The Salimetrics Salivary Melatonin kit is a competitive immunoassay specifically designed and validated for the quantitative measurement of salivary melatonin. It is not intended for diagnostic use. It is intended only for research use in humans and some animals. Salimetrics has not validated this kit for serum or plasma samples. Please read the complete kit insert before performing this assay. Failure to follow kit procedure and recommendations for saliva collection and sample handling may result in false values. For further information about this kit, its application, or the procedures in this insert, please contact the technical service team at Salimetrics or your local sales representative.
Storage
All components of this kit are stable at 2 - 8°C until the kit's expiration date.
Precision
The intra-assay precision was determined from the mean of 20 replicates each sample. The inter-assay precision was determined from the average of 20 replicates across 10 runs, replicates of 2 per run.
Sensitivity
Analytical Sensitivity: The lower limit of sensitivity was determined by interpolating the mean optical density minus 2 SDs of 10 sets of duplicates randomized across the plate within one run at the 0 pg/mL level. The minimal concentration of Melatonin that can be distinguished from 0 is 0.58 pg/mL. Functional Sensitivity: The functional sensitivity was determined as the concentration of 11 saliva samples, tested as 20 replicates each, resulting in a CV of 30% or less. The functional sensitivity of the Salivary Melatonin ELSIA is 1.9 pg/mL.
General Description
Melatonin (N-acetyl-5-methoxytryptamine) is a compound secreted mainly by the pineal gland, but synthesized also in many other tissues and cells. In humans, nocturnally peaking oscillations of melatonin are involved in sleep-wakefulness where melatonin concentrations are lower during the day. In recent years, the role of melatonin and its metabolites have been identified as potent, broad acting antioxidants and free radical scavengers in addition to playing a role in the upregulation of antioxidant enzymes. Melatonin levels in plasma are paralleled by corresponding variations in saliva where the saliva concentrations are about 30% of that found in plasma. Measurement of salivary melatonin is advantageous, especially to avoid invasive venipuncture procedures.
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Background
Melatonin plays an important role in coordinating circadian rhythms, helping to regulate various physiological processes in the body to synchronize them with circadian changes in the environment, and is the prototype for many drugs that affect physiological rhythms. It is also an effective cytoprotective agent with powerful antioxidant activity, helping to scavenge free radicals and reduce cellular damage from oxidative stress; anti-inflammatory action, helping to reduce inflammatory responses in the body; and immunomodulatory activity, regulating and balancing the function of the immune system. The biological clock is one of the indispensable functions of an organism that regulates the synchronization of the homeostatic system, which includes sleep and wakefulness, hormone secretion, immune function, and a wide range of other bodily functions, with the 24-hour cycle. Circadian rhythms in mammals are regulated by the suprachiasmatic nucleus (SCN) and melatonin is secreted by the pineal gland. The SCN, located in the hypothalamus, is a center that regulates physiological rhythms, and SCN nuclei receive light signals from the retina. Nonvisual photosensitive ganglion cells in the retina are not involved in visual processes but regulate biological rhythms and transmit light signals to the SCN via the retino-hypothalamic pathway when exposed to light.
Melatonin is synthesized from tryptophan in a multi-step enzymatic reaction. The human pineal parenchyma cells synthesize melatonin and secrete it into the bloodstream and cerebrospinal fluid. Chemically speaking, melatonin is an indoleamine compound named N-acetyl-5-methoxytryptamine. The first step in melatonin synthesis is the hydroxylation of tryptophan to 5-hydroxytryptophan by tryptophan hydroxylase, followed by decarboxylation of 5-hydroxytryptophan to serotonin, i.e., 5-hydroxytryptophan, in the presence of aromatic-L-amino acid decarboxylase. Aromatic amine N-acetyltransferase (AANAT) converts serotonin to N-acetylserotonin, and finally acetylserotonin O-methyltransferase (ASMT) converts N-acetylserotonin to melatonin.
Figure 1. Neural connection between SCN and pineal gland, and steps of melatonin synthesis in pineal gland (Source: Samanta S. 2022)
Diurnal variation of melatonin levels in body fluids is harmonized with the environmental light-dark cycle. Melatonin synthesis and secretion increase at night and remain low during the day due to the inhibition of melatonin secretion by external light. AANAT is one of the key enzymes in the synthesis of melatonin, and its activity increases 7- to 150-fold during the night. The diurnal variation of AANAT activity, together with the regulation of melatonin secretion by external light, determines the circadian rhythm of melatonin levels. This rhythm is synchronized with the environmental light/dark cycle and is regulated by the sympathetic nervous system. This mechanism ensures the circadian variation of melatonin levels and thus regulates physiological rhythms. For sighted individuals who live in environments that may induce free-running rhythms, taking 2-5 mg of melatonin per day advances the phase of their internal clocks, thus keeping their circadian rhythms on a 24-hour cycle. Over-the-counter melatonin is now widely used, but supplementation with melatonin requires attention to its dosage and timing of administration. Excessive melatonin concentrations may lead to desensitization of melatonin receptors, whereas melatonin produces the required response phase profile to exert a phase-shifting effect only at specific time points of administration.
Figure 2. Chronobiotic activity of melatonin (Source: Cardinali DP. 2021)
References
1. Anghel L, et al. Benefits and adverse events of melatonin use in the elderly (Review). Exp Ther Med. 2022 Mar;23(3):219.
2. Samanta S. Physiological and pharmacological perspectives of melatonin. Arch Physiol Biochem. 2022 Oct;128(5):1346-1367.
3. Cardinali DP. Melatonin and healthy aging. Vitam Horm. 2021;115:67-88.
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References
Tin disulfide-graphene oxide-beta-cyclodextrin mediated electro-oxidation of melatonin hormone: an efficient platform for electrochemical sensing
Here, we have developed an electrochemical sensor based on integrated 2D materials including tin disulfide (SnS2) nanoflakes, graphene oxide (GO), and beta-cyclodextrin (beta-CD) forming a ternary nanocomposite decorated on a screen-printed electrode (SPE) for the electrochemical detection of melatonin. Hydrothermally synthesized SnS(2)was mixed with GO/beta-CD to prepare the ternary compositeviaan ultra-sonication process. The nanocomposite was characterized using X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), and field emission transmission electron microscopy (FEG-TEM). The electrochemical performance of the modified electrode was investigated using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV). The electrochemical sensor exhibited a linearity range from 1 nM to 100 mu M with the lowest detection limit of 0.17 nM. The sensor was successfully applied for the detection of melatonin in commercial drugs and human saliva, which showed a consistent result with the Enzyme-Linked Immuno-Sorbent Assay (ELISA).
Evaluation the cardiopulmonary markers in cecal ligation and puncture-induced sepsis in Wistar rats
Objective: Sepsis is a clinical problem caused by host immune disability against pathogens. Rodent Cecal Ligation and Puncture (CLP) models mimic sepsis in humans. Gauges needle size in CLP is related to cytokine storm, inflammation, and organ failure. This study focus, for the first time, on precise and inexpensive biochemical markers to evaluate the difference of sepsis severity in the heart and lung tissues, one day after cecal ligation and puncture-induced sepsis with needle gauge 18 (G-18). Methods: Twelve adult male Wistar rats were randomly allocated into two groups of six animals. These groups include; sham operation as the control group and underwent CLP procedure with G-18. All rats were sacrificed 24 hours after CLP. Then lungs and heart samples were collected for biochemical and histological assessment. Following the procedure, reactive oxygen species (ROS), Myeloperoxidase Activity (MPO), Tumor Necrosis Factor-Alpha (TNF-alpha), High Mobility Group Box 1 (HMGB1), lactate generation, caspases (-3 and -9), gene expression of autophagy, cellular hypoxia, and pathological assessment of both tissues were measured. Results: Increased level of ROS, MPO, pro-inflammatory cytokines, hyperlactatemia, caspases production, overexpression of hypoxia (PRKAA1 gene), and autophagy (MAP1LC3B gene) in the lungs were higher compared to heart 24 hours after the procedure. Moreover, hyperplasia of pneumocyte and inflammatory cells, and myocardial necrosis were found in the pathological assessment. Conclusion: The purpose of study was to determine the severity of sepsis by means of cost-effective and precise inflammatory markers. Our findings demonstrated that injury-related indicators in lungs meaningfully increased compared to heart 24 hours after CLP.
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Figure 1. Neural connection between SCN and pineal gland, and steps of melatonin synthesis in pineal gland
Figure 2. Chronobiotic activity of melatonin
