RU2421732C2 - Method for prediction of state of newborns of women with gestosis of various severity levels - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: peripheral blood thrombocytes of women suffering gestosis of various severity levels on their 32-38 weeks of pregnancy are analysed for the activity of glutathione reductase (GY), NADF-dependent glutamate dehydrogenase (NADFGDG) and NADF-dependent isocitrate dehydrogenase (NADFICDG). A nicotine amide adenine dinucleotide phosphate transfer coefficient (NTC) represented by the relation of the GY activity to a product of the NADFGDG and NADFICDG activities is calculated. At the NTC value is equal to 1.3 and lower, the newborn's Apgar score is predicted to be equal to 6 and less, and the NTC value exceeding 1.3 provides the Apgar score being 7-10 points.

EFFECT: more accurate prediction of the newborn's state.

2 tbl, 5 ex

Description

The invention relates to medicine, namely to obstetrics and neonatology, and can be used for antenatal prediction of the condition of the newborn from women with gestosis of varying severity.

A known method for predicting the condition of newborn babies by determining the activity of succinate dehydrogenase and alpha-glycerophosphate dehydrogenase in the peripheral blood lymphocytes of a pregnant woman 1-3 days before delivery [5]. With succinate dehydrogenase activity values of 4.12-8.75 granules / lymph. and alpha-glycerophosphate dehydrogenase 3.00-5.15 granules / lymph. predict a severe condition of the newborn with severe metabolic disorders in the first three days of life, and with succinate dehydrogenase activity values of 13.50-18.25 granules / lymph. and alpha-glycerophosphate dehydrogenase 5.70-9.45 granules / lymph. predict a satisfactory condition with metabolic functional changes.

The known method is not accurate enough, since it does not take into account that a pregnant woman may have gestosis, which can affect the condition of a newborn baby. The cytochemical method used in the known method for determining the activity of enzymes is semi-quantitative, which further reduces the accuracy of prediction. In addition, the method is implemented only 1-3 days before delivery, as a result of which obstetrician-gynecologists do not have time to use medications aimed at improving the condition of newborns.

The objective of the invention is to create a new informative method for predicting the status of newborns from women with gestosis of varying severity in the first minutes of life.

The task is achieved by the fact that the activity of glutathione reductase (GR), NADP-dependent glutamate dehydrogenase (NADPHDG) and NADP-dependent isocitrate dehydrogenase (NADPH) is determined using the bioluminescent method in blood platelets of pregnant women with gestosis of various severity at a gestational age of 32-38 weeks. Then, the exchange coefficient of nicotinamidine nucleotide phosphate (KOH) is calculated, which is the ratio of the activity of GR to the product of the activities of NADPHDG and NADFITsDG.

That is: KOH = GR / (NADFGDG · NADFITZG).

With a KOH value of 1.3 or lower, an assessment of the condition of the newborn on the Apgar scale of 6 or less is predicted. If the KOH value is higher than 1.3, an assessment of the condition of the newborn is predicted on the Apgar scale of 7-10 points.

A value of 1.3 was obtained experimentally by comparing the results of a bioluminescent analysis of the activity of enzymes in platelets in pregnant women with varying degrees of gestosis and the subsequent clinical condition of newborns.

One of the reasons for the development of gestosis in pregnant women is a disorder of the hemostatic system, which is considered as one of the most important pathogenetic links in the development of this pathology [2, 7]. Pathological changes in the hemostatic system in pregnant women lead to serious clinical consequences, including for the fetus and newborn. Platelets play a key role in the hemostatic system. It is these cells that form the basis of the cellular link of the hemostasis system, and also a number of key factors of the plasma link of hemostasis, in particular fibrinogen, are synthesized in platelets. Platelet function is largely determined by the state of their intracellular metabolic system. It is as a result of the optimal ratio of the intensity level of various metabolic processes that the platelet performs its function, including the synthesis of biologically active substances involved in blood coagulation. One of the important metabolic parameters, including those regulating the intensity of synthetic processes, is the ratio of the reduced and oxidized forms of nicotinamide dinucleotide (NADPH / NADP ⁺ ). The value of this ratio is determined by the activity of NADP-dependent dehydrogenases that restore NADP ⁺ and oxidize NADPH.

Glutathione reductase (GR, EC 1.6.4.2) catalyzes the reduction of oxidized glutathione (disulfide form) due to NADPH. The enzyme is localized in the cytoplasmic compartment. It is believed that one of the main functions of glutathione is the preservation of enzymes, in the active center of which there are SH-groups, in an active reduced form. The coenzyme function of glutathione and its role in the transport of amino acids across the membrane are discussed [1].

NADP-dependent glutamate dehydrogenase (NADPHDG, EC 1.4.1.4) oxidatively deaminates L-glutamic acid using NADP ⁺ as a cofactor. The enzymatic reaction of glutamate dehydrogenase is reversible, respectively, ammonia in the presence of NADPH and α-ketoglutaric acid can participate in the synthesis of glutamate. Glutamate dehydrogenase is an oligomeric enzyme with a molecular weight of 312,000, which consists of 6 subunits. The enzyme shows its activity only in multimeric form. When glutamate dehydrogenase is dissociated into subunits, which occurs in the presence of NADPH, guanosine triphosphate and a number of steroid hormones, the enzyme loses its ability to oxidatively deamine glutamic acid, but it acquires the ability to deamine a number of other amino acids. A similar feature characterizes the allosteric mechanism of glutamate dehydrogenase regulation and defines this enzyme as regulatory in the amino acid metabolism system.

NADP-dependent isocitrate dehydrogenase (NADFITsDG, EC 1.1.1.42) carries out the dehydrogenation reaction of iso-citric acid in the presence of NADP ⁺ . In the course of this reaction, isolimonic acid is simultaneously decarboxylated. The enzyme is allosteric, adenosine diphosphate is required as a specific activator. The enzyme is localized in both the mitochondrial and cytoplasmic compartments of the cells. In mitochondria, NADFITsDG carries out an auxiliary dehydrogenase reaction for the tricarboxylic acid cycle [6].

The method is as follows.

Venous blood sampling in pregnant women with varying severity of gestosis with a gestational age of 32-38 weeks is carried out in the morning, on an empty stomach in the amount of 9 ml. A 3.8% sodium citrate solution is used as an anticoagulant. Platelets are isolated from venous blood according to the method proposed by Savchenko E.A. et al. (2006) [4]. For this, venous blood is mixed with a 3.8% solution of sodium citrate in a ratio of 9: 1. Platelet-rich plasma is obtained by centrifuging stabilized blood at 140 g for 10 minutes. The supernatant was carefully taken from the tube, transferred to a clean tube and adjusted to 10 ml with buffer No. 1 (90 mM NaCl, 5 mM KCl, 36 mM sodium citrate, 10 mM EDTA, pH = 7.2). The resulting mixture was centrifuged for 15 min at 400 g. The precipitate was resuspended in 10 ml of buffer No. 1 and centrifuged again for 1 min at 400 g. 9 ml of the supernatant are collected and centrifuged again at 400 g for 15 minutes. The supernatant is carefully drained and 10 ml of buffer No. 2 (0.13 M NaCl, 0.02 M Tris-HCl buffer, 0.03 M EDTA, 0.015 M glucose, pH = 7.4) is added to the precipitate and centrifuged in the same mode. Then the precipitate was diluted in 400 μl of buffer No. 2 and centrifuged for 50 s at 140 g. For further studies, 250 μl of the supernatant was collected. To determine the activity of NADPH and NADP-dependent dehydrogenases, a volume containing 10 ⁷ cells was selected from the collected platelet supernatant. Platelets are destroyed by osmotic lysis with a total volume of 2.5 ml (the final cell concentration is 4 × 10 ⁶ / ml). The activity of GR, NADPHDG, NADFITsDG is determined using the bioluminescent method. For this, in 50 μl of the incubation mixture containing the appropriate substrate and cofactor, add 50 μl of a suspension of destroyed platelets. Specific values of the concentrations of substrates and cofactors, as well as the pH of the medium for the determined enzymes are presented in table 1.

Table 1 Enzyme Substrate, mm Cofactor, mm PH buffer GR GSH-0.5 NADPH-0.0025 7.4 NADFGDG Glutamate - 0.5 NADF-1.65 9.8 NADFITSDG Isocytrate - 1,375 NADP-0.075 7.4 Note: medium with a pH of 9.8 is prepared on Tris-HCl buffer (ICN Biomedicals Inc., USA); with a pH of 7.4 - on K ⁺ , Na ⁺ - phosphate buffer (buffer prepared from K ₂ HPO ₄ and NaH ₂ PO ₄ (Reakhim, Russia)).

It should be noted that in the incubation mixture to determine the activity of NADFITsDG add ADP in concentrations of 2.15 mm.

After incubation of the test samples at 37 ° C for 30 minutes for NADPHIC and NADPHDG or 5 minutes for GR, 50 μl of flavin mononucleotide (FMN) at a concentration of 1.5 × 10 ^-5 M, 50 μl 0.0005% are added to 200 μl myristic aldehyde and 10 μl of the NADH enzyme system: FMN oxidoreductase-luciferase (all reagents of the bioluminescent system are diluted in 0.1 M K ⁺ , Na ⁺ - phosphate buffer with a pH of 7.0). After mixing the bioluminescent reagents and the incubation sample using a biochemiluminometer, for example brand "BLM-8803", measure the glow. Given that the cells have a certain number of substrates for the course of various metabolic reactions, including those catalyzed by the studied enzymes, the parameters conventionally called "substrate background of enzymes" are determined. The determination is made under the same conditions as for the above dehydrogenases, but a buffer is added to the incubation mixture instead of the corresponding substrate. As a result of measuring the glow on a bioluminometer, relative values of the activity of the studied enzymes are obtained. To obtain the absolute values of activity, build graphs of the dependence of the intensity of bioluminescence on the concentration of NADPH (calibration graph). For this, 200 μl of a standard solution of NADPH in the range of 10 ^-9 -10 ^-4 M is added to the bioluminometer cuvette containing FMN, myristin aldehyde and NADPH: FMN oxidoreductase-luciferase at the concentrations indicated above, after which the intensity of bioluminescence is measured. Due to the wide range of pH buffers used to determine dehydrogenase activity, as well as the pH dependence of the bioluminescence of the enzymatic system from luminous bacteria, calibration plots are plotted for each pH buffer. The activity of NADP-dependent dehydrogenases is calculated by the formula:

A = Δ [C] × V / T,

Where:

A - dehydrogenase activity, E per 2 × 10 ⁵ platelets (1E = 1 μmol / min [1]);

Δ [C] is the difference in the concentrations of NADPH in the samples "enzyme" and "background enzyme", mmol;

V is the sample volume, ml;

T - incubation time, min

Then, KOH is calculated in relation to the ratio of GR activity to the product of NADPHYDH and NADFITSDH activities. With a KOH value of 1.3 or lower, a low assessment of the condition of the newborn on the Apgar scale (6 or less points) is predicted and therapeutic measures are prescribed for the pregnant woman. With KOH values above 1.3, a satisfactory condition of the newborn is predicted with an Apgar score of 7-10 points.

A KOH value of 1.3 or lower indicates a pronounced prevalence of NADP ⁺ reduction over NADPH oxidation in dehydrogenase reactions, which increases the level of plastic processes and, accordingly, the synthesis of coagulation factors. It has been proven that with gestosis, the activity of the coagulation link of hemostasis increases, hypercoagulation and thrombinemia increase [3].

This method was tested on 76 pregnant women with preeclampsia, who are hospitalized in the Department of Pregnancy Pathology, MUZ of the Maternity Hospital No. 1 (Krasnoyarsk). The survey results are presented in table.2.

table 2 The value of KOH in pregnant women with preeclampsia, subsequently giving birth to children with normal and worsened clinical condition (Apgar scale) Statistical indicators Apgar score 7-10 points Apgar score of 6 or less The number of examined 59 17 Median 9.24 0.06 Minimum value 1.29 0,03 Maximum value 86.12 1.30

The activity of GR, NADPHDG, and NADFITsDG in platelets in pregnant women with gestosis was determined using the bioluminescent method. According to the survey results, 59 women subsequently gave birth to children with a normal clinical condition (Apgar score for 1 minute is 7-8, for 5 minutes - 8-10). In these women, the KOH value was 1.29-86.12. For one woman, the prognosis did not match. Her KOH was 1.29, but she gave birth to a clinically healthy child (Apgar score for 1 minute - 6, 5 minutes - 8). 17 women subsequently gave birth to children with a worsened clinical condition (Apgar score at 1 minute 5-6, at 5 minutes 3-6). In these women, the KOH value was 0.03-1.30. Thus, a coincidence of the forecast of 98.8% was noted.

Example 1. Pregnant J., 25 years old. The history of childbirth No. 1818. I was hospitalized in the pathology department of pregnant women at the MUZ of the Maternity Hospital No. 1 from 05.25.05 to 06.14.05 with a diagnosis of 38 weeks gestation, mild gestosis. When conducting bioluminescent analysis, the following enzyme activity in blood platelets was established: GR = 195.17 μE per 2 × 10 ⁵ platelets; NADPHDG = 2.15 μE per 2 × 10 ⁵ platelets; NADFITCG = 17.27 μE per 2 × 10 ⁵ platelets. The KOH value was 5.26. Forecast: the condition of the child at birth will be rated at 7-10 points on the Apgar scale (satisfactory clinical condition).

Self-delivery, occurred at 40 weeks. A child was born with a state assessment on the Apgar scale at 1 minute of life - 8 points, at 5 minutes - 9 points.

Example 2. Pregnant B., 22 years old. History of childbirth No. 833. I was hospitalized in the pathology department of pregnant women at the MUZ of the Maternity Hospital No. 1 from 05.06.06 to 05.24.06 with a diagnosis of 35 weeks gestation, mild gestosis. When conducting bioluminescent analysis, the following enzyme activity in blood platelets was established: GR = 993.0 μE per 2 × 10 ⁵ platelets; NADPHDG = 10.50 μE per 2 × 10 ⁵ platelets; NADFITsDG = 394.57 μE per 2 × 10 ⁵ platelets. The value of KOH was 0.24. Forecast: the condition of the child at birth will be rated below 7 points on the Apgar scale (poor clinical condition).

Delivery by emergency caesarean section occurred at 37 weeks. A child was born with a state assessment on the Apgar scale at 1 minute - 5 points, at 5 minutes - 6 points.

Example 3. Pregnant I., 25 years old. The history of childbirth No. 95. I was hospitalized in the pathology department of pregnant women at the MUZ of the Maternity Hospital No. 1 from 01/17/06 to 02/02/06 with a diagnosis of 38 weeks gestation, moderate gestosis. When conducting bioluminescent analysis, the following enzyme activity in blood platelets was established: GR = 4207.0 μE per 2 × 10 ⁵ platelets; NADPHDG = 1.05 μE per 2 × 10 ⁵ platelets; NADFIODG = 2336.04 μE per 2 × 10 ⁵ platelets. The value of KOH was 1.72. Forecast: the condition of the child at birth will be rated at 7-10 points on the Apgar scale (satisfactory clinical condition).

Self-delivery, occurred at 40 weeks. A child was born with a state assessment on the Apgar scale at 1 minute - 8 points, at 5 minutes - 8 points.

Example 4. Pregnant S., 24 years. The history of childbirth No. 875. She was hospitalized in the Department of Pregnancy Pathology, MUZ of the Maternity Hospital No. 1 from 05/12/06 to 06/03/06 with a diagnosis of 32 weeks gestation, moderate gestosis. When conducting bioluminescent analysis, the following enzyme activity in blood platelets was established: GR = 2615.09 μE per 2 × 10 ⁵ platelets; NADPHDG = 4.28 μE per 2 × 10 ⁵ platelets; NADFIODG = 890.18 MKE 2 × ¹⁰ May platelets. The value of KOH was 0.69. Forecast: the condition of the child at birth will be rated below 7 points on the Apgar scale (poor clinical condition).

Delivery by planned caesarean section occurred at 35 weeks. A child was born with a state assessment on the Apgar scale at 1 minute - 5 points, at 5 minutes - 3 points.

Example 5. Pregnant I., 24 years. The history of childbirth No. 953. I was hospitalized in the Department of Anesthesiology and Intensive Care at the Municipal Hospital of the Maternity Hospital No. 1 from 05.25.06 to 06.15.06 with a diagnosis of pregnancy of 35 weeks, severe gestosis. When conducting bioluminescent analysis, the following enzyme activity in blood platelets was established: GR = 3148.23 μE per 2 × 10 ⁵ platelets; NADPHDG = 157.17 μE per 2 × 10 ⁵ platelets; NADFITsDG = 14.74 μE per 2 × 10 ⁵ platelets. The value of KOH was 1.36. Forecast: the condition of the child at birth will be rated at 7-10 points on the Apgar scale (satisfactory clinical condition). Delivery by elective caesarean section occurred at 36 weeks. A child was born with a state assessment on the Apgar scale at 1 minute - 8 points, at 5 minutes - 8 points.

Example 6. Pregnant Valieva PM, 38 years old. The history of childbirth No. 877. She was hospitalized in the Department of Anesthesiology and Intensive Care at the Municipal Hospital of the Maternity Hospital No. 1 from 05/12/06 to 06/14/06 with a diagnosis of 32 weeks gestation, severe gestosis. When conducting bioluminescent analysis, the following enzyme activity in blood platelets was established: GR = 158.11 μE per 2 × 10 ⁵ platelets; NADPHDG = 1.87 μE per 2 × 10 ⁵ platelets; NADFITCG = 124.35 μE per 2 × 10 ⁵ platelets. The value of KOH was 0.68. Forecast: the condition of the child at birth will be rated below 7 points on the Apgar scale (poor clinical condition).

Delivery by emergency caesarean section occurred at 35 weeks. A child was born with a state assessment on the Apgar scale at 1 minute - 4 points, at 5 minutes - 3 points, which confirms the accuracy of the forecast.

The technical result obtained by the implementation of the proposed method:

- the forecast is carried out 2-6 weeks before birth, which allows the use of preventive measures aimed at improving the condition of the newborn;

- high level of coincidence of the forecast - 98.8%.

Thus, the proposed method allows 2-6 weeks prior to delivery with a high degree of accuracy to predict the state of newborns in women with gestosis of varying severity, which makes it possible to timely carry out therapeutic and preventive measures and, accordingly, reduce the frequency of pathological conditions in newborns.

Information sources

1. Berezov T.T., Korovkin B.F. Biological chemistry. - M.: Medicine, 1998 .-- 704 p.

2. Zhelev V.A., Kannskaya N.V., Belova N.G. et al. Organization of the work of the hemostatic laboratory in obstetric practice // Clinical laboratory diagnostics. - 2006. - No. 9. - S. 27-28.

3. Makatsaria A.D. Thrombotic conditions in obstetric practice. - M., 2004 .-- 35 p.

4. Savchenko E.A., Savchenko A.A., Gerasimchuk A.N., Grishchenko D.A. Assessment of the metabolic status of platelets in normal and coronary heart disease // Clinical laboratory diagnostics. - 2006. - No. 5. - S.33-36.

5. A method for predicting the condition of newborn children. RU 2265850 C2. Publ. 12/10/2005.

6. Stroyev EA Biological chemistry. - M .: Higher. school., 1986. - 479 p.

7. Shabanova E.Yu., Mindukshev I.V., Malakhovskaya E.A. et al. The cooperative nature of platelet hypersensitivity to ADP // Bull. an experiment. biol. and honey. - 2005. - T. 140, No. 9. - S.261-265.

Claims (1)

A method for predicting the condition of newborns by examining the activity of enzymes in the peripheral blood of pregnant women, characterized in that the activity of glutathione reductase (GR), NADP-dependent glutamate dehydrogenase is determined using a bioluminescent method in blood platelets of pregnant women with gestosis of varying severity with a gestational age of 32-38 weeks ( NADPHDH) and NADP-dependent isocitrate dehydrogenase (NADPHIC), the nicotinamidine nucleotide phosphate (KOH) exchange coefficient, which is the ratio ktivnosti GR activity to the product and NADFGDG NADFITSDG and KOH at a value equal to 1.3 and below, projected state estimation newborn Apgar score 6 or less, and a value above 1.3 KOH - 7-10 points.