RU2536268C1 - Method for measuring maximum allowable blood concentration of heavy metals in children after integrated exposure - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention aims at asserting the maximum allowable blood concentrations (MAC) of heavy metals in the children living in the dirty environment as shown by health risk criteria after the chronic integrated exposure. An environmentally neglected zone is selected; a representative sampling of the children for the examination is drawn that is a basic group with using biological, social and hygienic criteria; the same criteria are used to draw a representative sampling of the children to a reference group living in the environmentally friendly zone. In the territory of the above zones, the chronic exposure of the analysed heavy metal is qualitatively assessed by establishing its average daily concentration in the ambient environment; the derived value is used to calculate a total average daily doses of a heavy metal supplied from various sources into a child's body averaged over the annual exposure for the children of both groups. Blood is sampled from the children every three months for one year to determine the content of the analysed heavy metal and also to measure the biochemical values of blood plasma and serum characterizing body responses presented by actual or potential health problems that are response markers. That is followed by calculating the average blood concentration of the analysed heavy metal and comparing it to the reference for the same heavy metal with using a Student two-sample test, thereby stating whether the children were sampled from the main and reference groups adequately. A mathematical modelling procedure is used to establish a relation between the exposure that is the total average daily doses of the analysed metal, and the exposure marker that is the average blood metal concentration. A sliding window technology is used to assert the response markers selected. The maximum allowable concentration of the exposure marker and respective marker is determined by a technique based on ratio analysis.

EFFECT: enabled measurement of the blood MAC of the heavy metals in the children after the integrated exposure with using sparing techniques making it possible to avoid a health risk.

4 tbl, 2 dwg, 1 ex

Description

The invention relates to medicine and is intended to justify the maximum permissible concentrations (MPC) of heavy metals in the blood of children living in a polluted environment, according to health risk criteria for chronic multi-media exposure. The results can be used to improve the planning of control and supervision measures for environmental objects and make managerial decisions to ensure sanitary and epidemiological safety of the population and protect consumer rights.

There is a method of determining the standard maximum permissible concentration of pollutants in water bodies (RF Patent No. 2480747), which includes the analysis of water samples taken at sections located in areas with proven environmental well-being, determining the average sample concentration of pollutants, while determining the maximum permissible concentration pollutants are carried out taking into account the minimum pollution according to the formula:

where C _n is the maximum permissible concentration of pollutants;

C _f - background concentrations of chemicals in watercourses;

- средняя концентрация вещества; $${\overline{\mathrm{FROM}}}_{\mathrm{from}f}$$

- the average concentration of the substance;

S _sf - the standard deviation of the concentration;

t _st - Student's coefficient at P = 0.95;

n is the number of samples taken at the site.

The specified known method provides an increase in the accuracy of determination.

However, this method is not intended to determine the MPC in the blood of a child.

A number of methods are also known for determining the MPC of heavy metals in air, namely: The method for determining the MPC of metal compounds in the atmospheric air of populated areas (Aut. St. USSR No. 1660681) and the Method for determining the MPC of aerosol vanadium pentoxide in atmospheric air (Aut. St. USSR No. 1140789).

According to the first method, the experiment is carried out on two groups of pregnant rats, one of which serves as a control, and the other is exposed to a toxic agent in a concentration whose value is an order of magnitude higher than the approximate safe level of exposure, determine the biochemical parameters in the blood serum of white rats, calculate the pair entropy of the average the values of the indicators in the experimental and control groups, find the average value of the pair entropy of the indicators and determine the MPC by mathematical expression.

According to the second method, inhalation is carried out on laboratory animals and the biochemical parameters in biological material are examined, and the animal is affected by 3-5 concentrations of vanadium pentoxide, lipids, proteins and enzymes are simultaneously determined in cadaveric organs on all animal groups and the MPC is determined by the magnitude of their changes .

However, the disadvantage of both of these methods is the impossibility of their use to determine the MPC of heavy metals in the blood of children, because It provides for the impact on the body of a deliberately high concentration of such a metal, which can cause irreparable harm to the health of the child.

The prior art has not identified sources of information about inventions that are fully relevant to the claimed technical object, as a result of which there is no prototype.

The technical result achieved by the present invention is to provide the ability to determine the maximum permissible concentration of heavy metals in the blood of children with multi-media exposure using gentle methods that exclude harm to the health of the child.

The specified technical result is achieved by the proposed method for determining the maximum permissible concentration of heavy metals in the blood of children with multi-medium exposure, according to which an environmentally disadvantaged area with a high load of heavy metals in the environment is selected with a hazard index for public health greater than 1; from this territory a representative sample of children is made for research - the main group, using biological, social, and hygienic criteria; using the same criteria, a representative sample of children is taken into the control group from a territory that is ecologically safe; in these territories, using the annual exposure, a chronic exposure of the studied heavy metal is quantified to establish the average daily concentration in the environment, then using the indicated average daily values the concentration of heavy metal in environmental objects is calculated for children of both groups averaged n annual exposure is the total average daily dose of heavy metal coming from various sources into the child’s body, while simultaneously determining the leading route of entry and the priority medium, then blood samples are taken once every three months for one year to establish the following parameters: for determine the content of the studied heavy metal and to determine the level of biochemical parameters of plasma and blood serum, which characterize the responses in the form of actual or potential total health disorders - response markers, under the influence of this heavy metal coming from the priority exposure medium; then, from the four results obtained during the year, the average concentration of the studied heavy metal in the blood is calculated and compared with the reference level for this heavy metal, using the two-student student criterion with a significance level of 0.05, while establishing the adequacy of the choice of children of the main and control groups ; at the same time, the indicated adequacy criterion for the control group of children is the absence of significant differences in the average concentration of the studied metal in the blood with the reference level, and for the main group of children - the presence of significant differences in the average concentration of the studied metal in the blood with the average concentration of this metal in the blood of the children of the control group; then, using the mathematical modeling method for children of each group, a relationship is established between the exposure — the total average daily dose of the studied metal entering the body from environmental objects, and the exposure marker — the average concentration of heavy metal in the blood, determining the validity of the selected exposure marker by the presence of a reliable connection with the exposure ; then, using the “sliding window” technology, the selected response markers are substantiated by establishing and assessing the dependence of the probability of deviation of the previously established biochemical parameters of plasma and blood serum in children of the main group relative to similar indicators in children of the control group, and from the physiological norm; Further, using the method based on the analysis of the odds ratio, the maximum permissible concentration of the exposure marker and the corresponding response marker are determined based on the condition under which the odds ratio indicator characterizing the strength of the connection between the influence of a heavy metal and the body's response will be greater than or equal to one, for this for each observation, a data table is formed: the value of the average concentrations of the studied metal in the blood and the values of the response markers for each observation and conditionally divide the data into two parts: below the current observation - the level of the average concentration of heavy metal in the blood and above the current observation; then, for both parts, a value is calculated that characterizes the probability of deviation of the response marker of the main group from the response marker in the control group, as the ratio of the number of observations that differ from the control to the total number of observations, and the OR odds ratio for each daily average concentration of the metal in the blood is determined from ratios:

where i is the index reflecting the observation number;

- вероятность отклонения маркера ответа основной группы от маркера ответа в контрольной группе в таблице в области ниже текущего наблюдения; $$p_i^{-}$$

- the probability of deviation of the response marker of the main group from the response marker in the control group in the table in the area below the current observation;

- вероятность отклонения маркера ответа основной группы от маркера ответа в контрольной группе в таблице в области выше текущего наблюдения; $$p_i^{+}$$

- the probability of deviation of the response marker of the main group from the response marker in the control group in the table in the area above the current observation;

moreover, the reliability of the calculated odds ratio indicator is estimated at a 95% confidence interval within which the true value of the odds ratio indicator is located, and the connection is recognized to be reliably established with a lower limit of the confidence interval greater than or equal to 1; then, a model is built of the relationship between the level of the exposure marker and the indicated indicator of the odds ratio, and the model is built on the basis of data on the average concentration of the studied metal in the blood and data on the corresponding value of the ratio of the chances of deviation of the response marker in the main group relative to the value of the response marker in the control group according to every observation; moreover, to establish the adequacy of this model, and hence the reliability of the data obtained, use the analysis of variance based on the calculation of the Fisher criterion and coefficient of determination, taking into account that the differences are considered statistically significant at p≤0.05; then calculate the reference maximum inactive concentration of heavy metal in the blood x ₀ for each marker of response according to the formula:

where a _1, a ₀ - model parameters,

and also, when constructing the indicated model, 95% confidence limits of point estimates of reference concentrations of the studied metal in the blood are determined, and the value of the upper 95% confidence limit for each response marker is taken as the reference concentration of the studied heavy metal in the blood; and as the maximum permissible concentration of the studied heavy metal in the blood of children, the smallest concentration of the available series of 95% upper confidence limits of the reference concentration of metal in the blood is taken for each response marker.

The technical result is achieved due to the following.

For an unambiguous understanding of the terms and definitions of the present invention, the following is their essence:

Exposure marker - an exogenous chemical substance or its metabolite, the amount of which is determined in the biological environments of the body.

Response marker - an indicator that quantitatively characterizes a biochemical, physiological, behavioral or other change in the body, the degree of severity of which determines the actual or potential impairment of health or the development of the disease.

Reference level - daily exposure to a chemical throughout life, which is established taking into account all available modern scientific data and probably does not lead to an unacceptable risk to the health of sensitive populations.

The external human environment is a combination of objects, phenomena and environmental factors that determines the conditions of human life.

Daily Average Dose / Concentration - potential daily dose / concentration averaged over the entire period of exposure to a chemical (in the invention, annual exposure).

Exposure (exposure level) - contact of the body (receptor) with a chemical, physical or biological agent.

The maximum permissible concentration (MPC) of heavy metals in the blood of children is the maximum permissible concentration of heavy metals in a unit of blood volume of children, due to chronic multiexposure, which does not cause a significant (with probability p≤0.05) daily exposure for an unlimited time body responses determined by health risk criteria.

Due to the fact that when implementing the proposed method, an ecologically unfavorable region is selected with a high load of chemical environmental factors with a criterion of the HI hazard index for public health greater than 1 (the criterion for the HI hazard index for public health is determined by the Guidelines for assessing public health risk when exposed to chemical substances polluting the environment ”(R 2.1.10.1920-04), the definition of residential areas in which the children’s population develops health disorders is provided ry from exposure to heavy metals is most likely.

The use of several criteria when selecting children for the main and control groups: biological, hygienic, social, allows you to create the most representative samples, the main factor of the difference in which is the presence of multi-media exposure of heavy metals.

The quantitative assessment of the chronic exposure of the studied heavy metal, during which the quantitative intake of metal into the body of children is established in various ways: by oral, inhalation, cutaneous, as a result of contact with various environmental objects (water, air, soil, food), is necessary in order to correctly calculate the value of the average daily dose of the studied metal.

Calculation of the total average daily dose, averaged over the annual exposure, for various routes of administration: inhalation, oral administration with drinking water, food, the studied heavy metal into the body using standard values of exposure factors and specific values of body weight and age of children included in the main and a control group, allows you to set the level of exposure to multi-media exposure of the studied metal during chronic exposure, taking into account the exposure time, individual Nost of children and priority admission path, which increases the information content of the proposed method. The specified calculation is performed according to the formulas presented in Appendix 3 of the Guidelines for assessing public health risk when exposed to chemicals that pollute the environment (R.2.1.10.1920-04).

A comparative analysis of the average daily doses of the test metal for children of the main and control groups, while establishing the contribution of each route of entry of the test metal to the total average daily dose, is necessary to determine the leading route of entry and the priority exposure environment, to determine the affected organs and systems at each route of entry and with complex admission simultaneously in several ways. This ensures almost complete consideration of priority chemical risk factors for impaired child health, which allows you to take into account their negative impact and increase the reliability of the determination.

Thanks to the use of venous blood samples as the test material, the simplicity and reliability of research, as well as obtaining the necessary information content, are ensured. The determination of the content of a chemical contaminant - a heavy metal in the blood is due to the fact that blood is the most homeostatic medium (controllability and controllability of the concentrations of its constituent components) and the only one with referenced constants (reference level) in relation to technogenic chemicals. And taking a blood sample from children from the main and control groups for 1 year with a frequency of 1 time in 3 months and determining the content of the studied metal in it allows you to quantitatively characterize its level and calculate the average concentration of the studied heavy metal in the blood, which makes the method more accurate and trustworthy.

Comparison of the obtained value of the average daily concentration of the studied metal in the blood sample for each observation (for each child in the sample) in the experimental and control groups with the reference level (the source of information on the values of the reference level is the “Guide to assessing public health risk when exposed to chemicals polluting habitat "R.2.1.10.1920-04), using the two-tailed student criterion (t) with a significance level of 0.05, ensures the establishment of an adequate choice of children and control groups. In this case, the indicated adequacy criterion for the control group of children is the absence of significant differences in the average concentration of the studied metal in the blood with a reference level, and for the main group of children - the presence of significant differences in the average concentration of the studied metal in the blood with the average concentration of this metal in the blood of the children of the control group,

Establishing a relationship between exposure — the total average daily dose of the studied metal from external factors and the average concentration of heavy metal in the blood, by mathematical modeling, is due to the need to calculate the exposure marker, which indicates the presence of a reliable relationship between the certified average concentration of metal in the blood and exposure, which increases the reliability of this way.

The justification of response markers is necessary in order to establish the degree of negative impact of the studied metal on critical organs and systems. And the implementation of this justification by establishing and assessing the dependence of the probability of deviation of the investigated laboratory (biochemical) indicators of responses in children of the main group relative to a similar indicator in children of the control group on the average concentration of the metal in the blood (exposure marker) using the “sliding window” technology simplifies way. For this, for each value of the concentration of the studied metal in the blood (x _i ), the probability of the deviation of the laboratory (biochemical) indicator from the value in the control (p _i ) calculated for the range (“sliding window”) is calculated:

x _i -δ <x≤x _i + δ,

where δ is the width of the "sliding window", which is determined from the ratio:

where N is the total number of studies for the entire population;

x _max - maximum concentration of the studied metal in the blood;

x _min - the minimum concentration of the studied metal in the blood.

The probability p _{i of the} deviation of the laboratory (biochemical) indicator of the main group from the control and from the physiological norm is estimated using the classical probability formula:

where m _i is the number of studies on the i-th laboratory parameter deviating from the value in the control for the range xi-δ <x≤xi + δ;

n _i is the total number of studies on the i-th laboratory parameter for the range xi-δ <x≤xi + δ.

A graphic illustration of the process of assessing the probability of deviation of a laboratory (biochemical) indicator from a physiological norm using a sliding window ”is presented in Figure 1.

The estimation of the parameters of the dependence of the probability of deviation of the laboratory parameter, relative to the physiological norm, on the average daily concentration of heavy metal in the blood is carried out by the method of constructing a logistic regression model:

where p is the probability of deviation of the laboratory parameter from the physiological norm;

x is the concentration of the chemical in the blood, mg / DM ³ ;

b ₀ , b ₁ - parameters of the mathematical model.

Due to the fact that the blood in each child from both groups determines the set of laboratory (biochemical) indicators characterizing specific and non-specific response of the body, taking into account the risk of illness of critical organs and systems with possible routes of entry of the studied metal into the body, the negative effect of the studied metal is justified to critical organs.

и $$p_i^{+}$$

соответственно) как отношение числа наблюдений, отличающихся от контроля, к общему числу наблюдений.The determination of the maximum permissible concentration of the exposure marker is carried out on the basis of the analysis procedure for calculating the odds ratio, based on the condition under which the odds ratio indicator OR, which characterizes the strength of the relationship between the effect of the chemical compound and the body's response, will be greater than or equal to one. Moreover, to calculate the odds ratio indicator for each observation, first create a data table: the average concentration of the studied metal in the blood and the values of the response markers for each observation (1 ... n), and conditionally divide the data into two parts in the indicated data table: below the current observations - the level of the average concentration of the test metal in the blood and above the current observation - the level of the average concentration of the test metal in the blood, for both parts calculate the value characterizing the probability of marker response deviations from laboratory values similar in the control group ( $$p_i^{-}$$

and $$p_i^{+}$$

respectively) as the ratio of the number of observations that differ from the control to the total number of observations.

A measure of the odds ratio for each average concentration of the studied metal in the blood is determined from the ratio:

where i is the index reflecting the observation number.

Moreover, the reliability of the calculated odds ratio indicator is estimated at a 95% confidence interval within which the true value of the odds ratio indicator is located, and the connection is recognized to be reliably established with a lower limit of the confidence interval greater than or equal to 1.

Due to the fact that we further construct a model of the relationship between the level of the exposure marker and the indicated indicator of the odds ratio, the model is constructed on the basis of data on the average concentration of the studied metal in the blood and data on the corresponding value of the indicator of the ratio of the chances of deviation of the response marker relative to the value in the control group for to each observation, the reliability of the obtained connection is ensured.

Moreover, to establish the adequacy of the model used, and hence the reliability of the data obtained, the analysis of variance is used, based on the calculation of the Fisher criterion (F) and the determination coefficient (R ² ), taking into account that the differences are considered statistically significant at p≤0.05.

Next, calculate the reference maximum inactive concentration of the studied metal in the blood (x ₀ ) for each response marker according to the formula:

where a _1, a ₀ - model parameters,

and when constructing the indicated model, 95% confidence limits of the point estimates of reference concentrations of the test metal in the blood are determined, and the value of the upper 95% confidence limit for each response marker is taken as the reference concentration of the test metal in the blood, and as the maximum the permissible concentration of the metal under study in the blood of children accept the lowest concentration of the available series of 95% upper confidence limits of reference metal concentrations in the blood for each response markers.

Thus, the achievement of the technical result is achieved due to the combination of all the features of the proposed invention and the exclusion of any will lead to the inability to implement the purpose of the method.

The proposed method is as follows, implementing it on a specific example:

1) Choose an ecologically unfavorable territory for a high aerogenic load with heavy metals. The city of Chusovoy was chosen as such a territory, characterized by the presence of priority components of emissions from industrial enterprises - manganese compounds in the atmospheric air of residential areas. According to the results of a previous risk assessment procedure for public health in a given territory, exposure to metals under aerogenic exposure forms an unacceptable risk with respect to the respiratory system, central and autonomic nervous system (health hazard index HI = 8-44 with an acceptable level of ≤1.0).

2) Generate comparable samples of children on the basis of biological criteria: I-II health group, which is established on the basis of the results of the analysis of medical records (form 26 / U) and a clinical examination; physiological course of pregnancy and childbirth in the mother; lack of pathology of the perinatal period; lack of burdened hereditary history; Ketle index weight-growth indicators, not exceeding ± 15%; the absence of acute infectious diseases for at least 3 weeks before the start of the study; an infectivity index of 0.2-0.5; based on social criteria: income level, family lifestyle, quality of life; the parents of the children included in the sample must have secondary and higher education, which determines the family lifestyle as a comparable, average level of material security, housing conditions that comply with established hygiene standards; based on hygienic criteria: the samples are distinguished by the presence of manganese in the atmospheric air of the territory of the children in the study sample and the absence of a sample of the control group in the territory of residence; according to the presence of common compounds, territories are comparable. The main group was selected 80 children from an organized team living in the city of Chusovoy. As a control group, 50 children from an organized team living in the town of Ilyinsky were selected.

3) In the selected territories, the chronic exposure of the studied heavy metal is quantified using the annual exposure to establish the average daily concentration of it in the air and drinking water based on monitoring observations, according to field studies. The average daily concentration of manganese in the atmospheric air of an ecologically unfavorable territory was 0.0011 ± 0.0003 mg / m ³ (1.1 percent of MAC), in the comparison territory it was 0.00003 ± 0.00001 mg / m ³ (0.03 share MPC). The average daily concentration of manganese in drinking water in an ecologically unfavorable territory was 0.02 ± 0.003 mg / m ³ (0.2 percent of MPC), in the comparison territory it was 0.01 ± 0.002 mg / m ³ (0.1 percent of MPC).

4) Using the formula given in Appendix 3 of the Guidelines for assessing public health risk when exposed to chemicals that pollute the environment (R.2.1.10.1920-04), the total average daily dose of heavy metal from various sources in the body is calculated children's population. In an ecologically unfavorable territory, the average daily dose of manganese when ingested with atmospheric air was 9.0 · 10 ^-4 mg / (kg · day), with drinking water - 1.1 · 10 ^-4 mg / (kg · day). The total average daily dose was 0.001 mg / (kg · day), and atmospheric air is a priority route of entry. In the comparison territory, the average daily dose of manganese upon intake with atmospheric air was 2.9 · 10 ^-5 mg / (kg · day), with drinking water - 5.5 · 10 ^-5 mg / (kg · day). The total average daily dose of 8.4 · 10 ^-5 mg / (kg · day).

5) In children from these groups, venous whole blood is sampled in one test tube once every 3 months for 1 year (i.e. four times a year) to determine the manganese content in whole blood. In the last sampling of blood, three test tubes are used: the first to determine the metal content, the second to determine the level of biochemical parameters of blood serum, the third to determine the level of biochemical parameters of blood plasma, a list of which is established taking into account the identified affected organs and systems characteristic of the negative impact of the studied heavy manganese metal and the priority route of its entry.

6) In whole blood, the level of manganese is determined on a Perkin Elmer 3110 atomic absorption spectrophotometer using an acetylene-air mixture as an oxidizing agent with flame atomization detection.

In blood serum, taking into account critical organs during chronic inhalation of manganese, the level of lipid hydroperoxides, the level of total immunoglobulin E (IgE), specific IgE specific for manganese, the level of activity of cytoplasmic superoxide dismutase Cu / Zn-SOD, immunoglobulins G (IgG), A (IgA) are determined cyclic adenosine monophosphate c-AMP; cyclic gluanidine monophosphate c-GMP. In the blood plasma determine the level of malondialdehyde (MDA), an indicator of antioxidant activity (AOA).

7) Calculate the average concentration of manganese in the blood of children during the year (i.e., the sum of the four indicators obtained over the years is divided into four). The data obtained are compared with the reference level. The results are presented in table 1.

The data shown in table 1 indicate the validity of the sample of children of the main and control groups, because in the control group there are no significant differences in the concentration of manganese in the blood with a reference level (p = 0.065, i.e., more than 0.05), and in the main group, compared with the control group, p = 0.001, i.e. less than 0.05.

8) Next, a reliable relationship is established between the exposure - the total average daily dose of the studied metal entering the body from environmental objects, and the exposure marker - the average concentration of heavy metal in the blood, determining the validity of the selected exposure marker, using mathematical modeling, by the presence of a reliable connection with the exposure . Identification and evaluation of the parameters of this dependence made it possible to obtain adequate models (F≥3.96, p≤0.05) of the relationship between the average concentration of manganese in the blood and the total average daily dose for inhalation and oral administration to the body (in the range of the studied concentrations for the same period) observations) (F = 2055.12, R ² = 0.64, p = 0.0001). The dependence of the average concentration of manganese in the blood of children on the total average daily dose of manganese during a chronic complex exposure in the territory with the metallurgical production is shown in Figure 2. Based on the data obtained, the average concentration of manganese in the blood of children of the main group is taken as a marker of exposure to chronic exposure to manganese.

9) To substantiate the response markers, a laboratory examination of the children of the main and control groups is performed (through the determination of biochemical parameters of plasma and blood serum). The results are presented in table 2.

Analysis of the results shown in table 2, allowed to establish an increase in nonspecific sensitivity of the body (sensitization) by a reliable increase of 1.3 times the average level of total IgE in blood serum (103.2 ± 15.6 IU / cm ³ ) relative to the indicator in children control group (79.4 ± 4.38 IU / cm ³ , p = 0.001), in 94% of cases exceeding the physiological norm. The initial activation of the metabolic oxidation process was established by the level of lipid hydroperoxides in blood serum (362.11 ± 8.6 μmol / dm ³ ), which was 1.2 times higher than in children in the control group (301.7 ± 5.23, p = 0.02) and in 93% of cases exceeded the physiological norm limit. These deviations of indicators characterize the development of negative effects - initial sensitization and activation of metabolic oxidation. A change in the activity of intracellular regulatory intermediaries in increasing c-AMP in serum (9.2 ± 0.24 μmol / dm ³ ) was 1.2 times relative to this indicator in the control group (7.73 μmol / dm ³ , p = 0.005 ) and a decrease in c-GMF in blood serum (2.8 ± 0.11 μmol / dm ³ ) 1.2 times relative to the control group (3.35 ± 0.14 μmol / dm ³ p = 0.005). This indicates the activation of the elements of sympathetic regulation at the molecular level. A reliable 1.4-fold excess of the average level of Cu / Zn SOD (102.4 ± 2.42 ng / cm ³ ) in blood serum relative to that of the control group (72.00 ± 2.43 ng / cm ³ ) was recorded. In 21% of cases, this indicator exceeded the physiological norm, which indicates the activation of the antioxidant link.

10) Perform the analysis procedure for calculating the odds ratio, an indicator that characterizes the strength of the relationship "exposure marker - marker response adverse effects." The results are presented in table 3.

11) A model is built of the relationship between the level of the exposure marker and the established indicator of the odds ratio with the procedure for calculating the maximum inactive average metal concentration in the blood for each response marker. The results are presented in table 4.

Of the obtained series of permissible concentrations of manganese in the blood for each probable negative response, the lowest concentration (at the lower 95% confidence limit) is 0.015 mg / dm ³ (the limiting indicator is the probability of an increase in total immunoglobulin E in blood serum). This concentration can be recommended as a daily allowable concentration of manganese in the blood of children for conditions of chronic multiple exposure.

Thus, the proposed method provides a reliable determination of the maximum permissible concentration of heavy metals in the blood of children. The knowledge of this information will increase the efficiency of the complex of sanitary and hygienic measures in the territories with the placement of metallurgical and machine-building production facilities and will make it possible to assess the real exposure of the population.

Table 1 The average concentration of manganese in the blood of children throughout the year Metal Group The concentration of manganese (M ± m), mg / DM ³ Reference level (RL), mg / dm ³ RL Shares Significance of differences (p≤0,05) with a control group with reference level control 0.010 ± 0.002 0.9 - 0,065 Manganese the main 0.042 ± 0.006 0.011 ± 0.002 3.8 0.001 0.0001

table 2 Laboratory (biochemical) indicators in children with a high content of manganese in the blood, p≤0,05 Structural level Indicator Control group Main group Significance of differences (p) Mean value and error of the mean (M ± m) The frequency of registration of samples with a deviation from the physiological norm,% Mean value and error of the mean (M ± m) The frequency of registration of samples with a deviation from the physiological norm,% below above below above Molecular c-AMP in serum, pmol / cm ³ 9.28 ± 0.24 0,0 8.0 7.73 ± 0.73 6.8 9.0 0.01 c-GMF in serum, pmol / cm ³ 2.82 ± 0.11 0,0 0,0 3.35 ± 0.14 6.8 2,3 0.05 IgE total in blood serum, ME / cm ³ 103.2 ± 15.6 0,0 94.0 79.38 ± 4.38 0,0 33.5 0.001 IgE spec. serum manganese, ME / cm ³ 1.61 ± 0.03 0,0 14.0 1.61 ± 0.05 0,0 16 > 0.05 Antioxidant activity of blood plasma,% 37.11 ± 1.03 4.0 14.6 42.02 ± 1.95 15.0 45.0 > 0.05 Cu / Zn superoxide dismutase in serum, ng / cm ³ 102.36 ± 2.42 0,0 21.0 72.0 ± 2.43 0,0 12.3 0.001 Plasma malondialdehyde, μmol / cm ³ 1.84 ± 0.15 0,0 14.0 2.59 ± 0.11 25.0 0,0 > 0.05 Hydroperoxide of lipids in blood serum, mmol / dm ³ 362.11 ± 8.61 0,0 93.0 301.7 ± 5.23 19.3 19.3 0.02 IgG in serum, g / dm ³ 12.53 ± 0.21 2.0 3.0 10.22 ± 0.25 13.1 18.5 > 0.05 IgA in serum, g / dm ³ 1.45 ± 0.1 5,0 7.0 1.34 ± 0.07 18.5 19,2 > 0.05

Table 3 An odds ratio indicator characterizing the relationship between deviations in response marker indicators and the average concentration of manganese in the blood of children Response Marker Score Group The average concentration of manganese in the blood, mg / DM ³ Odds Ratio (OR) 95% confidence interval (CI) Lowering serum c-AMP control 0.010 ± 0.002 0.17 0.104 ÷ 0.34 the main 0.042 ± 0.006 1.43 1.24 ÷ 1.90 Increased serum c-GMF control 0.010 ± 0.002 0.10 0,094 ÷ 0,11 the main 0.042 ± 0.006 0.52 0.494 ÷ 0.56 Increased total IgE in serum control 0.010 ± 0.002 0.57 0.554 ÷ 0.60 the main 0.042 ± 0.006 2.17 2.044 ÷ 2.31 Increased IgE spec. to serum manganese control 0.010 ± 0.002 0.27 0.154 ÷ 0.35 the main 0.042 ± 0.006 1.82 1,564 ÷ 2,45 decrease in antioxidant activity of blood plasma control 0.010 ± 0.002 0.27 0.124 ÷ 0.42 the main 0.042 ± 0.006 1,56 0.934 ÷ 1.83 Lowering Serum Cu / Zn Superoxide Dismutase control 0.010 ± 0.002 0.30 0.284 ÷ 0.35 the main 0.042 ± 0.006 0.66 0.624 ÷ 0.69 Increased plasma malondialdehyde control 0.010 ± 0.002 0.36 0,054 ÷ 0,40 the main 0.042 ± 0.006 1.45 1.244 ÷ 1.90 Increased serum lipid hydroperoxides control 0.010 ± 0.002 0.82 0.754 ÷ 0.95 the main 0.042 ± 0.006 1.10 1,054 ÷ 1,15

Table 4 Parameters of models of the relationship of the odds ratio of deviations of response markers from the average daily concentration of manganese in the blood of children of the main group (p≤0.05) Response marker Direction change indicator Model Parameters Fisher test (F) Coefficient of determination (R ² ) The concentration of manganese in the blood, mg / DM ³ a ₀ a ₁ reference level (upper 95% confidence boundary of the model) lower 95% confidence limit of the model Biochemical and immunological parameters Total serum IgE increase -0.92 57.50 8956.5 0.63 0.015 0.017 Serum lipid hydroperoxides increase -0.15 7.89 1789.1 0.35 0.017 0,021 IgE spec. to serum manganese increase -0.22 6.47 4094.3 0.63 0,032 0,036 serum c-AMP decline -1.51 41.94 1437.77 0.68 0,034 0,038 MDA in blood plasma increase -0.04 1,03 3876.4 0.71 0,036 0,042 AOA in plasma decline -0.37 7.55 652.5 0.66 0,050 0.051

Claims (1)

A method for determining the maximum permissible concentration of heavy metals in the blood of children with multi-media exposure, characterized in that an environmentally disadvantaged area with a high load of heavy metals in the environment is selected with a criterion of the hazard index for public health greater than 1; from this territory a representative sample of children is made for research - the main group, using biological, social, and hygienic criteria; using the same criteria, a representative sample of children is taken into the control group from a territory that is ecologically safe; in these territories, using the annual exposure, a chronic exposure of the studied heavy metal is quantified to establish the average daily concentration in the environment, then using the indicated average daily values the concentration of heavy metal in environmental objects is calculated for children of both groups averaged n annual exposure is the total average daily dose of heavy metal coming from various sources into the child’s body, while simultaneously determining the leading route of entry and the priority medium, then blood samples are taken once every three months for one year to establish the following parameters: for determine the content of the studied heavy metal and to determine the level of biochemical parameters of plasma and blood serum, which characterize the responses in the form of actual or potential total health disorders - response markers, under the influence of this heavy metal coming from the priority exposure medium; then, from the four results obtained during the year, the average concentration of the studied heavy metal in the blood is calculated and compared with the reference level for this heavy metal, using the Student’s two-sample criterion with a significance level of 0.05, while establishing the adequacy of the choice of children of the main and control groups ; at the same time, the indicated adequacy criterion for the control group of children is the absence of significant differences in the average concentration of the studied metal in the blood with the reference level, and for the main group of children - the presence of significant differences in the average concentration of the studied metal in the blood with the average concentration of this metal in the blood of the children of the control group; then, using the mathematical modeling method for children of each group, a relationship is established between the exposure — the total average daily dose of the studied metal entering the body from environmental objects, and the exposure marker — the average concentration of heavy metal in the blood, determining the validity of the selected exposure marker by the presence of a reliable connection with the exposure ; then, using the “sliding window” technology, the selected response markers are substantiated by establishing and assessing the dependence of the probability of deviation of previously established biochemical parameters of plasma and blood serum in children of the main group relative to similar indicators in children of the control group and from the physiological norm; Further, using the method based on the analysis of the odds ratio, the maximum permissible concentration of the exposure marker and the corresponding response marker are determined based on the condition under which the odds ratio indicator characterizing the strength of the connection between the influence of a heavy metal and the body's response will be greater than or equal to one, for this for each observation, a data table is formed: the value of the average concentrations of the studied metal in the blood and the values of the response markers for each observation and conditionally divide the data into two parts: below the current observation - the level of the average concentration of heavy metal in the blood and above the current observation; then, for both parts, a value is calculated that characterizes the probability of deviation of the response marker of the main group from the response marker in the control group, as the ratio of the number of observations that differ from the control to the total number of observations, and the OR odds ratio for each daily average concentration of the metal in the blood is determined from ratios:
$$O{R}_i=\frac{p_i^{+}}{\mathrm{one}-p_i^{+}}/\frac{p_i^{-}}{\mathrm{one}-p_i^{-}}$$

where i is the index reflecting the observation number;
$$p_i^{-}$$

- the probability of deviation of the response marker of the main group from the response marker in the control group in the table in the area below the current observation;
$$p_i^{+}$$

- the probability of deviation of the response marker of the main group from the response marker in the control group in the table in the area above the current observation;
moreover, the reliability of the calculated odds ratio indicator is estimated at a 95% confidence interval within which the true value of the odds ratio indicator is located, and the connection is recognized to be reliably established with a lower limit of the confidence interval greater than or equal to 1; then, a model is built of the relationship between the level of the exposure marker and the indicated indicator of the odds ratio, and the model is built on the basis of data on the average concentration of the studied metal in the blood and data on the corresponding value of the ratio of the chances of deviation of the response marker in the main group relative to the value of the response marker in the control group according to every observation; moreover, to establish the adequacy of this model, and hence the reliability of the data obtained, use the analysis of variance based on the calculation of the Fisher criterion and coefficient of determination, taking into account that the differences are considered statistically significant at p≤0.05; then calculate the reference maximum inactive concentration of heavy metal in the blood x ₀ for each marker of response according to the formula:
$$x_0=\frac{a_0}{a_{\mathrm{one}}}$$

mg / dm ³
where a _1, a ₀ - model parameters,
and also, when constructing the indicated model, 95% confidence limits of point estimates of reference concentrations of the studied metal in the blood are determined, and the value of the upper 95% confidence limit for each response marker is taken as the reference concentration of the studied heavy metal in the blood; and as the maximum permissible concentration of the studied heavy metal in the blood of children, the smallest concentration of the available series of 95% upper confidence limits of the reference concentration of metal in the blood is taken for each response marker.

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