Enzyme immunoassay for the quantitative determination of Melatonin-Sulfate (synonyms: 6-hydroxymelatonin sulfate, 6-sulfatoxymelatonin) in human urine.
Contents of Kit
1. Microtiter Plate Break apart strips. 1 × 12 × 8 MTP (12 strips of 8 wells each) coated with goat anti-rabbit antibody in solution containing bovine serum albumin. Vacuum dried. 2. Melatonin Sulfate Antiserum. Ready to use. 1 × 6 mL Rabbit anti-melatonin sulfate antibody in Tris buffer containing bovine serum albumin and < 0.1 % Thimerosal(w/w). 3. Enzyme Conjugate,Concentrate (40×). 1 × 0.2 mL Contains melatonin sulfate conjugated to peroxidase containing Thimerosal < 0.1% (w/w). 4. Standard A-F. Ready to use. 1 × 6 × 0.1 mL 0; 2.0; 6.0; 18.0; 54; 162 ng/mL 0; 6.1;18.3, 54.9; 164.7, 494.1 nmolI/L Contains melatonin sulfate in Tris buffer, bovine serum albumin and Thimerosal < 0.1 %(w/w). 5. Control 1+2. Ready to use. 1 × 2 × 0.1 mL Contains melatonin sulfate in Tris buffer, bovine serum albumin and Thimerosal < 0.1%(w/w) Concentrations / acceptable ranges see QC certificate. 6. Assay Buffer. Ready to use. 1 × 80 mL Red colored. Contains Tris buffer, bovine serum albumin and Thimerosal < 0.1% (w/w). 7. Wash Buffer, Concentrate (20×). 1 × 50 mL 0.2 M phosphate buffer containing 0.2 % Tween 20 and Thimerosal < 0.1%(w/w). 8. TMB Substrate Solution. Ready to use. 1 × 15 mL Contains 3,3',5,5' Tetramethylbenzidine solution 9. TMB Stop Solution. Ready to use. 1 × 15 mL Contains 1 M sulfuric acid. 10. Adhesive Foil. 3
Storage
The kit is shipped at ambient temperature and should be stored at 2 - 8°C. Keep away from heat or direct sunlight. The storage and stability of specimens and prepared reagents is stated in the corresponding chapters. The microtiter strips are stable up to the indicated expiry date after the kit is opened. Make sure that the opened bag is tightly closed when stored at 2 - 8°C.
Precision
The intra-assay and inter-assay study was conducted for 20 days using one reagent lot. Two runs were performed per day with high and low kit controls and with a panel of 5 urine samples. Each sample was run in duplicate. The intra-assay precision calculated from five human urine samples showed a mean CV of 9.0 % and range of 5.8 % - 15.7 %. The inter-assay precision calculated from four human urine samples showed a mean CV of 11.6 % and of range 9.6 % - 15.3 %. The inter lot precision study was conducted during 5 days of testing. Each run was performed with high and low kit controls and with a panel of 5 urine samples. Each sample was tested five times per run with 3 different reagent lots. The inter lot precision showed a mean CV of 12.8 % and a range of 10.4 % - 17.8 %.
Detection Limit
Limit of Blank (LoB) The LoB study was performed with five blank samples (Standard A), measured in 4 replicates per sample over three days with two kit lots. Limit of Blank: 1.27 ng/mL Limit of Detection (LoD) The LoD study was performed using five different low concentrated samples, measured in 4 replicates per sample over three days with two kit lots. Limit of Detection: 2.18 ng/mL Limit of Quantitation (LoQ as functional sensitivity) The LoQ (functional sensitivity) study was performed using five low and three higher concentrated samples, measured in 4 replicates over three days with two kit lots. Limit of Quantitation (Functional Sensitivity): 2.18 ng/mL
General Description
Melatonin is an intermediate product of tryptophan metabolism and is released into the bloodstream originating from the pineal gland. Melatonin production is regulated by the circadian timing system. In healthy individuals, it is produced in synchrony to the light/dark cycle, being tightly restricted to the night, provided it is dark. Light stimulus (mainly in the blue range) inhibits melatonin synthesis. The role as a physiological marker has been documented in review articles and textbooks. Most of the circulating melatonin is metabolized in the liver to 6-hydroxymelatonin and subsequently to 6- sulfatoxymelatonin which is excreted into the urine. The concentration of 6-hydroxymelatonin sulfate in urine correlates well with the total level of melatonin in plasma during the collection period. Melatonin concentrations play an important role in the regulation of sleep patterns. The start of melatonin production in the human body can we assessed via the timing of the acrophase and/or the dim light melatonin onset (DLMO). Disruption to healthy sleep patterns (exposure to bright light at nighttime or the participation in night shift work) can alter the DLMO and lead to a phase advance or phase delay of the melatonin concentration in the sample. Urinary melatonin sulfate is a useful biomarker in circadian dysregulation through shift work or exposure to bright light at night. Furthermore, time-resolved quantification of melatonin levels can provide information about diurnal type (morning versus evening). Melatonin and melatonin sulfate concentrations in circulation are highly variable. During the day, melatonin sulfate levels are very low. At night, melatonin sulfate concentrations rise. A large variety of methods to assess the onset of melatonin production has led to a formation of an expert group to establish recommendations for the calculation beginning of melatonin secretion. Laboratories should follow consensus guidelines on the generation of data for circadian pace making. Measurements of Melatonin-sulfate in urine show the accumulated amount of 6-sulfatoxymelatonin during an interval of time; overnight Melatonin sulfate levels can be calculated from the first morning void and compared to daytime collected urine fraction. Clinicians should consider the effects of non-modifiable factors (e.g. age), modifiable factors (lighting, seasonal change, physical activity), comorbidities (ophthalmic diseases, spinal cord injuries, liver diseases and disorders, kidney diseases and disorders) and drugs or nutrition supplements that either increase (melatonin, antidepressants, MAO inhibitors) or decrease (1-adrenergic blockers alpha-2 adrenergic agonist, benzodiazepines) on the measurement of melatonin and its metabolite melatonin sulfate. Enzyme immunoassay for the quantitative determination of Melatonin Sulfate in human urine. Quantification of melatonin sulfate indicates physiological functions or states of the pineal gland that regulates sleep-wake cycles. Patient population includes healthy adults and adults suspected to be affected by sleep disorders. The assay should not be performed with patient samples that might be affected by interference from exogenous melatonin or samples that might be affected by drugs that interact with melatonin metabolism. The Melatonin-Sulfate Urine ELISA is a solid phase enzyme-linked immunosorbent assay (ELISA), based on the principle of competitive binding and measured on an absorbance reader. The assay is semi-automated requiring general purpose laboratory instruments and consumables such as absorbance microplate reader/washer, vortexer and pipettes to execute the test. Test results may be calculated manually from a standard curve and compared to laboratory established reference ranges from healthy adults (i.e., normal ranges). The test kit is intended for professional laboratory use only. The test kit is not for self-testing. The Melatonin-Sulfate Urine ELISA is NOT intended for near-patient testing.
Standard Curve
(Example. Do not use for calculation!) The measuring range for the Melatonin-Sulfate Urine ELISA is between 2.18 ng/mL (LoQ as functional sensitivity) and 162 ng/mL (Standard F).
Citations
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Background
Melatonin is named for its ability to accumulate melanin granules in melanocytes. Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine that is synthesized and secreted primarily by the pineal gland, which hydroxylates essential amino acids to form 5-hydroxytryptophan, primarily through the enzyme tryptophan hydroxylase. Decarboxylation of 5-hydroxytryptophan produces a key neurotransmitter, 5-hydroxytryptophan, which is acetylated by arylalkylamine N-acetyltransferase to produce the immediate precursor (N-acetylhydroxytryptophan) of melatonin. Subsequently, acetylserotonin methyltransferase (ASMT) converts N-acetylserotonin to melatonin through the process of methylation.
Figure 1. Pathway of melatonin synthesis in humans (Source: Ahmad SB, et al. 2023)
Melatonin is synthesized primarily at night and is bound in the bloodstream primarily to albumin, with peak plasma concentrations occurring around 3:00 to 4:00 AM. Due to its high fat and water solubility, it easily passes through most cell membranes, including the blood-brain barrier, and enters different body fluids and tissues. Melatonin is not produced in children until 3 months of age, peaks at 3-4 years of age, and gradually decreases by 80% in adulthood.
Melatonin exhibits diverse and pleiotropic effects, not only playing a dominant role in resistance to oxidative stress, but also influencing biological rhythms and reducing inflammatory states. Different species have evolved different pathways for melatonin biosynthesis, and the genes encoding the enzymes involved in these pathways vary.
References
1. Cipolla-Neto J, et al. Melatonin as a Hormone: New Physiological and Clinical Insights. Endocr Rev. 2018 Dec 1;39(6):990-1028.
2. Ahmad SB, et al. Melatonin and Health: Insights of Melatonin Action, Biological Functions, and Associated Disorders. Cell Mol Neurobiol. 2023 Aug;43(6):2437-2458.
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References
Melatonin improves skin barrier damage caused by sleep restriction through gut microbiota
It is widely known that lack of sleep damages the skin. Therefore, it is necessary to explore the relationship between sleep deprivation and skin damage and to find effective treatments. We established a 28-day sleep restriction (SR) mice model simulating continuous long-term sleep loss. We found that SR would damage the barrier function of mice's skin, cause oxidative stress damage to the skin, weaken the oscillations of the skin's biological clock, and make the circadian rhythm of Bacteroides disappear. The circadian rhythm of short-chain fatty acids (SCFA) receptors in the skin was disordered. After melatonin supplementation, the skin damage caused by SR was improved, the oscillations of the biological clock were enhanced, the circadian rhythm of Bacteroides was restored, and the rhythm of the receptor GPR43 of propionic acid was restored. We speculated that the improving effect of melatonin may be mediated by propionic acid produced by the gut microbiota. We verified in vitro that propionic acid could improve the keratinocytes barrier function of oxidative damage. We then consumed the gut microbiota of mice through antibiotics and found that oral melatonin could not improve skin damage. Moreover, supplementing mice with propionic acid could improve skin damage. Our research showed that lack of sleep impaired skin barrier function. Oral melatonin could improve skin damage by restoring the circadian rhythm of Bacteroides and its propionic acid metabolite.
Melatonin Engineering M2 Macrophage-Derived Exosomes Mediate Endoplasmic Reticulum Stress and Immune Reprogramming for Periodontitis Therapy
Adv Sci (Weinh)
Authors: Cui Y, Hong S, Xia Y, Li X, He X, Hu X, Li Y, Wang X, Lin K, Mao L.
Periodontitis is a chronic infectious disease caused by bacterial irritation. As an essential component of the host immunity, macrophages are highly plastic and play a crucial role in inflammatory response. An appropriate and timely transition from proinflammatory (M1) to anti-inflammatory (M2) macrophages is indispensable for treating periodontitis. As M2 macrophage-derived exosomes (M2-exos) can actively target inflammatory sites and modulate immune microenvironments, M2-exos can effectively treat periodontitis. Excessive endoplasmic reticulum stress (ER stress) and unfolded protein response (UPR) are highly destructive pathological characteristics during inflammatory periodontal bone loss. Although melatonin has antioxidant and anti-inflammatory effects, studies focusing on melatonin ER stress modulation remain limited. This study fabricates engineered M2-exos loading with melatonin (Mel@M2-exos) for treating periodontitis. As a result, M2-exos drive an appropriate and timely macrophage reprogramming from M1 to M2 type, which resolves chronic inflammation and accelerated periodontal healing. Melatonin released from Mel@M2-exos rescues the osteogenic and cementogenic differentiation capacity in inflammatory human periodontal ligament cells (hPDLCs) by reducing excessive ER stress and UPR. Injectable gelatin methacryloyl (GelMA) hydrogels with sustained-release Mel@M2-exos accelerate periodontal bone regeneration in rats with ligation-induced periodontitis. Taken together, melatonin engineering M2 macrophage-derived exosomes are promising candidates for inflammatory periodontal tissue regeneration.
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Figure 1. Pathway of melatonin synthesis in humans
