HOW RELIABLE ARE CALIBRATORS FOR URINARY MELATONIN SULFATE ?

Melatonin is a versatile physiological agent that relating to the control of numerous physiologic processes owing to the multiplicity of sites of formation and the presence of melatonin receptors in various places of a human body. The melatonin metabolite 6-sulphatoxymelatonin (alias aMT6s or 6-SMT) infiltrates in urine. Therefore, there is a sense to find the sensitive and selective express-methods for the precise detection of melatonin metabolites in body fluids. The calibrators are proper tools for the signal processing. In the present research work that is the set of few calibers with well-defined concentrations of analyte. Comparison of the measured response of actual sample with reactions of calibers permits to interpret the real signal. Such a response can be either the transmittance or the optical density of sample that are measured by a photometer. The calibration of dose-response relation has been described by logistic Hill’s curve with four parameters. The photometric transmittances of analyzed solutions were considered as the response. This paper describes the evaluation and comparison of sets of Hill’s parameters for aMT6s-calibrators of independent producers. Reliable calibrators could help to simplify the analysis of urinary melatonin-sulfate dose. Such express-analysis could be more accessible in addition. Hence, the problem of creation of reliable calibrators is still unsolved and challenging.

Introduction.Melatonin is a versatile physiological agent that relating to the control of numerous physiologic processes owing to the multiplicity of sites of formation and the presence of melatonin receptors in various places of a human body [4].Existing data suggest that melatonin is affecting human physiology overall, including the sleep-wake cycle, via the body's internal clock [5].It's established fact that melatonin is present in saliva or blood plasma, and the melatonin metabolite 6-sulphatoxymelatonin (alias aMT6s or 6-SMT) infiltrates in urine [4,5].Therefore, the search of sensitive and selective express-methods for the precise detection of melatonin metabolites in body fluids looks as worthy aim.
One of the most popular modern methods of melatonin detection in the urine is enzyme-linked immunosorbent assays (ELISA) [2,[7][8][9].Such a more or less complicated biochemical test measures the presence and concentration of a macromolecule in a solution with high sensitivity.The package of reagents for urinary Melatonin-Sulfate analysis is quite expensive yet, and this test requires the special qualification of staff.
Often, the calibrators are proper tools for the signal processing.In our case, that is the set of few solutions (calibers) with well-defined concentrations of analyte.Comparison of the measured response of actual sample with reactions of calibers provides possible to interpret the real signal.Such a response can be either the transmittance or the optical density of sample [2,[6][7][8][9] that are measured by a photometer.It would certainly simplify the analysis of urinary melatonin-sulfate dose, if we had reliable calibrators.Such express-analysis could be more accessible in addition.
The calibration of dose-response relationship has been described most frequently by logistic Hill's curve with four parameters [1,3].The goal of this paper is the computing and comparison of sets of Hill's parameters for aMT6s-calibrators of independent producers.
Materials and methods.Two different methods were used for preparation and testing of calibers with known dosage of aMT6s in human urine.The first of them is above mentioned enzyme-linked immunosorbent assays (ELISA) [2,[8][9].Another is radioimmunoassay (RIA) that commonly recognized as extremely sensitive assay technique [6].
Here should be underlined that a number of authors are using the same set of calibers tested by ELISA within different laboratories [2,3,7,8].Other authors are using different sets of calibers that tested by different methods (ELISA and RIA) but within one laboratory [6,9].Therefore, all of them were dealing just with three different calibrators.
Theory and calculations.The Hill's logistic equation looks so [1]: Here Y is the expected response at dosage x.Parameter a is the minimum asymptote or the response when x -> ∞.Whereas b is the maximum asymptote or the stabilized response for a zero dosage x -> ∞.Parameter c defines the point of inflection in the dosage -response curve and is denoted by various terms (e. g.EC 50 , ED 50 , LD 50 , IC 50 ).The last parameter d is the slope at the steepest part of the curve.This factor is also known as the Hill's slope [1].The Fig. 1 demonstrates the Hill's logistic curve with descriptions of these parameters.

МЕДИЧНА ІНФОРМАТИКА МЕДИЧНА ІНФОРМАТИКА ТА ІНЖЕНЕРІЯ
Equation (1) describes ascending or descending logistic curve depending on sign of Hill's slope.We expected the descending type and so probably d < 0 [3].
There are different ways of determination of Hill's parameters [1,3].We used the computing technique [3] as the program package Statistics of Maple 17.That allowed us to fit of above Hill's parameters to experimental data of different producers.
The Fig. 2 presents three dosage-response curves where the response means the optical transmittance of analyte solution (B/B0).All logistic curves are descending (that is d < 0).
Both, Fig. 2 and Table 1, show the significant divergence between data [2,7,8] and data [6,9].The third coefficient c (alias EC50, ED50, LD50, IC50) causes mainly this large difference.Its value varies from 5.1 to 30.9 (about six times).Obviously, the difference of ELISA and RIA methods of dose measuring is «too much».The reasons of such mismatch are still unexplained.Note that the results of RIA and ELISA demonstrated first-rate accordance if we dealt with data of one laboratory [6,9].
The rest three coefficients of    [2,7,8] the diamonds present data [9] and the boxesdata [6] МЕДИЧНА of them was close to zero, the second one was near to 100 and the third coefficient was negative.Obtained results were expected.
Conclusions.There is the evident mismatch among calibrators of [2,7,8] and those of [6,9].The main Hill's parameter c that denoted also as EC50, ED50, LD50, IC50 is responsible for this variance, and the difference looks unexplainably large.
The set of calibers as well as the method of calibrating (i.e. ELISA) are the same despite on different sources [2,7,8].Opposite, the methods (ELISA and RIA) and sets of calibers were different as for competing results of [6,9] though both are from the same laboratory.These two methodically independent results of one laboratory are in good agreement.
Therefore, the titling question of this paper is still open because photometry of the human urine may give very different results as for Melatonin-sulfate dose nowadays.In the same time, the results of measurements should not depend on the calibrating set that would be in use.Thus, the reliable calibrators of urinary Melatonin-Sulfate are still wanted.Table 1 Hill's parameters