Yuan Y. Preparation and identification of anti-melatonin monoclonal antibodies. J Pineal Res. 2006;40:350–354. [PubMed]82. Soukhtanloo M, Ansari M, Paknejad M, Parizadeh MR, Rasaee MJ. Preparation and characterization of monoclonal antibody against melatonin. Hybridoma. 2008;27:205–209. [PubMed]83. Silva SO, Snieckus V. Indole-N-alkylation of tryptamines via dianion and phtalimido intermediates. New potential indolealkylamine hapten. Can J Chem. 1978;56:1621–1628.84. Kawashima K, Nagakura A, Wurzburger RJ, Spector S. Melatonin in serum and the pineal of spontaneously hypertensive rats. Clin Exp Hypertens A. 1984;6:1517–1528. [PubMed]85. Blair IA, Seaborn CJ. The synthesis of melatonin antigens. Aust J Chem. 1979;32:399–403.86. Kennaway DJ, Gilmore TA, Seamark RF. Effects of melatonin implants on the circadian rhythm of plasma melatonin and prolactin in sheep. Endocrinology. 1982;110:2186–2188. [PubMed]87. Manz B, Seidel A, Alexander H, Vollrath L, Wagner B, Zimmermann G, Wiedemann K, Pollow K. Development and validation of a radioimmunoassay for serum melatonin. J Clin Chem Clin Biochem. 1989;27:797–802. [PubMed]88. Wilkinson M, Arendt J, Bradtke J, Ziegler D. Determination of a dark-induced increase of pineal N-acetyl transferase activity and simultaneous radioimmunoassay of melatonin in pineal, serum and pituitary tissue of the male rat. J Endocrinol. 1977;72:243–244. [PubMed]89. Vakkuri O, Leppaluoto J, Vuolteenaho O. Development and validation of a melatonin radioimmunoassay using radioiodinated melatonin as tracer. Acta Endocrinol. 1984;106:152–157.[PubMed]90. Rollag MD, Niswender GD. Radioimmunoassay of serum concentrations of melatonin in sheep exposed to different lighting regimens. Endocrinology. 1976;98:482–489. [PubMed]91. Levine L, Riceberg LJ. Radioimmunoassay for melatonin. Res Commun Chem Pathol Pharmacol. 1975;10:693–702. [PubMed]92. Wurzburger RJ, Kawashima K, Miller RL, Spector S. Determination of rat pineal gland melatonin content by a radioimmunoassay. Life Sci. 1976;18:867–877. [PubMed]93. Lynch HJ, Ozaki Y, Shakal D, Wurtman RJ. Melatonin excretion of man and rats: effect of time of day, sleep, pinealectomy and food consumption. Int J Biometeorol. 1975;19:267–279. [PubMed]94. Grota LJ, Brown GM. Antibodies to indolealkylamines: serotonin and melatonin. Can J Biochem. 1974;52:196–202. [PubMed]95. Kennaway DJ, Frith RG, Phillipou G, Matthews CD, Seamark RF. A specific radioimmunoassay for melatonin in biological tissue and fluids and its validation by gas chromatography-mass spectrometry. Endocrinology. 1977;101:119–127. [PubMed]96. Arendt J, Bojkowski C, Franey C, Wright J, Marks V. Immunoassay of 6-hydroxymelatonin sulfate in human plasma and urine: abolition of the urinary 24-hour rhythm with atenolol. J Clin Endocrinol Metab. 1985;60:1166–1173. [PubMed]97. Fraser S, Cowen P, Franklin M, Franey C, Arendt J. Direct radioimmunoassay for melatonin in plasma. Clin Chem. 1983;29:396–397. [PubMed]98. English J, Middleton BA, Arendt J, Wirz-Justice A. Rapid direct measurement of melatonin in saliva using an iodinated tracer and solid phase second antibody. Ann Clin Biochem. 1993;30(4):415–416.[PubMed]99. Miles A, Philbrick D, Tidmarsh SF, Shaw DM. Direct radioimmunoassay of melatonin in saliva. Clin Chem. 1985;31:1412–1413. [PubMed]100. Miles A, Philbrick DR, Grey JE. Salivary melatonin estimation in assessment of pineal-gland function. Clin Chem. 1989;35:514–515. [PubMed]101. Vakkuri O. Diurnal rhythm of melatonin in human saliva. Acta Physiol Scand. 1985;124:409–412.[PubMed]102. Lewy AJ, Sack RL. The dim light melatonin onset as a marker for circadian phase position. Chronobiol Int. 1989;6:93–102. [PubMed]103. Leibenluft E, Feldman-Naim S, Turner EH, Schwartz PJ, Wehr TA. Salivary and plasma measures of dim light melatonin onset (DLMO) in patients with rapid cycling bipolar disorder. Biol Psychiatry. 1996;40:731–735. [PubMed]104. Poeggeler B, Huether G. Versatile one-tube scintillation proximity homogeneous radioimmunoassay of melatonin. Clin Chem. 1992;38:314–315. [PubMed]105. Markey SP, Higa S, Shih M, Danforth DN, Tamarkin L. The correlation between human plasma melatonin levels and urinary 6-hydroxymelatonin excretion. Clin Chim Acta. 1985;150:221–225.[PubMed]106. Nowak R, McMillen IC, Redman J, Short RV. The correlation between serum and salivary melatonin concentrations and urinary 6-hydroxymelatonin sulphate excretion rates: two non-invasive techniques for monitoring human circadian rhythmicity. Clin Endocrinol. 1987;27:445–452. [PubMed]107. Cavallo A, Dolan LM. 6-Hydroxymelatonin sulfate excretion in human puberty. J Pineal Res. 1996;21:225–230. [PubMed]108. Martin PR, Higa S, Burns RS, Tamarkin L, Ebert MH, Markey SP. Decreased 6-hydroxymelatonin excretion in Korsakoff’s psychosis. Neurology. 1984;34:966–968. [PubMed]109. John TM, Brown MC, Brown GM. An oral melatonin replacement regimen that re-establishes the normal circadian levels of urinary 6-sulphatoxymelatonin in functionally pinealectomized rats. J Pineal Res. 1992;13:145–150. [PubMed]110. Fideleff HL, Boquete H, Fideleff G, Albornoz L, Pérez Lloret S, Suarez M, Esquifino AI, Honfi M, Cardinali DP. Gender-related differences in urinary 6-sulfatoxymelatonin levels in obese pubertal individuals. J Pineal Res. 2006;40:214–218. [PubMed]111. Ferrua B, Masseyeff R. Immunoassay of melatonin with enzyme-labeled antibodies. J Immunoassay. 1985;6:79–94. [PubMed]112. Yie SM, Johansson E, Brown GM. Competitive solid-phase enzyme immunoassay for melatonin in human and rat serum and rat pineal gland. Clin Chem. 1993;39:2322–2325. [PubMed]113. Shavali S, Samejima M, Uchida K, Morita Y, Fukuda A. Improved enzyme immunoassay method for melatonin: application to the determination of serum melatonin in rats, sheep, and humans. Clin Chem. 1999;45:690–692. [PubMed]114. Chegini S, Ehrhart-Hofmann B, Kaider A, Waldhauser F. Direct enzyme-linked immunosorbent assay and a radioimmunoassay for melatonin compared. Clin Chem. 1995;41:381–386. [PubMed]115. Yamada H, Chiba H, Amano M, Iigo M, Iwata M. Rainbow trout eyed-stage embryos demonstrate melatonin rhythms under light-dark conditions as measured by a newly developed time-resolved fluoroimmunoassay. Gen Comp Endocrinol. 2002;125:41–46. [PubMed]116. Peniston-Bird JF, Di WL, Street CA, Edwards R, Little JA, Silman RE. An enzyme immunoassay for 6-sulphatoxy-melatonin in human urine. J Pineal Res. 1996;20:51–56. [PubMed]117. Vitale AA, Ferrari CC, Aldana H, Affanni JM. Highly sensitive method for the determination of melatonin by normal-phase high-performance liquid chromatography with fluorometric detection. J Chromatogr B Biomed Appl. 1996;681:381–384. [PubMed]118. Iinuma F, Hamase K, Matsubayashi S, Takahashi M, Watanabe M, Zaitsu K. Sensitive determination of melatonin by precolumn derivatization and reversed-phase high-performance liquid chromatography. J Chromatogr A. 1999;835:67–72. [PubMed]119. Yang S, Zheng X, Xu Y, Zhou X. Rapid determination of serum melatonin by ESI-MS-MS with direct sample injection. J Pharm Biomed Anal. 2002;30:781–790. [PubMed]120. Simonin G, Bru L, Lelievre E, Jeanniot JP, Bromet N, Walther B, Boursier-Neyret C. Determination of melatonin in biological fluids in the presence of the melatonin agonist S 20098: comparison of immunological techniques and GC-MS methods. J Pharm Biomed Anal. 1999;21:591–601. [PubMed]121. Motoyama A, Kanda T, Namba R. Direct determination of endogenous melatonin in human saliva by column-switching semi-microcolumn liquid chromatography/mass spectrometry with on-line analyte enrichment. Rapid Commun Mass Spectrom. 2004;18:1250–1258. [PubMed]122. Almeida EA, Klitzke CF, Martinez GR, Medeiros MHG, Mascio P. Synthesis of internal labeled standards of melatonin and its metabolite N1-acetyl-N2-formyl-5-methoxykynurenin for their quantification using an on-line liquid chromatography-electrospray tandem mass spectrometry system. J Pineal Res. 2004;36:64–71. [PubMed]123. Eriksson K, Ostin A, Levin JO. Quantification of melatonin in human saliva by liquid chromatography-tandem mass spectrometry using stable isotope dilution. J Chromatogr B Anal Technol Biomed Life Sci. 2003;794:115–123. [PubMed]124. Kim YO, Chung HJ, Chung ST, Kim JH, Park JH, Han SY, Kil KS, Cho DH. Determination of melatonin in biological samples by capillary electrophoresis. J Chromatogr A. 1999;850:369–374.[PubMed]125. Pobozy E, Michalski A, Sotowska-Brochocka J, Trojanowicz M. Determination of melatonin and its precursors and metabolites using capillary electrophoresis with UV and fluorometric detection. J Sep Sci. 2005;28:2165–2172. [PubMed]126. Musijowski J, Pobozy E, Trojanowicz M. On-line preconcentration techniques in determination of melatonin and its precursors/metabolites using micellar electrokinetic chromatography. J Chromatogr A. 2006;1104:337–345. [PubMed]127. Chen G, Cheng J, Ye J. Application of a novel micro-injector in the determination of indole derivatives in the rat pineal gland by capillary electrophoresis with electrochemical detection. Fresenius J Anal Chem. 2001;370:930–934. [PubMed]128. Wu X, Wu W, Zhang L, Xie Z, Qiu B, Chen G. Micellar electrokinetic capillary chromatography for fast separation and sensitive determination of melatonin and related indoleamines using end-column amperometric detection. Electrophoresis. 2006;27:4230–4239. [PubMed]129. Hevia D, Botas C, Sainz RM, Quiros I, Blanco D, Tan DX, Gomez-Cordoves C, Mayo JC. Development and validation of new methods for the determination of melatonin and its oxidative metabolites by high performance liquid chromatography and capillary electrophoresis, using multivariate optimization. J Chromatogr A. 2010;1217:1368–1374. [PubMed]
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can any one knows what solubility of melatonin in 0.1 N HCL?
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The EXPERIMENTAL intrinsic solubility of melatonin was reported by Benet LZ, Broccatelli F, Oprea TI. BDDCS applied to over 900 drugs. AAPS J. 13 (2011) 519-547 as 0.43 mM. I have not been able to find any other references to an experimental value. Since melatonin is generally considered a non-ionizable molecule, its solubility in 0.1 M HCl medium is expected to be 0.10 mg/mL. Using pDISOL-X v.3 (in-ADME Research), the PREDICTED value is 0.32 (25`C) and 0.38 (37`C) mg/mL.
Andrei - How did you calculate the value you suggested?
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Melatonin levels are found to be lowered before epileptic episode and increased after epilepsy. Is these some relationship between melatonin levels and epileptic activity?
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Dear Rimple
yes definitely there are some relationships between epilepsy and melatonin. there are different interfaces between epilepsy and melatonin. One is sleep- epilepsy and melatonin. Melatonin inproved sleep, epileptic activities impairs sleep. Melatonin can modulate circadian rhythm, sleep, mood, ageing, tumour growth, etc.
some studies showed melatonin can be used for treatment of some types of epilepsy. The below is the link for some good papees.
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I'm looking for either an RIA or ELISA kit that works reliably with mouse plasma. Max blood per sample is 1 mL (500uL plasma). ELISA would be preferred. Any recommendations?
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Thank you!
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I implanted a number of passerines with melatonin packed silastic capsules (subq). However, the birds showed no increase in plasma melatonin levels at the end of the experiment (implants were in place for 1-1.5months). The implants also still appeared to contain melatonin when explanted (they weren't empty). Anyone have any advice on what might have gone wrong or possible adjustments to make?
Thank you in advance
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I appreciate the multifaceted physiological role melatonin plays in an animal, but disrupting the circadian rhythm is the point of the implant.
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I want to make solution of melatonin for my research( doses of 20mg/kg ip injection) please guide me for making this solution what should I do?
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The usual way is: take 1 mg of melatonin and add 40 ul of ethyl alcohol; shake and and 960 ul of water or saline as your convenience. That is a mother solution. From there you go to the final solution you need.
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I am looking for an appropriate literature source regarding the halftime (lifespan) of gut mucosal cells in mammals.
Does anyone can suggest a paper or book section, respectively?
Thank you very much in advance!
Best wishes,
Daniel.
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Many sources state ~4 days for the epithelium. Paneth cells may be different.
search at bio numbers.hms.harvard.edu using these numbers as search terms: 107812 and 109231.
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A simple, rapid protocol for determination of melatonin in plant tissues?
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Please check these useful PDF attachments.
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I want to check the effect of melatonin on TK6 cells. I do not want to use DMSO in experiment.
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I think there was once a paper or part of a paper describing that melatonin was first dissolved in ethanol (in a tube), let the ethanol evaporate, add water, vortex it for some time, and at the end you had melatonin in water at the desired concentration. Indeed, I looked it up: http://onlinelibrary.wiley.com/doi/10.1111/j.1600-079X.1994.tb00102.x/full
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There is some research indicating possible mitochondrial dysfunction in Psychosis.  Currently a client with psychosis has been given melatonin  for sleep in combination with therapies done by myself - Cognitive restructuring, talking to voices and cognitive therapy. This was after ALL normal (medical and psychotherapeutic) treatments had been applied for a 2 year period with little success before I received the client. The only two differences in treatment was talking to voices and melatonin prescribed by the doctor. Naturally this is not enough to propose that melatonin may have an impact, however there was a distinct improvement in cognitive processing abilities. Has anyone done any trials or research on the use of melatonin. Studies suggest that melatonin may or may not partially or fully repair mitochondria, whilst no research has been found on the possible utilisation of Gh.
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Some research from my area of interest (gynecologic psychosomatics): Anderson G. The role of melatonin in post-partum psychosis and depression associated with bipolar disorder. J Perinat Med. 2010 Nov;38(6):585-7. doi: 10.1515/JPM.2010.085 and from the area of general psychiatry: van Beveren NJ, Schwarz E, Noll R, Guest PC, Meijer C, de Haan L, Bahn S. Evidence for disturbed insulin and growth hormone signaling as potential risk factors in the development of schizophrenia. Transl Psychiatry. 2014 Aug
26;4:e430. doi: 10.1038/tp.2014.52. FULL TEXT)
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melatonin is a hormon and secreted by the pineal gland and is antioxidant so may not reverse at the over dose level
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Hi fellows,
Does any body know if the synthesis of melatonin differs between diurnal and nocturnal mammals? Diurnal species receive and configure their circadian system by interpreting the changes from diurnal to nocturnal levels of light, but what about nocturnal species? Do they follow the same path?
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Melatonin is synthesized and secreted during the dark period of the daily light/ dark  cycle, independent of whether the animal is diurnally or nocturnally active, and the duration of the nocturnal production is proportional to the length of the night. Generally, melatonin synthesis and its excretion increased in darkness in both humans and experimental animals. Constant light leads to diminishing activity of HIOMT in the rats and a reduction in the melatonin production of the pineal gland.
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Hi, may I know which part of the gene region you are targeting to control the circadian rhythm? What is the model organism used?
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Not a single gene, not a Gene but several genes (Per, Tim, Clock, Cry .... ) working in a very complex way.
The SCN generates circadian rhythms by means of a transcriptional-translational
feedback loop. In short, the mechanism is formed by the basic helix-loop-helix (bHLH) Per-Arnt-Sim (PAS) domain containing transcription factors circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), which activate the expressionof three Period (Per 1–3) and two Cryptochrome (Cry 1–2) genes by
binding to their E-box (5′-CACGTG-3′) promoter elements. The PERIOD
(PER 1–3) and CRYPTOCHROME (CRY1–2) proteins rhythmically accumulate,
heterodimerize, and translocate to the nucleus to suppress
their own transcription by interactionwith the CLOCK:BMAL1 complex.
CLOCK/BMAL1 also rhythmically control the expression of nuclear receptors,
such as REV-ERBα/β (reverse transcript of erythroblastosis
gene) and RORα/β/γ (retinoic acid related (RAR) orphan receptor),
which in turn repress and activate Bmal1 expression, respectively, conferring
amplitude and robustness to the oscillations in the molecular
clockwork. From a molecular point of view, light activates the expression
of several genes in SCN with different expression patterns
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I am curious from the point of view of those who may take sleep-aiding/soporific drugs to hasten adaptation to new time-zones?
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Thanks Tomás!
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Agomelatine is a melatonin (MT1/MT2) receptor agonist and a serotonin 5HT2C antagonist (increasing dopamine and norepinephrine in the frontal cortex). It is an antidepressant and it can also reduce anxiety and re-synchronize circadian rhythms. De Berardis et al (2012, Ann Clin Psychiatry) informed about a female patient with PTSD with substantial improvement after agomelatine treatment. In my clinical practice, I have successfully used agomelatine in the treatment of female patient with PTSD and comorbidity with a psychotic disorder. It is therefore possible that anxiolytic (and many other) properties of agomelatine can help to alleviate symptoms of PTSD.  
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I would advise to ask Servier Labs (they produce this drug) their bibliography on the theme.
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Is it possible that the value of circadian amplitude higher than circadian MESOR? or depends upon the variable such as physiological (blood pressure) or biochemical (ex. melatonin)?
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Hi, Babita 
Yes, it is quite a usual thing. The amplitude of a rhythm can be a higher value then mesor. The mesor is usually an estimate of statics of a wave and derived from the average of all variable samples, whereas amplitude is specific to a acrophase of the curve. I would suggest reading this paper would give better insight. 
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I mean is melatonin a direct cause in such issues or it is a result? Many papers and reviews reported a decrease in this neurohormone's concentration in patients suffering neurodegenrative diseases!  
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It seems that the deficiency of melatonin may be one of the factors increasing the risk of neurodegenerative disease development. Melatonin may be one of the main antioxidant factors present in the nervous system, so if it is in decreased levels there is not enough protection for the nervous tissue.
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Could you tell me if light stimulation might be an adequate treatment for sleep-related disorders?
For instance blue light influences melatonin production, what might be helpful to re-entrain the circadian rhythm. Do you know about some light treatment that is nowadays used clinically?
I will be most grateful if someone could give me some information!
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Dear Ta-wei Guu,
thank you for your answer! Do you think that light theraphy might be beneficial for people, who suffer from chronic fatigue syndrome, as well? Is it reasonable to apply a light therapy, just as a support to, for instance Cognitive Behavioral Therapy?
Kind regards
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Melatonin is derived from amino acid L-tryptophan. Is there any difference between ELISA specificity to the endogenous (naturally occurring) and the exogenous (drug or implants) melatonin?
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A melatonin implant will result in 24h melatonin levels and by that interfere with the endogenous circadian melatonin rhythm. This can have a negative influence on several metabolic processes that also have a 24h circadian rhythm.
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Hi, I am doing research on light, melatonin and the effects of melatonin on health. I couldn't find any systematic review or recent review on this topic. Can anyone please advise?
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Negative health effects of low melatonin levels are numerous. There are several indications that low melatonin levels are a CVD risk factor. Russel Reiter has published many papers on this subject:
Melatonin is a small, highly conserved indole with numerous receptor-mediated and receptor-independent actions. Receptor-dependent functions include circadian rhythm regulation, sleep, and cancer inhibition. The receptor-independent actions relate to melatonin's ability to function in the detoxification of free radicals, thereby protecting critical molecules from the destructive effects of oxidative stress under conditions of ischemia/reperfusion injury (stroke, heart attack), ionizing radiation, and drug toxicity, among others. Melatonin has numerous applications in physiology and medicine. (Reiter RJ et al. Melatonin: exceeding expectations. Physiology (Bethesda). 2014 Sep;29(5):325-33. doi: 10.1152/physiol.00011.2014.)
Also several other papers are on this topic:
Statistically significant inverse correlations were observed between urinary 6-sulfatoxymelatonin (aMT6s), the major metabolite of melatonin in urine, and WBC count, uric acid, and high-sensitivity C-reactive protein, being established independent risk factors for CVD, such as WBC, UA, and hs-CRP. Endogenous melatonin may have implications for the risk of CVD. (Masue T et al. Associations of urinary 6-sulfatoxymelatonin with biomarkers related to cardiovascular disease in Japanese women. Metabolism. 2012 Jan;61(1):70-5. doi: 10.1016/j.metabol.2011.05.021. Epub 2011 Aug 3.)
Women working rotating night shifts for ≥5 years have a modest increase in all-cause and CVD mortality; those working ≥15 years of rotating night shift work have a modest increase in lung cancer mortality. These results add to prior evidence of a potentially detrimental effect of rotating night shift work on health and longevity. (Gu F et al. Total and cause-specific mortality of U.S. nurses working rotating night shifts. Am J Prev Med. 2015 Mar;48(3):241-52. doi: 10.1016/j.amepre.2014.10.018. Epub 2015 Jan 6)
Another relation is low melatonin and breast cancer risk (i.e. nurses and night shift):
The results showed that melatonin not only inhibits breast cancer cell
growth, but also is capable of inhibiting angiogenesis, cancer cell invasion, and
telomerase activity. Interestingly this hormone is able to induce apoptosis
through the suppression or induction of a wide range of signaling pathways.
Moreover, it seems that the concomitant administration of melatonin with other
conventional chemotherapy agents had beneficial effects for patients with breast
cancer, by alleviating unfavorable effects of those agents and enhancing their
efficacy. (Nooshinfar E et al. Melatonin, an inhibitory agent in breast cancer. Breast Cancer. 2016 Mar 26. [Epub ahead of print])
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High or low level of melatonin has higher risk for CVD. Is there any optimal range for endogenous melatonin?
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It is rather easy: the optimal level is the one that you find in a healthy young adult individuum. Nature has found it over millions of years.
As you might know, the level changes drastically with day-time. So the optimal level has to adapt continuously to remain optimal.
Beware of too much melatonin at the wrong time of the day! Only during a certain phase of the night you really will need the melatonin: shortly after the first slow wave sleep period. This SWS phase is the time, when the immune cells produce their radical oxygen against noxious and cancer cells . Melatonin is a most effective anti-oxidant catching the free radicals that did not find their goal and would endanger healthy cells if they remained in the tissues. Earlier production of melatonin would catch the radicals before they can be active - so the right timing, just as in a healthy indiviual, is crucial.
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Hello!
As part of a laboratory project, my group and I are studying the effects of cytochrome p450 inhibitors (like insulin, grapefruit juice, etc.) on drug metabolism in Hepa1c1c7 cells. Are there any alternatives to expensive ELISA's that we could use to compare levels of estradiol in these cells? We would also be open to using other substrates such as melatonin, naproxen, caffeine, etc.
Thanks!
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The US EPA and NTP / ICCVAM have several assays available for estradiol testing.  Check out the following links and see which system is best for you.
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Rehabilitation normally consists of different types of therapies, neuropsychological rehabilitation and pharmacological treatments.
Has there been any new developments in the use of stem cell therapy in the hippocampus, use of the endocrine system to buffer against further deterioration (such as the use of melatonin), or other forms of advanced treatment that have occurred or are under way?
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Dear Cheryl,
I do agree with Geoffrey with regard to the effect of stem cells; there are recent reports about it. However, the problem, in TBI, for instance, or Diffuse Axonal Injury occurring as a consequence of it or due to Cardiac Arrest is the time needed for harvesting autologous stem cells, expanding them until arising a significant number and injecting them back to the patient. During this time the cascade of inflammatory signals turned on by diffuse axonal injury is progressively increasing, making the brain damage stronger. In my opinion, and in my experience, the best urgent solution for blocking pro-inflammatory cytokines and avoiding the dead cascade is to administer melatonin, at doses no lower than 10 mg, it can be increased through 200-400 mg though. 
Best regards
Jesús Devesa, MD, PhD
Professor of Physiology
Specialist in Endocrinology and Magister in Advanced Regenerative Therapies
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I want to give a group of elderly aerobic exercise and melatonin supplements؛ How much and when I complete?
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Useful re melatonin and exercise:  Miller JC. Melatonin. Pages 183–184 in LM Castell, SJ Stear, LM Burke (ed.), Nutritional Supplements in Sport, Exercise and Health: An A-Z Guide. London: Routledge, 2015.
And circadian effects:  Paul MA, Gray G, Lieberman H, Love RJ, Miller JC, Trouborst M, Arendt J. Phase advance with separate and combined melatonin and light treatment. Psychopharmacology (Berl) 214(2):515-23, 2011. Selected for publication in Best of Sleep Medicine for 2012.
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Melatonin and Circadian Typology
What are the causes behind the late secretion (i.e. 2-3 hours) of melatonin hormone in owls as compared to that of larks?
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Hello Amrit,
The neural mechanisms regulating  circadian changes in melatonin secretion are well described. A number of studies suggested a role of the  clock genes  in the control of the phase, and therefore circadian rhythms in melatonin secretion. At the same time, the synthesis of melatonin is inhibited directly by light, and the duration of melatonin release is reported to be related to the length of the dark period. These evidences suggest that there likely multiple mechanisms which drive melatonin secretion in both owls and larks.
Best wishes, Tatyana 
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Melatonin signal transduction in skeletal muscle during  aerobic exercise and Prevention of DNA damage in The elderly?
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This is a very interesting question. Melatonin has anti-inflammatory effects in different situations (see our review Mauriz et al, 2013). In aging, melatonin has anti-oxidative and anti-apoptotic effects in liver (see our papers Mauriz et al 2007, and Molpeceres et al 2007). Moreover, in exercised aging animals changes in AMK and GLUT have been described by Mendes et al (2013).
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I would like to measure the content/activity of tryptophan decarboxylase that is the first enzyme in melatonin biosynthesis catalyzing the conversion of tryptophan to tryptamine. I can currently measure tryptophan, melatonin and tryptamine contents in plant tissues with HPLC fluorescent detection but hard time finding complete method for analysis of this enzyme. Any help on this matter will be greatly appreciated.
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This is a very common enzyme and many researchers have done lot of work on quantitation of this enzyme. You can search either PubMed or Medline and you will get a lot of information. However, if you are using HPLC then there is no need to use the FD, you can simply detect it with UV sensor that comes with it. The compound that might give you a good and accurate result is Tryptamine.
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I am trying to identify genes involved in the biosynthesis of melatonin in yeast. I am not too sure which is the best approach to use. 
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If you don't know it already the attached paper is very informative. You can find which enzymes convert tryptophan to melatonin in Fig. 3. The next step would be a database search to find sequence information of the corresponding biosynthesis genes. Yeastgenome.org should contain the information you seek.
Cheers, Christian
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We know that melanopsin is sensitive to blue light with a peak around 480 nm and that melatonin suppression is most sensitive to blue light with a peak around 460 nm. Furthermore, it seems that melanopsin is important but not essential to circadian photoentrainment. I have found some older reports showing that circadian phase shifts are more sensitive to short wavelength light.
Does anyone have any idea of how important blue light is to circadian photoentrainment, preferably based on recent research?
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Lots of experimental evidence are available to support that monochromatic light shift the clock, especially blue light compared to red light. Blue light shifts the clock (circadian locomotor activity rhythm)  but not red light. When we keep animals in constant DD, people have been using constant red light.  if you keep the animal in DD, blue light exposure definitely shifts the clock. 
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Everywhere is clear that melatonin is produced mainly at nighttime, and its production decreases with age almost half from 25 to 50 years old. However the absolute amount of produced melatonin remains unclear for me. I would appreciate very much any information on that. Maximum concentrations in blood in young or old individuals would help also!
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All the answers are correct, but the body’s melatonin levels maybe different according the dietary, for instance, the people, who uses to eat rice, have a higher level of melatonin, because the tryptophan content is higher in this grain, the principal precursor of melatonin.
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malaria researchers
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Thank you very much  Pandi-Perumal Seithikurippu Ratnas 
For this useful  site
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In an ongoing study, we have exposed freshwater fish Clarias gariepinus and Giant African Land Snail Archachatina marginata to varying monochomatic lights of different colours - red, blue, green, yellow. Our intention is to establish the physiological impact of light on these animals viz their growth, production of acetylcholinesterase and melatonin. We seek to understand the likely pathway of these impacts and the ecological implications 
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I'm not sure about snail and fish, but earthworms have eye cells on their skins that are sensitive to light waves except blue. In my past research I used blue light chambers to observe earthworms at the soil surface. 
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Dear All,
I am planning to study the effect of melatonin or H2O2 on different treating time periods to the cells, for example, treating at t0 (starting time) and t12 (after 12hr), then collect all samples at t24 (after 24hr).
There two treating solutions: (1) melatonin solution (soluble in ethanol then diluted with complete growth media) and (2) H2O2 solution (diluted with complete growth media), and then store them one or two days at 4 degree C. before treating to adherent cells such as SH-SY5Y. To prepare melatonin and H2O2, a day or two before instead of prepare fresh, could help me reduce workload on the treating day.
My questions are:
(1) Is it ok to prepare melatonin or H2O2 a day or few day, instead of fresh to treat to cells?
(2) If I prepare melatonin or H2O2 stock solution fresh and treat to cells at t0, Can I keep this stock solution at 4degreeC and use it again to treat to cells at t12? Or I should prepare another fresh solution with the same concentration at t12?
Thank you so much
Best regards,
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Thank you all of you. Your answers are very helpful to me.
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I want to check the effect of melatonin on RAW264.7 cells.I have used ethanol 10 microliter/mL, but this ethanol has some effect and I found the effect of melatonin very few or nothing. So, what solvent and in which concentration will be good for my experiments? Do I need to make molar solution of melatonin or 10, 1, 0.1, 0.01 microgram/mL is ok? Please suggest me.
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The Molecular weight of melatonin is 232.28. Solubility of melatonin in DMSO is 50 mM. That means if you dissolve 11.61 mg in 1 ml of DMSO it will give you a 50mM solution. So the standard range of exposure for vehicle (DMSO) is 0.1%. This means if you add 1 ul in 1ml that will give you a 0.1% solution. So if you add 1 ul of 50 mM solution in 1 ml of DMEM, the final strength of the solution will be 50 uM. This can be your starting point. In case you don't see an effect at 50 uM exposure. You can increase the vehicle percentage upto 0.5% but not higher than that. In my experience RAW264.7 cells are fully viable and healthy at 0.5% exposure to DMSO. At 0.5% you will get a working solution of 250 uM. Don't forget to expose the cells with vehicle only and vehicle + RANKL as negative and positive control.
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I want to now how some circadian process change with ageing. For example, the variables like the body temperature, melatonin level exhibit circadian rhythms with characteristic amplitude and time-period. I will like to know whether the amplitude is a constant or does it change with ageing. I have read that melatonin level variations during day and nigh are larger in infants (larger amplitude) as compared to the adults. Is it true for all other circadian processes? What references should I see to understand the variation of the amplitude of the circadian process with ageing. I will be intrested in comparing the circadian processes in children, adults and old people. 
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Many variables change their circadian characteristics with age. In many cases, including melatonin, the circadian amplitude decreases. In variables such as blood pressure and heart rate, the variability in the circadian acrophase increases. The circadian waveform can also undergo changes: for instance, in blood pressure, the second harmonic with a period of 12 hours increases in prominence with advancing age, resulting in a more pronounced post-prandial dip in the elderly. Other changes are also taking place, such as an increase in the relative prominence of the biologic week versus the circadian variation, seen for blood pressure and heart rate.
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I mean are there any effects on sleep in patients with Alzheimers with melatonin?
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See these two recent reviews:
Sleep and Alzheimer's disease.
Peter-Derex L, Yammine P, Bastuji H, Croisile B.
Sleep Med Rev. 2015 Feb;19:29-38. doi: 10.1016/j.smrv.2014.03.007.
Pharmacotherapies for sleep disturbances in Alzheimer's disease.
McCleery J, Cohen DA, Sharpley AL.
Cochrane Database Syst Rev. 2014 Mar 21;3:CD009178. doi: 10.1002/14651858.CD009178.pub2.
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Recently, I want to check the expression of melatonin receptor on B6 on mRNA level. Frustrately, I could not detect the mRNA both of the MT1 and MT2 on C57BL/6J background mice.
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See this useful papers:
Psychopharmacology (Berl). 2015 Feb 24. [Epub ahead of print]
Melatonin administration alters nicotine preference consumption via signaling through high-affinity melatonin receptors.
Horton WJ1, Gissel HJ, Saboy JE, Wright KP Jr, Stitzel JA.
Neuropharmacology. 2012 Nov;63(6):966-73. doi: 10.1016/j.neuropharm.2012.06.065. Epub 2012 Jul 20.
Diurnal variation in nicotine sensitivity in mice: role of genetic background and melatonin.
Mexal S1, Horton WJ, Crouch EL, Maier SI, Wilkinson AL, Marsolek M, Stitzel JA.
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I want to work with the macrophage cell line with melatonin. Which solvent and how can I use it for dissolving melatonin minimizing solvents adverse effects? Can you answer elaborately with its required concentration, side effects, preparation method and also something else need to consider for this experiment?
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Hi,
As ethanol tends to evaporate changing the concentration I preferred to use DMSO. I prepared melatonin 1000 times more concentrated in DMSO to finally dilute it 1000 times in the buffer. In that way DMSO is used safely (0.1%) and your melatonin final concentration is well controlled.
Good luck!
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If you have 4 blood samples to measure melatonin, what is the best stat program that can be used to best delineate the rhythm?
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Actually  for measurement of melatonin rhythm in the blood samples you might need more then four samples collected several hours apart. For rhythm assement use of rhythmometric analysis (Cosinor) would be more suitable. 
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BAT = brown adipose tissue
Fexaramine is an investigational compound which acts as an agonist of the farnesoid X receptor (FXR), which is a bile acid-activated nuclear receptor that controls bile-acid synthesis. It induces enteric FGF15, changing the composition of biliary acids, thus enhancing BAT, thermogenesis, and decreasing glucose hepatic production and weight in mice.
See Fang S, et al. Intestinal FXR agonism promotes adipose tissue browning and reduces obesity and insulin resistance. Nat Med. 2015 Jan 5. doi: 10.1038/nm.3760.
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Fexaramine reduced diet-induced weight gain, body-wide inflammation and hepatic glucose production, while enhanced thermogenesis and promoted browning of white adipose tissue (WAT) in diet induced obesity (DIO) mice. Its effects in humans are not known yet.
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Hello,
I would like to know what is the equivalent concentration of 1 mM melatonin for use in humans. How can I make these calculations?
Several in vitro studies have shown that the pharmacologic concentrations of melatonin is 1 mM, but the physiological concentration in humans is about 70 pM, which is 14,285,714-times lower compared to 1 mM. Please, could you comment this?
Thank you!
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I) Patrick: please note that 232  mg of melatonin corresponds to 1 mmol (1 millimol) and not to 1 mM (1 millimol/L or 1 millimol per liter). 1 mmol is the amount of matter and 1 mM is the concentration. So one cannot say that 232 mg of melatonin is 1 mM of melatonin, unless these 232 mg were dissolved exactly in 1 L of water, buffer or any other solvent
II) Bruna: 4 mg of melatonin corresponds to 17 umol (micromol) of melatonin, because 0.004g/232 g.mol-1 = 17.10(-6) mol = 17 umol (or 17 micromol). If we dissolved this amount in 1 liter of any solvent, we would get the concentration of 17 uM (or 17 umol/L). However, these 4 mg of melatonin were not dissolved in 1 liter of something but they were administered orally to a group of adults. The mean peak blood concentration of melatonin (which depends on several pharmacokinetics parameters, including the volume of distribution) was determined empirically  as 3999 pg/mL. If you re-calculate the mass concentration to molar concentration, you will get the value 17.2 nmol/L (or 17.2 nM). For the data reference see PUBMED (PMID: 22348451).
Your last question was whether 20 mg dose is equivalent to 0.085 mM in vitro. Again, if you dissolved these 20 mg in 1 L, it would correspond to 0.085 mM, but this is not the right way to assess the concentrations that would be achieved if 20 mg were administered orally to an individual. If we consider volume of distribution for melatonin roughly 1602 L (the same reference as above), this dose would correspond to peak plasma level of melatonin ~ 54 nM (or 54 nmol per liter). That would be the estimated concentration for testing in vitro.
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1 mM of melatonin equivalent to 0.23 mg/mL
- 20 mg/mL of melatonin equivalent to 86 mM and similarly 1mM of melatonin equivalent to 0.23 mg/mL.
The approximate molecular weight of melatonin is 232,278 g/mole.
So, to convert mg/mL to mM, I divided the concentration in mg/mL (20 mg/mL) by the molecular weight of the sample (232,278 g/mole) and multiplied by 1,000.
If this calculation is incorrect, please, how could I know how many mg are equivalent to 1mM of melatonin?
Thank you.
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1 mg/ml = 1 g/L
(1 g/L) x (1 mole/232.278 g) = 0.004305 mole/L = 4.305 mM
20 mg/ml = 20 x 4.305 mM = 86.1 mM
In the other direction:
1 mM = 0.001 mole/L
(0.001 mole/L) x (232.278 g/mole) = 0.2323 g/L = 0.2323 mg/ml
86 mM = 86 x 0.2323 mg/ml = 20 mg/ml
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How melatonin from plants can be used by species such as sheep? There are some papers describing analytical procedures, but which one is more accurate?
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I am adding one more article, hope it will be useful for your work
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