Quantification of melatonin in human saliva by liquid chromatography-tandem mass spectrometry using stable isotope dilution

A method for the determination of melatonin in human saliva has been developed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS). Saliva was collected in plastic tubes. 7-D-Melatonin was added as internal standard and the samples were cleaned and concentrated by solid-phase extraction. The limit of detection was 1.05 pg x ml(-1) and the limit of quantification was 3.0 pg x ml(-1). The accuracy of the method was +/-14% at 5.60 pg x ml(-1) and +/-9% at 19.6 pg x ml(-1). The precision was +/-13% at 6.18 pg x ml(-1) and +/-11% at 31.2 pg x ml(-1), respectively. Our HPLC-MS-MS method shows a high sensitivity and specificity for melatonin and more reliable results compared with a radioimmunoassay. The chromatographic method has been used to determine the circadian rhythm of melatonin among three nurses working the night shift and a patient suffering from an inability to fall asleep at night.     

High-performance liquid chromatographic analysis of melatonin in human plasma and rabbit serum with on-line column enrichment

This paper describes the development of a simple and sensitive analytical method for the quantification of melatonin in human plasma and rabbit serum, using standard analytical equipment and on-line column enrichment without prior extraction, clean-up or derivatization. The analytical procedure was found to be accurate, precise and linear. For human plasma, the accuracy was 101% (range 89–106%), and the mean precision was 5% (range 2–9%) for all concentrations (0, 2, 10, 50 and 200 ng/ml) tested (n=6). The accuracy in rabbit serum was 101% (range 90–112%), and the mean precision was 13% (range 8–19%) for all concentrations (0, 2, 10, 50, 200 and 500 ng/ml) tested (n=6). The retention time of melatonin was about 8 min and the total recoveries were found to be approximately 65 and 85%, respectively, for human plasma and rabbit serum. The limit of detection was found to be lower than 1 ng/ml for human plasma and around 2 ng/ml for rabbit serum. The method is, therefore, found to be suitable for melatonin bioavailability studies in rabbits and presumably also in humans.     

The profile of melatonin production in tumour-bearing rats

The pineal gland is involved in the regulation of tumour growth through the anticancer activity of melatonin, which presents immunomodulatory, anti-proliferative and anti-oxidant effects. In this study we measured melatonin content directly in the pineal gland, in an attempt to clarify the modulation of pineal melatonin secretory activity during tumour growth. Different groups of Walker 256 carcinosarcoma bearing rats were sacrificed at 12 different time points during 24h (12h:12h light/dark cycle) on different days during the tumour development (on the first, seventh and fourteenth day after tumour inoculation). Melatonin content in the pineal gland was determined by high-performance liquid chromatography with electrochemical detection. During tumour development the amount of melatonin secreted increased from 310.9 ng/mg of protein per day from control animals, to 918.1 ng/mg of protein per day 14 days after tumour implantation, and there were changes in the pineal production profile of melatonin. Cultured pineal glands obtained from tumour-bearing rats turned out to be less responsive to noradrenaline, suggesting the existence, in vivo, of putative factor(s) modulating pineal melatonin production. The results demonstrated that during tumour development there is a modification of pineal melatonin production daily profile, possibly contributing to cachexia, associated to changes in pineal gland response to noradrenaline stimulation.     

Knock-down of RGS4 and beta tubulin in CHO cells expressing the human MT1 melatonin receptor prevents melatonin-induced receptor desensitization

Previously, it has been shown that chronic melatonin exposure in MT1-CHO cells results in receptor desensitization while at the same time producing drastic morphological changes. The addition of a depolymerizing agent during the melatonin pretreatment period prevents MT1 receptor desensitization and the changes in cellular morphology. The lack of morphological change in the presence of a depolymerizing agent is easily explained by the inability of the microtubules to polymerize, however, the prevention of receptor desensitization is a little more complex and may involve G-protein activation. The goal of this study was to determine whether melatonin-induced MT1 receptor desensitization is regulated by proteins known to regulate G-protein activation states, beta-tubulin and RGS4,using anti sense knockdown approaches. The expression of RGS4 mRNA in CHO cells was confirmed using RT PCR and successful knockdown of each was confirmed by western blot analysis or quantitative PCR. Pretreatment of MT1-CHO cells, transfected with the nonsense probes and exposed to melatonin, resulted in a desensitization of the receptor, an increase in forskolin-induced cAMP accumulation, an increase in 2-[125I]-iodomelatonin binding and no change in the affinity of melatonin for the MT1 receptor. However, knockdown of either beta-tubulin or RGS4 in MT1-CHO cells followed by pretreatment with melatonin attenuated the desensitization of melatonin receptors, decreased total 2-[125I]-iodomelatonin binding, and did not affect neither the forskolin response nor the affinity of melatonin for the MT1 receptor. Perhaps RGS4 and beta-tubulin modulate Galpha-GDP and Galpha-GTP states thus modulating MT1 melatonin receptor function.     

Isolation of melatonin by immunoaffinity chromatography

A single-step, highly specific and easy-to-use method was developed for isolation and purification of melatonin from complex biological matrices. Polyclonal antibodies highly specific against melatonin (with cross-reactivities with related compounds below 0.02%, except for 6-hydroxymelatonin) were raised, characterised by enzyme-linked immunosorbent assay (ELISA) and used for preparation of immunoaffinity gel. Melatonin recovery by the immunoaffinity method was approximately 95%, allowing single-step processing of samples prior to electrospray HPLC-MS analysis (with detection limit 10 fmol). The method was successfully used for determining melatonin in human serum and turned out to be better than the non-specific solid-phase extraction published earlier.     

Some Perturbations That Disturb the Circadian Melatonin Rhythm

The circadian melatonin rhythm is highly reproducible and generally not easily altered. The few perturbations that are capable of significantly changing either the amplitude or the pattern of the 24-h melatonin rhythm are summarized herein. Aging alters cyclic melatonin production by decreasing the amplitude of the nocturnal melatonin peak in all species in which it has been studied. The best known acute suppressor of nocturnal melatonin is light exposure. The brightness of light required to acutely depress pineal melatonin production is species dependent; of the visible wavelengths, those in the blue range (∼500-520 nm) seem most effective in suppressing melatonin production. Nonvisible, nonionizing radiation in the extremely low frequency range (e.g., 60 Hz) seems also capable of altering pineal melatonin synthesis. Hormones have relatively little influence on the circadian production of melatonin, although either adrenalectomy or hypo-physectomy does attenuate the amplitude of the melatonin cycle. Exercise at the time of high melatonin production rapidly depresses pineal concentrations of the indole without influencing its synthesis; the mechanism of this suppression remains unknown.     

New melatonin (MT1/MT2) ligands: Design and synthesis of (8,9-dihydro-7H-furo[3,2-f]chromen-1-yl) derivatives

Herein we describe the synthesis of novel tricyclic analogues issued from the rigidification of the methoxy group of the benzofuranic analogue of melatonin as MT₁ and MT₂ ligands. Most of the synthesized compounds displayed high binding affinities at MT₁ and MT₂ receptors subtypes. Compound 6b (MT₁, K_i = 0.07 nM; MT₂, K_i = 0.08 nM) exhibited with the vinyl 6c and allyl 6d the most interesting derivatives of this series. Functional activity of these compounds showed full agonist activity with EC₅₀ in the nanomolar range. Compounds 6a (EC₅₀ = 0.8 nM and E_max = 98%) and 6b (EC₅₀ = 0.2 nM and E_max = 121%) exhibited good pharmacological profiles.     

Melatonin modulates the ERG circadian rhythm in crayfish

One of the most important functions modulated by melatonin is the synchronization of circadian rhythms. In crayfish (Procambarus clarkii), we have obtained evidence that the amplitude of the electrical response to light of the retinal photoreceptors the receptor potential, is modified by the action of melatonin and that the magnitude of this action depends on the circadian time of melatonin application. In contrast, the electroretinogram (ERG) circadian rhythm can be synchronized by either single or periodic melatonin application. In this work we hypothesized that, in crayfish, melatonin acts on effectors and on pacemaker of ERG circadian rhythm as a non-photic synchronizer. Melatonin could be a hormone that sends a signal of darkness to the ERG circadian system.     

褪黑素和褪黑素受体对下丘脑-垂体-肾上腺轴作用的研究进展

松果体于儿童中期可发育至最高峰,普遍在7岁之后开始呈逐渐萎缩,并在成年后逐渐有钙盐沉着.褪黑素主要是由松果体进行合成和分泌所形成,存在较好的昼夜节律性,且通常是通过下丘脑的视交叉上核进行控制,并与环境中的光-暗呈现的周期改变存在密切关联.此外,褪黑素具有极其广泛的生物学作用,且其发挥作用的首站便是与特异性褪黑素受体相关...     

Melatonin Receptor-Mediated Inhibition of Cyclic AMP Accumulation in Chick Retinal Cell Cultures

Abstract: Melatonin receptors were characterized in cultured neurons and photoreceptors prepared from chick embryo retina. Cultured cells contained high-affinity 2-[¹²⁵I]iodomelatonin binding sites (K_D = 41.6 pM), similar to those in intact retina. The effects of melatonin and related indoles on cyclic AMP accumulation were examined. Melatonin (10^?7M) had no effect on basal or K⁺-stimulated cyclic AMP accumulation, but inhibited forskolin-stimulated cyclic AMP accumulation by approximately 50%. Melatonin inhibited forskolin-stimulated cyclic AMP accumulation in the presence or absence of the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, suggesting an effect on cyclic AMP synthesis rather than degradation. Half-maximal inhibition was observed at 5.9 × 10^?10M melatonin. The relative order of potency among melatonin analogues was 2-iodomelatonin > melatonin ≈ 6-chloromelatonin ≥ 6-hydroxymelatonin > N-acetylserotonin ≈ 5-methoxytryptophol > serotonin. The EC₅₀ value for inhibition of cyclic AMP accumulation by 2-iodomelatonin (36.7 pM) was comparable to the K_D value for binding of the radioligand, suggesting that the binding sites represent functional receptors. The inhibitory effect of melatonin was antagonized by the putative melatonin antagonists luzindole, N-acetyltryptamine, and N-(2,4-dinitrophenyl)-5-methoxytryptamine, with estimated K_B values of 0.12, 0.17, and 1 µM, respectively. At a concentration of 10 µM, N-(2,4-dinitrophenyl)-5-methoxytryptamine significantly inhibited forskolin-stimulated cyclic AMP accumulation when added alone; at 30 µM, luzindole and N-acetyltryptamine also had significant inhibitory effects. The inhibitory effect of melatonin was blocked by pretreatment with pertussis toxin. The results of this study indicate that melatonin receptors on retinal cells are coupled via inhibitory G proteins to cyclic AMP accumulation. Thus, some of the effects of melatonin on retinal physiology may be related to regulation of cyclic nucleotide metabolism.     

Low,but not high,doses of melatonin entrained a free-running blind person with a long circadian period

In a previous report, we were unable to entrain one out of seven totally blind people with free-running endogenous melatonin rhythms to 10 mg of exogenous melatonin. This person had the longest circadian period (24.9 h) of the group. We now find that this person can be entrained to 0.5 mg of melatonin, but not to 20 mg. These results are consistent with the idea that too much melatonin may spill over onto the wrong zone of the melatonin phase-response curve.     

Entrainment of Melatonin Rhythms in Rams By Symmetrical Light-Dark Cycles of Different Period Length

The objective of this study was to investigate the entrainment of melatonin rhythms in rams using symmetrical light-dark cycles of different period length. Five groups of six He de France rams were kept in 12L: 12D for 7 weeks and then (i) 12L: 12D, (ii) 11L: 11D, (iii) 10L: 10D, (iv) 13L: 13D and (v) 14L: 14D for a further 3 weeks. Environmental factors other than the light dark cycle were not controlled. The onset and offset of the plasma melatonin rhythm in DD after 3 weeks of the respective light treatments was assessed for 48 hr, immediately after transferring to DD. The duration of secretion in DD was positively related to the length of the previous dark phase. The phase of the melatonin rhythm with respect to the anticipated dark phase suggested entrainment with no change in phase-relationship to the zeitgeber by 12L: 12D and 13L : 13D. Entrainment with a phase-delay or a phase-advance was apparent after 11L: 11D and 14L: 14D, but the individual rhythms were not all synchronized with respect to each other after 10L: 10D. Activity recordings for 2-3-week periods during 12L: 12D, 10L: 10D and 14L: 14D all showed a major 24-hr component at all times, with activity during the light phase in 12L: 12D. It appears that melatonin may be readily desynchronized from overt activity-rest cycles in sheep. The upper and lower entrainment limits are probably greater than 28 hr and close to 20 hr cycles, respectively.     

des-Tyr1-gamma-endorphin and haloperidol increase pineal gland melatonin levels in rats

The effect of subcutaneously injected DT gamma E (beta-endorphin, (beta E)2-17) on the pineal melatonin level was compared with that of closely related peptides and the neuroleptic drug haloperidol. As found previously, DT gamma E (3 ng/rat and 300 ng/rat) increased the melatonin levels. Similar doses of DT alpha E (beta E 2-16), DT beta E (beta E 2-31), gamma E (beta E 1-17), alpha E (beta E 1-16) and beta E failed to significantly change the melatonin levels in both the dark and the light phase. Haloperidol in a dose of 300 ng/rat exhibited a similar effect as DT gamma E.     

A review of the molecular conformations of melatonin ligands at the melatonin receptor

This review examines the 1992-2000 literature on studies of the molecular conformations of melatonin ligands at the melatonin receptor. In order to investigate quantitative structure-affinity relationships between different chemical classes of melatonergic ligands binding to the melatonin GPCR, CoMFA has been applied to extended sets of compounds, to obtain 3D-QSAR agonist/antagonist models. The results of several authors have suggested that the active conformation of the C-3 aminoethyl side chain of melatonin and related compounds is in a folded form, orthogonal to the aromatic ring. Positive steric potentials were found in the C-2 region, surrounding the C-5 methoxy group and near the N -acyl group of the side chain, while substituents in positions C-6 and C-7 cause a decrease in affinity. Negative steric regions were found between indole N-1 and C-2. Receptor binding affinities have been predicted for a range of structurally diverse compounds for the sheep brain melatonin receptor considering steric, electrostatic and lipophilic fields.     

Zeitgeber hierarchy in humans: resetting the circadian phase positions of blind people using melatonin

Four blind individuals who were thought to be entrained at an abnormal circadian phase position were reset to a more normal phase using exogenous melatonin administration. In one instance, circadian phase was shifted later. A fifth subject who was thought to be entrained was monitored over four years and eventually was shown to have a circadian period different from 24 h. These findings have implications for treating circadian phase abnormalities in the blind, for distinguishing between abnormally entrained and free-running blind individuals, and for informing the debate over zeitgeber hierarchy in humans.