Free Melatonin Research Articles

Alterations in the circadian rhythm of salivary melatonin begin during middle-age.

To investigate whether free melatonin may be better suited to reveal age-related changes, we studied the circadian rhythm alterations in saliva melatonin levels during aging. Special attention was paid to the question as to how the free melatonin rhythms change in aging and when such changes take place. A total of 52 healthy volunteers participated in the study consisting of young, middle-aged, old and the oldest groups. In each subject, a total of 12 time-point salivary melatonin samples was taken over 24 hr. Of the 52 data sets, 51 exhibited significant circadian rhythm over 24 hr by using the base cosine function analysis to fit the data. A clear circadian rhythm of salivary melatonin was present in all age groups. The decline in nocturnal peak levels (amplitude) in salivary melatonin was found in old and the oldest subjects. Both the old and the oldest subjects showed an increased daytime (baseline) melatonin levels. The off-set melatonin levels were more than two times higher in the oldest group than that in the other groups indicating a delayed phase of salivary melatonin. Most strikingly, we found that a step-wise decrease in the circadian rhythms of saliva melatonin occurred early in life, around 40 yr of ages. The middle-aged subjects had only 60% of the amplitude of the young subjects. In addition, the middle-aged subjects showed the longest peak levels duration and the lowest daytime melatonin levels. The present study showed that the alterations in the circadian rhythms of salivary melatonin begin during middle-age. Our results showed that salivary melatonin measurement is a reliable, sensitive and easy method to monitor changes in the circadian rhythms of melatonin during the course of aging.

Determination of free and total (free plus protein-bound) melatonin in plasma and cerebrospinal fluid by high-performance liquid chromatography with fluorescence detection

A simple, sensitive and accurate method for the estimation of free and total (free plus protein-bound) melatonin (MLT) in human plasma and cerebrospinal fluid (CSF) is described. Via Chem-Elut cartridges, free and total MLT (the latter obtained after a deproteinization step) were quantified in dichloromethane-extracted samples and analyzed in one chromatographic run by high-performance liquid chromatography (HPLC) with fluorimetric detection. The column used was an Extrasil ODS-2 (3 μm, 150×4.6 mm I.D.), while the mobile phase consisted of 75 m M sodium acetate–acetonitrile (72:28, v/v) (pH 5.0). Repeatability and reproducibility of the method were 3.24 and 9.4%, respectively. The recovery of melatonin from plasma and CSF was 99.9±4.0% for non-deproteinized samples and 93.2±4.8% for deproteinized samples. The detection limit of the assay was 0.5 pg/ml. In human plasma, the mean±SD concentrations in the darkness period were 23.18±7.44 pg/ml for free melatonin and 82.5±36.48 pg/ml for total melatonin, while the lowest concentrations detected during daytime were 2.23±2.22 and 7.40±5.68 pg/ml, respectively. Detection of MLT in CSF was 5.01±2.31 and 28.55±6.95 pg/ml for the free and total fraction, respectively.

Detection of specific protein bands with melatonin-like immunoreactivity in different cell lines and human brain regions.

The pineal hormone melatonin regulates various neural and endocrine processes involved in mammalian circadian rhythms. To understand how melatonin mediates these functions, we investigated melatonin-like immunoreactivity (MLI) in cell extracts and human brain. In Western immunoblots, we detected high-molecular-mass protein bands (85-135 kDa) that specifically reacted with the anti-melatonin antibody. The specific protein bands were present in cell extracts from the human brain and cell lines of different origins. The immunoreactive signal of the 135-kDa protein band was highest in a neuroendocrine PC12 cell line, which was 10-fold higher than the signal observed in any cell extracts used. The commercial antibody employed in the Western blots was further purified against serum proteins and thyroglobulins. We have previously reported that the antibody against melatonin recognizes MLI as detected by a sensitive RIA. In the present report we have detected the putative melatonin-specific binding proteins, which could contribute to the MLI. Our results suggest that melatonin binds with specific proteins in different cellular and brain extracts, the protein(s) being maximally synthesized in PC12 cells. These results may indicate a group of yet unknown proteins sharing a melatonin-like epitope or the presence of melatonin-binding protein(s) that regulate availability of free melatonin, or both.

Circadian Rhythm of Free Melatonin in Human Plasma

Circadian Rhythm of Free Melatonin in Human Plasma

Radioimmunoassay of bound and free melatonin in plasma

We describe a nonextraction procedure, and two extraction procedures, for RIA of melatonin in human plasma. All procedures showed a diurnal rhythm of melatonin in human subjects, with interindividual differences greater than interprocedure differences. However, further investigations demonstrated considerable variability of recovery in the nonextraction procedure, suggesting a variability of binding proteins between samples. Combining recovery and dialysis experiments in the extraction procedures, we demonstrated that chloroform was unable to extract albumin-bound melatonin from a human serum albumin solution but, paradoxically, was able to extract bound and free melatonin from a plasma sample. The methanol extraction procedure extracted free and bound melatonin from all sources. These results indicate that albumin binding can substantially affect the RIA procedures. We conclude that assays should be validated against free and bound melatonin and that the two forms should be independently investigated when assessing bioactivity.

Gas-chromatographic determination of melatonin and 6-hydroxymelatonin

Gas-chromatographic methods for the determination of melatonin and 6-hydroxymelatonin have been developed and retention times established for one non-polar and three polar columns. Efforts to detect melatonin and 6-hydroxymelatonin (free or conjugated as the glucuronide or sulphate) in normal human urine and in the urine of two patients with parenchymatous pinealoma were unsuccessful. The conditions of the analysis were such that the normal excretion of free melatonin and free 6-hydroxymelatonin was estimated to be less than 10 μg per day.