Melatonin (N-acetyl-5-methoxytryptamine) is a naturally occurring hormone found in animals, plants, and microbes. In humans, melatonin is produced primarily by the pineal gland, located in the center of the brain. Melatonin forms part of the system that regulates the sleep-wake cycle by chemically causing drowsiness and lowering the body temperature. Production of melatonin by the pineal gland is inhibited by light and permitted by darkness. Secretion of melatonin, as well as its level in the blood, peaks in the middle of the night and gradually falls during the second half of the night, with normal variations in timing according to an individual’s chronotype. (1,2) Melatonin is also important for its ability to scavenge free radicals and to regulate the activity and expression of antioxidant and pro-oxidant enzymes. (3,4,5)
In human plasma 61-85% of melatonin is weakly bound to proteins, and there is a close relationship between circulating free and salivary melatonin levels. (6) Melatonin enters saliva from blood either by passive diffusion or active transport. (7) Melatonin is measurable in saliva, and the acrophases of saliva and plasma melatonin rhythms are significantly correlated. (8,9) Plasma and salivary melatonin concentrations increase when moving from a supine to a standing position, and decrease when these positions are reversed, due to changes in plasma volume. (10) Inflammatory processes such as periodontitis trigger an increase in plasma melatonin, which then increases melatonin levels in the oral cavity, where it may increase antioxidant protection. (11)
Due to a strong correlation with serum melatonin concentrations, analysis of salivary melatonin effectively enables researchers to design non-invasive studies that assess circadian timing and evaluate the degree of circadian phase abnormalities in association with major diseases, conditions, administration of pharmaceutical compounds, seasonal affective disorder, jet-lag and night shift work, etc. It also pertains to the clinical aspect of improving diagnosis and treatment of irregular sleep-wake disorders via chronotherapy using exogenous melatonin or altered light exposure. Research of melatonin dysregulation affects multiple body-wide systems and often results in chronic sleep deficits with symptoms that can be misdiagnosed as depression, attention deficit hyperactivity disorder, fibromyalgia, or chronic fatigue.
With saliva as a biological sample, researchers can non-invasively monitor melatonin levels without an overnight sleep clinic stay (polysomnogram). This eliminates the rhythm perturbation caused by a clinical sleep center laboratory, can be performed in 4-6 hrs (as opposed to 24 hrs), and offers the convenience of collecting samples at home. Salivary DLMO is determined by measuring salivary melatonin levels over several time-points prior to and just after habitual sleeptime. Calculating the time that the nocturnal melatonin concentration is significantly elevated from daytime samples (+/- 2 deviations from baseline levels)- DMLO is the most sensitive and direct index for identifying an individual’s biorhythm which may be entrained (synchronized) to a 24 hr light/dark cycle or can be in a free-running state. A misaligned DMLO may indicate delayed sleep phase syndrome (DSPS) or advanced sleep phase disorder (ASPD) and its relationship to various metabolic conditions, diseases, disorders and genetic and environmental components are of significant interest in current research.
The effects of freeze thaw on most biological measures, regardless of biospecimen type, can be dramatic. Analytes in oral fluid are not distinct or different in this way. As a general rule, multiple freeze-thaws should be avoided. The most practical way to address this concern is by aliqouting samples after collection. Some analytes are more resistant to freeze thaw than others. We recommend that investigators consult the literature for the analytes of interest. If there is freeze thaw data for a specific biological measure in traditional biospecimens, it is reasonable to assume this would also be true for saliva.
No, but the literature is rather extensive on this subject for several salivary analytes; especially for salivary alpha-amylase and cortisol. We do not track that information internally.
With modern search tools online, we no longer maintain records of this type. We suggest that you use Pubmed or Psychlit to search the literature or you can Ask An Expert and we will be happy to assist you in your search.
If the analyte you are interested in is not noted in our website, please contact Dr. Granger at [email protected] to find out what developments are in the pipeline.
Salimetrics does not release products for sale if the quality control (QC) testing shows any issues. Here are some probable causes:
1) Can timing of adding reagents be off? For instance with a multichannel you can pipette the conjugate and TMB so many times before you refill, but you can pipette the stop twice as fast because it is a smaller volume. If you do this, it shortens the time the bottom rows have with TMB.
2) Can your washer be uneven and sheering off antibody in the bottom corner? Aspirate and check the amount of fluid left. It should be even in all wells and no wells should be completely dry.
3) Are you mixing faster than recommended? Or slower?
4) Are all reagents completely at room temperature? A bottle of assay diluent takes 2 hours to come to RT. You can pour some off into a smaller tube to warm up quicker for the zero and nsb.
5) Are you leaving the plate come to room temperature BEFORE opening the bag? (Otherwise moisture due to humidity may form in the wells and this is particularly a problem in this high humidity weather)
6) Are your multichannel pipettes dispensing the same amount each time reliably? We discard the first and last dispenses as they are not as reliable.
7) Are you incubating with TMB in the dark? (We no longer recommend aluminum foil.)
8) Are you testing one plate at a time? For example, do not put the standards on 5 plates then go back and fill in with samples. This delays the addition of conjugate to the plates.
9) Clean your plate reader filter. Dust from the lab can collect on the filter.
10) Are you adding assay diluent to the zero in sequence after the standards and not the last thing?
11) Never put the multichannel pipette tips into the wells as you can drag down standard from the wells above it causing lower readings in other wells.
12) Thoroughly blot all wells just before adding TMB but do not let the plate dry out.