RU2574937C1 - Method for assessing effect of cytomegaloviral infection on nad-dependent malate dehydrogenase activity in villous syncytiotrophoblast in third trimester of pregnancy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: peripheral blood is analysed to determine an anti-cytomegalovirus antibody titre; a histochemical NAD-dependent malate dehydrogenase test is conducted, and if the anti-cytomegalovirus IgG antibody titre is 1:1600 in a combination with a decrease in NAD-dependent malate dehydrogenase activity up to 41.6±2.81 standard units, an energy metabolic imbalance in the villous syncytiotrophoblast having a negative effect on the foetal formation during the gestation are detected.

EFFECT: more accurate assessment.

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Description

1. The purpose of the invention is to identify the effect of cytomegalovirus infection in the third trimester of gestation on the activity of NAD-dependent malate dehydrogenase in the placenta syncytiotrophoblast, leading to disruption of energy processes in the fetoplacental barrier.

2. The invention relates to medicine, namely to the development of a histochemical method for diagnosing a decrease in the activity of one of the stages of the tricarboxylic acid cycle, a NAD-dependent malate dehydrogenase, which proves the violation of cellular energy exchange processes in the syncytiotrophoblast of the placental villi during exacerbation of the cytomegalovirus infection in the third trimester of pregnancy.

3. Disclosure of invention

Malate dehydrogenase is a key enzyme in the tricarboxylic acid cycle, catalyzing the redox reaction of the conversion of malate to oxaloacetate [7, 8]. The tricarboxylic acid cycle is considered the central metabolic process that combines carbohydrate, lipid and amino acid metabolism. Thanks to it, the formation of reducing equivalents occurs, which enter the respiratory chain, during the functioning of which ATP is synthesized. Malate dehydrogenase deserves special attention as the most active source of NADH and a participant in the shuttle cycle of transfer of reducing equivalents through mitochondrial membranes impervious to NADH.

A decrease in the activity of this enzyme indicates a decrease in the intensity of the tricarboxylic acid cycle and, accordingly, aerobic energy processes [6] in the placenta, which, in turn, affects the growth and development of the fetus.

4. New technical challenge

Determine the level of activity of NAD-malate dehydrogenase, indicating the nature of the tricarboxylic acid cycle during exacerbation of cytomegalovirus infection in pregnant women in the third trimester of gestation in order to diagnose the state of the placental barrier during pregnancy, which determines the normal development of the fetus.

5. In the available literature there are references to a method for predicting the severity of exacerbation of atopic bronchial asthma by the activity of NAD-dependent malate dehydrogenase in peripheral blood lymphocytes [5], a method of differential diagnosis of heart rhythm disturbances in children [2] by the activity of NAD-dependent malate dehydrogenase in peripheral blood lymphocytes , a method for determining the activity of malate dehydrogenase in the homogenate of biological tissues [3]. Features of the functioning of NAD-dependent malate dehydrogenase are studied in myocardial ischemia [4]. All of them are performed by biochemical methods.

In contrast to the above prototypes, this work has significant differences: 1. The object of study is the placenta. 2. Changes in the activity of NAD-dependent malate dehydrogenase occur under the influence of exacerbation of cytomegalovirus infection during pregnancy. 3. Assessment of the activity of NAD-dependent malate dehydrogenase is performed by the histochemical method. The method is simple to implement, does not require expensive reagents, has good resolution and can be recommended for research and laboratory diagnostics.

6. The implementation of the invention

6.1. We examined 25 pregnant women aged 18-25 with exacerbation of chronic cytomegalovirus infection in the third trimester of gestation, the control group consisted of 20 pregnant women with chronic cytomegalovirus infection in the latent stage. In pregnant women, symptomatic cytomegalovirus infection manifested itself as an acute respiratory disease, accompanied by rhinopharyngitis. The clinical diagnosis (exacerbation of chronic cytomegalovirus infection) is established by a comprehensive study of peripheral blood for the presence of cytomegalovirus DNA (CMV), JgM or an increase in JgG antibody titer in paired sera (4 times or more) over time after 10 days, as well as avidity index ( more than 65%). The studies are carried out taking into account the requirements of the Helsinki Declaration of the World Medical Association “Ethical Principles for Conducting Research with the Human Participation” as amended in 2008 and the rules of clinical practice in the Russian Federation, approved by order of the Ministry of the Russian Federation No. 266 of 06/19/2003.

6.2. CMV verification, determination of type-specific antibodies, avidity index is carried out by ELISA on a Stat Fax-2100 spectrophotometer using test systems of Vector-Best CJSC (Novosibirsk), CMV DNA is detected by PNR methods on a DT-96 apparatus using NPO kits DNA technology ”(Moscow).

6.3. Malate dehydrogenase activity is histochemically determined on cryostat sections of freshly frozen non-fixed placental tissue using the following method. The method is based on the reduction of the tetrazolium salt into formazan by electrons accepted from the substrate through the coenzyme NAD [1]. The substrate is a 0.1 M solution of sodium salt of malic acid (Sigma). The incubation solution is prepared on the basis of 0.1 M phosphate buffer pH 7.2-7.4 with the addition of nitro-blue tetrazolium manufactured by ICN Biomedicals (USA) at a final concentration of 5 mM; NAD ⁺ from Applichem (Germany) - 1 mM, the presence of Mg ^{2+ ions} (ODM MgCl ₂ · 6H ₂ O).

The composition of the incubation medium:

- 0.1 M phosphate buffer pH 7.2-7.4 - 1 ml;

- nitrosine tetrazolium - 1 mg;

- NAD ⁺ - 1 mg;

- MgCl ₂ · 6H ₂ O - 2 mg;

- sodium salt of malic acid - 17.8 mg.

6.4. Freshly prepared tissue sections are placed in 1 ml of incubation solution. Incubation is carried out at 37 ° C for 30 minutes. At the end of the incubation, the sections are washed with distilled water, fixed in 10% neutral formalin at room temperature, washed again with distilled water, dried and enclosed in glycerol-gelatin. Control sections were incubated in a medium containing, instead of sodium malate solution, an adequate amount of phosphate buffer. The obtained histological preparations are studied by cytophotometric computer method using the Scion program (USA) on an MT digital microscope (Japan).

6.5. Statistical analysis and data processing is carried out using the software package “Statistica 6.0”. Characteristic of the variation series: arithmetic mean (M), standard error (m), coefficient of variation 95%, confidence interval for average values M ± m, where t was determined from the table of boundary values of the Student criterion at a confidence level of 95% and the number of degrees of freedom F = n ₁ + n ₂ -2. To determine the significance of differences, unpaired parametric student criterion is used. P <0.05;0.01; 0.001. To determine the significance of differences in the case of non-Gaussian distributions, non-parametric Kolmogorov-Smirnov and Mann-Whitney criteria are used.

7. Received data

During the study, the following results were obtained. The histochemical reaction of detecting the activity of NAD-dependent malate dehydrogenase is quite specific, since in the absence of a substrate in the incubation medium, precipitation of formazan granules in tissue structures is not observed. On histochemical preparations of the placenta of women who had an exacerbation of chronic cytomegalovirus infection in the third trimester of gestation, when the titer of class G antibodies to cytomegalovirus was registered to 1: 1600, the activity of NAD-dependent malate dehydrogenase decreased to 41.6 ± 2.81 conv. units (control - 65.4 ± 2.98 srvc. unit) (figure 1, 2).

Thus, a decrease in malate dehydrogenase activity with an increase in the titer of class G antibodies to cytomegalovirus 1: 1600 indicates a violation of energy exchange in the placenta syncytiotrophoblast, which affects the growth and development of the fetus.

The figure 2 shows the histochemical reaction to NAD-malate dehydrogenase in the placenta of a woman in the control group (not ill during the entire gestation). The intensity of the histochemical reaction is 65.4 ± 2.98 srvc. units The magnification is 10 × 100.

The figure 1 shows the histochemical reaction to NAD-malate dehydrogenase in the placenta of a woman who underwent exacerbation of cytomegalovirus infection with an antibody titer of class G 1: 1600 in the third trimester of gestation. A decrease in the activity of the histochemical reaction to 41.6 ± 2.81 conv. units The magnification is 10 × 100.

Literature

1. Loyda Z., Gossrau R., Schiebler T. Histochemistry of enzymes. Laboratory methods: Per. from English M .: Mir, 1982, 272 p.

2. Myakishev E.V., Savchenko A.A., Manchuk V.T. A method for the differential diagnosis of cardiac arrhythmias in children. Patent No. 2270448 of the Russian Federation. Publ. 02/20/2006. Bull. No. 5.

3. Pakhomova V. A., Kryukova G. I., Kozlyanina N. P. A method for determining the activity of malate dehydrogenase in biological tissues. Patent A.S. 930122 USSR. MKI G01 33/48. Publ. 05/23/82. Bull. No. 19. 2 sec

4. Safonova O.A., Popova T.N., Artyukhov V.G., Matasova L.V. Features of the functioning of the cytoplasmic NAD malate dehydrogenase from rat myocardium under ischemic conditions // Bulletin of Experimental Biology and Medicine. 2005. No. 7. S. 31-34.

5. Sobko EA, Bulygin GV, Kamzalakova N.I. A method for predicting the severity of exacerbation of atopic bronchial asthma. Patent No. 2190217 of the Russian Federation. Publ. 09/27/2002.

6. Daniš P., Farkas R. Hormone-dependent and hormone-independent control of metabolic and developmental functions of malate dehydrogenase - review // Endocrine regulations. 2009. Vol. 43.39-52.

7. Minárik P., Tomaskova N., Kollárová M., Antalík M. Malate dehydrogenases-structure and function // Gen Physiol. Biophys. 2002. Vol. 21, No. 3. P. 257-265.

8. Musrati R.A., Kollárová M., Mernik N., Mikulásová D. Malate dehydrogenase: distribution, function and properties // Gen Physiol. Biophys. 1998. Vol. 17, No. 3. P. 193-210.

Claims (1)

A method for detecting impaired energy metabolism in pregnant women’s placenta syncytiotrophoblast during exacerbation of cytomegalovirus infection in the third trimester of gestation, including determining the titer of antibodies to cytomegalovirus in peripheral blood, performing a histochemical reaction to NAD-dependent malate dehydrogenase, and when titer of class G antibodies to 1600 cytomegirus is reduced activity of NAD-malate dehydrogenase up to 41.6 ± 2.81 srvc. units reveal a violation of energy metabolism in the syncytiotrophoblast of the placenta, adversely affecting the formation of the fetus during gestation.

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