Method for determination of manganese toxicity in children being residents of technogenically-loaded territories

FIELD: medicine.

SUBSTANCE: whole blood is examined for a manganese level and two laboratory values, namely: a plasma antioxidant activity level and a plasma malonic dialdehyde content. Further, the correlation analysis of the specified laboratory values and the blood manganese level is performed, and if observing the authentic dependences simultaneously: the high blood manganese level - the low blood plasma antioxidant activity level, the high blood manganese level - the high blood plasma malonic dialdehyde level, the low blood plasma antioxidant activity level - the high blood plasma malonic dialdehyde level, manganese action is considered to be toxic.

EFFECT: higher accuracy and information value of the determination procedure.

2 tbl

 

The invention relates to medicine, in particular to methods of laboratory diagnostics of the toxic effect of manganese on the body of children living in conditions of anthropogenic load on the environment, and can be used for early detection of violations of children's health, as well as in the formation of sanitary measures for the prevention and elimination of harmful chemical substances that lead to the formation of environmentally caused diseases in children.

Habitat industrial cities characterized mnogoshagovyi pollution by chemical substances. One of the priority pollutants from a number of heavy metal is manganese, the accumulation of which in the body has a toxic effect, starting the process of lipid peroxidation and the pathogenic cascade of reactions that lead to the development of environmentally caused diseases, manifested in increased morbidity, in particular the violation of the endocrine system and metabolism, decreased immunity, damage to the respiratory and digestive system.

The prior art a number of patents on methods of assessing the impact of chemical technological factors, in particular metals, on the health of the population.

For example, known methods relating to the diagnosis of specific diseases pathologies, with whom according to which concentration is determined macromicrosimulation in biological media and on this content is judged on the disease (Patent RF №2340284 "a Method for predicting preclinical manifestation of multiple sclerosis in patients with optic neuritis"; Patent RF №2229711 "Method of determining the function of the thyroid gland in autoimmune thyroiditis"). However, all these known methods do not install the toxic effect of the designated metals.

Also known a number of ways to diagnose specific diseases, through the definition of laboratory clinical indicators that are affected by heavy metals (RF Patent No. 2160446 "method for determining the risk of formation of amabokoboko nephropathy in children; RF Patent №2137422 "Method for the diagnosis of environmentally caused diseases in the population"). However, these methods are aimed at identifying the existing problems are costly and time-consuming in execution.

The prior art also known methods for determining the toxic effects of heavy metals on human health.

For example, there is a method of determining the sensitivity of erythrocytes to the effects of heavy metals (RF Patent No. 2332668).

The invention lies in the fact that studies trying to enter 0,02 ml of capillary blood, placed in chilled mixture consisting of 0.5 ml of 0.15 M phosphate buffer with pH 7.4 and 0.5 ml of 0.25% solution of glutaraldehyde in the ratio of 1:1, resuspending, leaving in the refrigerator at 4°C for 60 min for fixation of cell shape, then after fixing the contents of the tube t see you a again resuspended and putting 0.01 ml of the cell suspension on a glass slide, count 250 cells and calculate the number of discocytes in native drug, take 0,08 ml of chilled cell suspension is poured into four test tubes: one tube add 0,02 ml of 0.01% solution of ZnSO4in another test tube of 0.02 ml of 0.1% MnSO4in the third test tube of 0.02 ml of a 0.005% solution with NISO4in the fourth test tube of 0.02 ml of 0.01% Pb(NO3)2and at room temperature, 18-20°C and incubated for 30 minutes after incubation the contents of the test tubes again resuspended and by placing 0.01 ml of the cell suspension on a glass slide, count 250 cells, calculate the number of discocytes and the sensitivity coefficient of erythrocyte membranes

where KFIFA world Cupis the coefficient of sensitivity of erythrocyte membranes; DN is the number of discocytes in native smear, %; DM is the number of discocytes when exposed to specific heavy metals, %, if KCHM less than 95% of the initial level, then the cell membrane sensitive to this heavy metal.

This known method determines gemotoksicescoe effect of heavy metals and the sensitivity of membranes to specific heavy metal. Expedient will be finding an effect that is caused membranotoksicheskoe properties of heavy metals forecast for the financing of pathological changes.

Closest to the proposed method is a method of determining the toxicity effect of heavy metals on the body (Patent RF №2138816), according to which determine the content of heavy metals in serum and additional laboratory indicator - catalase activity in erythrocytes, and decreasing (at a rate of 72.5-106,1 with-1/g of hemoglobin) in combination with elevated levels of heavy metals in comparison with the normal action of the latter is defined as toxic.

The disadvantage of this known method is not informative, because the level of catalase does not reflect the whole spectrum of reactions antioxidant system and is only an indirect criterion of activity of lipid peroxidation.

In addition, this method is verified only with the nature of the flow gastroenterological diseases, making it a means to identify other psycho-physiological forms of violations of children's health.

The known method is not sufficiently precise, since its implementation is not installed, the dependence of the level of laboratory indicator - catalase - level content in the blood of heavy metals.

The technical result achieved by the invention, is to improve the accuracy and informativeness due to the expansion of informational relations Midwesterner manganese directly in the biological medium (blood) and the proposed laboratory parameters, comprehensively reflecting the work of the antioxidant system of the organism, at their simultaneous determination.

This technical result is achieved by the proposed method for determining the toxicity of the actions of manganese on the body of children living in conditions of anthropogenic load on the environment, including the determination of manganese in whole blood and determination of laboratory performance, while, according to the invention, as laboratory parameters determine the level of antioxidant activity and the content of malondialdehyde in plasma, additional conduct correlation analysis between laboratory parameters and the level of manganese in blood, and at the same time establishing reliable dependency: elevated levels of manganese in blood, decreased levels of antioxidant activity and/or increased level of malondialdehyde in plasma, the effect of manganese defined as toxic.

The technical result is achieved due to the following.

It should be clear that the measure of antioxidant activity of blood plasma characterizes the total work of all antioxidants in the body to protect from the negative action of free radicals produced in the lipid peroxidation, a process which started in the financial p is Tata membranotoksicheskoe action of manganese.

The level of malondialdehyde in plasma characterizes the activity of free-radical oxidation, as is the final product of this process.

Due to the fact that in the proposed method as a laboratory characteristics (indicators) are not used, as in the prototype, the rate - catalase, reflecting only primary antioxidants, and take into account the aggregate of all groups of antioxidants - antioxidant activity of blood plasma in combination with an indicator of the activity of free-radical oxidation level of malondialdehyde in plasma using correlation analysis provides additional information link laboratory data with the content of manganese in the blood and at the same time informational link between these laboratory parameters, which makes the method more accurate and reliable.

Use as indicators of antioxidant activity and the level of malondialdehyde allows you to enhance the usefulness of the method due to the complex characteristics of the pathological process caused by the action of manganese, and to establish the influence of heavy metal - manganese on various psycho-physiological forms of violation of children's health, living in conditions of anthropogenic chemical load, the conditions of the habitat, without restrictions, only gastroenterological diseases, as in the prototype.

Thus, the proposed method at an early stage, even before the manifestation of clinical signs to predict the probability of diseases of the respiratory system, the digestive system, the immune system in children caused membranotoksicheskoe the effects of manganese.

All this will increase the efficiency of planning of sanitary measures for the prevention and elimination of harmful chemical substances, in particular manganese, lead environment-related pathology in children.

The proposed method is as follows, in the specific example:

- choose environmentally disadvantaged areas based on high load of chemical factors in the habitat. As this site was selected by the city of Perm, characterized by the presence of environmentally harmful substances in the atmosphere and in surface water reservoirs, and in particular manganese is a priority component of the emissions of industrial enterprises;

- select a random group of children living on this territory. Was selected 168 children from organized team, living on the territory of Perm. In the survey participated children, the last morning of the filter, sledovatel is no clinical manifestations of disease;

- in this group of children shall conduct sampling of venous whole blood in two test tubes. One tube is used for the determination of manganese in whole blood, the second test tube to obtain blood plasma;

in whole blood in the first tube define the levels of manganese by atomic absorption spectrophotometer Perkin Elmer 3110 using as oxidant acetylene-air mixture with detection mode flame atomization;

in blood plasma, obtained from the second test tube, determine the level of antioxidant activity and the level of malondialdehyde;

for comparative analysis as the comparison group were examined 100 children from an area with relatively favorable sanitary-hygienic situation;

next they calculate the probability of changes of the studied parameters including linear correlation analysis (based indicators of children from Perm: elevated levels of manganese in blood, decreased levels of antioxidant activity and/or increased level of malondialdehyde in plasma).

Tests of statistical hypotheses concerning the obtained correlation dependences carried out using student's criterion. Differences are considered statistically significant when the probability p<0,05.

The analysis determined which of oncentrate manganese in the blood during the examination 168 children were allowed to establish a statistically significant elevated levels of manganese (average concentration exceeded the reference level 2.1 times, p≤0.001) and 74% of patients. Differences with the comparison group were statistically significant (p=0.003). In the comparison group, the average concentration of manganese in the blood did not exceed the reference level, which is equal to 0.011±0.002 ml/DM3;

As a result of laboratory examination of children living in industrialized Perm revealed reduced levels of the antioxidant activity of blood plasma in 62,5% and its increase with 28% of the children with the increased level of malondialdehyde in plasma at 64.3% of children. The difference was significant as with the physiological norm (antioxidant activity (p=0.003), malonic dialdehyde (p≤0,001), and indicators in the comparison group (antioxidant activity (p=0.004), malonic dialdehyde (p≤0,003)). In the comparison group reported increased levels of antioxidant activity of blood plasma in only 25.3 per cent and a decrease of 5.6% of children, with differences from the norm were unreliable;then compare the level of manganese in whole blood with the level of antioxidant activity in plasma and the level of malondialdehyde in plasma. When the level value of antioxidant activity of blood plasma below the norm (reference level equal to 36.2%) in combination with increased levels of malondialdehyde (reference level equal to 1.8 ml/DM3) in the blood plasma due to high level of manganese in whole the Noi blood, when establishing reliable dependency: elevated levels of manganese in blood, decreased levels of antioxidant activity and/or increased level of malondialdehyde in plasma, the effect of manganese is defined as toxic (namely, embryotoxic).

Evaluation of the results of mathematical modeling of causal relationships has allowed to establish the dependence of the lower levels of the antioxidant activity of blood plasma and increase the level of malondialdehyde in blood plasma from increased blood concentrations of manganese was also the dependence of the lower levels of the antioxidant activity of blood plasma and increased malondialdehyde plasma. The data are given in tables 1 and 2.

Table 1
The results of correlation analysis between manganese and specific laboratory parameters
Substance in the bloodLaboratory indicator of blood plasmarp
Manganeseantioxidant activity-0,7420,0
malonic dialdehyde0,6520,02

Table 2
The results of correlation analysis between the level of laboratory data
Laboratory indicator of blood plasmaLaboratory indicator of blood plasmarp
antioxidant activitymalonic dialdehyde0,8340,03
r - correlation coefficient; p - significance of differences

Children in the comparison group correlation between the decline in antoxidants activity of blood plasma and increased malondialdehyde plasma from increased blood concentrations of manganese was observed (p>0,05).

The accuracy of the proposed method in comparison with the prototype due to the use of an aggregate measure of all groups of antioxidants - antioxidant activity of blood plasma in combination with a performance indicator of antioxidant si the topic - the level of malondialdehyde in plasma, using a correlation analysis of the relationship of laboratory parameters and their probability changes from the levels of manganese in the blood of children.

After examination of children the proposed method, it was found that 79 of 168 children living in industrialized Perm, there is a risk of development of environment-related diseases associated with toxic effects of manganese on the body.

To prove such a conclusion of the above 79 children (diagnosed at the same time three reliable dependencies: elevated levels of manganese in blood, decreased levels of antioxidant activity in plasma, elevated levels of manganese in blood increased level of malondialdehyde in plasma, decreased levels of antioxidant activity - increased level of malondialdehyde in plasma), after 6 months was the analysis of morbidity. Of these children, 11 were identified bronchial asthma, 21 - gastroduodenal pathology, the rest is a frequent manifestation of infection in combination with chronic ENT diseases. According to the Toxicological properties of the manganese all of these diseases due to exposure to high levels of manganese in the body.

The same analysis has been disease is TB 45 children from the same group with a high content of manganese, but without regulation (i.e. without proof) the correlation between the levels of manganese in whole blood and laboratory parameters of the antioxidant system. It is estimated that 8 children established pathology tonsils and adenoids identified 1 case of bronchial asthma, have 16 registered more than 2 cases of SARS during this period, 11 children on one episode of SARS, 9 children clinical manifestation of the disease is not established. That is, the incidence is much lower than that of the above 79 children.

Analysis of the incidence of 44 children with levels of manganese in blood, not exceeding the reference value, showed that 20 children registered on 1 occasion ARI, 5 revealed a chronic pathology of ENT-organs, the rest of the children clinical manifestation of the disease is not installed.

The above data prove that only the proposed relationship of the content of manganese in the blood and the stated level of laboratory performance is ensured accuracy and effectiveness of the proposed method through which you can determine toxic (membranotoksicheskoe) effect of manganese on the child's body.

Thus, the use of the proposed method in the examination of children living in ecologically unfavorable conditions, allows you to:

- to provide moznosti detection with high accuracy at an early stage membranotoksicheskoe action of manganese;

to provide a forecast of the occurrence and nature of the disease different nosologies;

- to justify the need for in-depth clinical examination of the child against diseases typical for the high content of manganese.

The method of determining the toxicity of the actions of manganese on the body of children living in conditions of anthropogenic load on the environment, including the determination of manganese in whole blood and determination of laboratory parameters, characterized in that the quality of laboratory parameters determine the level of antioxidant activity and the content of malondialdehyde in plasma, additional conduct correlation analysis between laboratory parameters and the level of manganese in blood, and at the same time establishing reliable dependency: elevated levels of manganese in blood, decreased levels of antioxidant activity and/or increased level of malondialdehyde in plasma, the effect of manganese is defined as toxic.



 

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