Method for prediction of state of newborns of women with gestosis of various severity levels

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

 

The invention relates to medicine, namely to obstetrics and neonatology, and can be used for prenatal prediction of the state of newborns from women with preeclampsia varying severity.

A known method for predicting the state of new-born children by determining the activity of succinate dehydrogenase and alpha-glitserofosfatdegidrogenazy in peripheral blood lymphocytes of pregnant for 1-3 days before the birth [5]. When values of the activity of succinate dehydrogenase 4,12-8.75 granules/limp. and alpha glitserofosfatdegidrogenazy of 3.00-5.15 granules/limp. forecast heavy condition of the newborn with severe metabolic disorders in the first three days of life, and when values of the activity of succinate dehydrogenase 13,50-18,25 granules/limp. and alpha glitserofosfatdegidrogenazy 5,70-9.45 granules/limp. predict satisfactory condition with metabolic changes of a functional character.

The known method is not sufficiently accurate, as it does not take into account that a pregnant woman can be preeclampsia, which may affect the condition of a newborn baby. Cytochemical method, used in the known method for determining the activity of enzymes is semiquantitative, which further reduces the accuracy of prediction. Furthermore, the method is implemented only for 1-3 days before delivery, the consequence obstetricians do not have time to apply medications, aimed at improving the status of newborns.

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

The task is achieved by using a bioluminescent method in platelets blood of pregnant women with gestosis of varying severity during pregnancy 32-38 weeks to determine the activity of glutathione reductase (GR), NADP-dependent glutamate dehydrogenase (ADFGD) and NADP-dependent izotsitratdegidrogenazy (NADRICH). Then calculate the rate of nikotinamidadenindinukleotida (CON), representing the ratio of the activity of GR to the work activities ADFGD and NADRICH.

Ie: CON=GR/(ADFGD·NADRICH).

When the value of the STAKE, equal to 1.3 and below, predict the assessment of the newborn on a scale Apgar of 6 or less points. When is the CON above 1,3 forecast assessment of the newborn on Apgar scale 7-10 points.

The value of 1.3 is obtained empirically based on the mapping results bioluminescent analysis of enzyme activity in platelets in pregnant women with varying degrees of preeclampsia and subsequent clinical condition of the newborn.

One of the reasons for the development of preeclampsia in pregnant disorders is the system of hemostasis, which is considered as one of the most important pathogenetic links in the development of this disease [2, 7]. Pathological changes in the hemostatic system in pregnant women can lead to serious clinical consequences, including for the fetus and newborn. A key role in the hemostatic system plays platelets. These cells form the basis of cellular link of the system of hemostasis, as well as in platelets synthesized a number of key factors plasma hemostasis, in particular fibrinogen. Platelet function is largely determined by the status of their intracellular metabolic system. As a result of the optimum ratio of the intensity level of various metabolic processes platelet function, including synthesis of biologically active substances involved in blood clotting. One of the important metabolic parameters, including regulatory and intensity of synthetic processes, is the ratio of the reduced and oxidized forms of nicotineinduced (NADPH/NADP+). The value of this ratio is determined by the activity of NADP-dependent dehydrogenases, reducing NADP+and oxidizing NADPH.

The glutathion reductase (GR, EC 1.6.4.2) catalyzes the recovery of oxidized glutathione (disulfide form) due to N IS DFN. 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 centre which has SH-group, active in restored form. Discusses the coenzyme function of glutathione and its role in amino acid transport across the membrane [1].

NADP-dependent glutamate dehydrogenase (ADFGD, EC 1.4.1.4) carries out the oxidative deamination of L-glutamic acid, using as a cofactor NADP+. Enzyme dehydrogenase reaction is reversible, respectively ammonia in the presence of NADPH and α-Ketoglutarate acid may participate in the synthesis of glutamate. Glutamate dehydrogenase is an oligomeric enzyme with a molecular mass 312000, which consists of 6 subunits. The enzyme exhibits its activity only in a multimeric form. When dissociation of glutamate dehydrogenase on subunit, which occurs in the presence of NADPH, GTP-independent and some steroid hormones, the enzyme loses its ability to carry out oxidative deamination of glutamic acid, but gains the ability to carry out the deamination of a number of other amino acids. This feature characterizes the allosteric mechanism of regulation of glutamate dehydrogenase and defines this enzyme as a regulator of the first in the system of amino acid metabolism.

NADP-dependent izotsitratdegidrogenazy (NADRICH, EC 1.1.1.42) carry out the dehydrogenation reaction of solimano acid in the presence of NADP+. During this reaction solimena acid and simultaneously decarboxylated. The allosteric enzyme is, as a specific activator necessary agencydivision. The enzyme is localized in the mitochondrial and cytoplasmic compartments of cells. In mitochondria NADRICH provides auxiliary dehydrogenase reaction for the tricarboxylic acid cycle [6].

The method is as follows.

The sampling of venous blood in pregnant women with various degrees of severity of preeclampsia during pregnancy 32-38 weeks exercise in the morning, on an empty stomach in the amount of 9 ml of an anticoagulant use of 3.8% solution of sodium citrate. Platelets isolated from venous blood by the method proposed Savchenko E.A. et al. (2006) [4]. This venous blood is mixed with a 3.8% solution of sodium citrate in the ratio of 9:1. Platelet-rich plasma obtained by centrifugation stable blood at 140 g for 10 minutes. From the tube carefully screened supernatant transferred to a clean tube and adjusted to 10 ml of buffer No. 1 (90 mm NaCl, 5 mm KCl, 36 mm sodium citrate, 10 mm EDTA, pH 7.2). The resulting mixture was centrifuged 15 min at 400 g. Precipitate resus enshroud in 10 ml of buffer No. 1 and re-centrifuged for 1 min at 400 g. Select 9 ml of supernatant, which again centrifuged at 400 g for 15 minutes the Supernatant is carefully poured, and to the precipitate, add 10 ml of buffer No. 2 (with 0.13 M NaCl, 0.02 M Tris-HCl buffer, 0.03 M EDTA, 0,015 M glucose, pH=7,4) and centrifuged in the same mode. Then the residue is dissolved in 400 ál buffer # 2 and centrifuged for 50 s at 140 g. For further research take 250 ál of supernatant. To determine the activity of NADPH and NADP-dependent dehydrogenases taken from the supernatant with platelets select the volume containing 107cells. Destroy platelets method of osmotic lysis with bringing the total volume to 2.5 ml (the final concentration of cells is 4×106/ml). The activity of GR, ADFGD, NADRICH determined by bioluminescent method. For this purpose, 150 μl of the incubation mixture containing the appropriate substrate and cofactor, make a 50 μl suspension of damaged platelets. Specific values of the concentrations of substrates and cofactors, as well as pH-defined enzymes are presented in table 1.

Table 1
EnzymeThe substrate, mmCofactor, mmThe PH of the buffer
GR GSH-0,5NADPH-0,00257,4
ADFGDGlutamate - 0,5NADP-1,659,8
NADRICHIsocitrate - 1,375NADP-0,0757,4
Note: an environment with a pH of 9.8 prepared in Tris-HCl buffer (ICN Biomedicals Inc., USA); pH 7.4 - K+, Na+phosphate buffer (buffer prepared from the K2HPO4and NaH2PO4(Reakhim, Russia).

It should be noted that in the incubation mixture for determining the activity of NADRICH additionally add ADP at concentrations of 2.15 mm.

After incubation of samples tested at 37°C for 30 minutes for NADRICH and ADFGD or 5 minutes for GR to 200 μl of the incubation mixture are added 50 μl of playmonopolyonline (FMN) in a concentration of 1.5×10-5M, 50 μl of 0.0005% myristic aldehyde and 10 ál of enzyme system NADH: Flexibilityto-luciferase (all reagents bioluminescent system diluted in 0.1 M+, Na+phosphate buffer with pH 7.0). After mixing bioluminescent reagents and incubation of the sample with biochemiluminescence, such as the brand "BL-8803", measure the luminescence. Given that in the cells of meets a certain number of substrates for the various metabolic reactions including catalyzed studied enzymes, identify indicators, conventionally called the "substrate background of enzymes". The determination performed in the same conditions as for the above dehydrogenases, but in the incubation mixture instead of the appropriate substrate contribute buffer. In the result of measuring luminescence on bioluminometer get the relative values of activity of the investigated enzymes. To obtain the absolute value of the activity, build graphs of the intensity of bioluminescence on the concentration of NADPH (calibration). For this, 200 µl of a standard solution of NADPH in the range of 10-9-10-4M contribute in the cell of bioluminometer containing FMN, maristany aldehyde and NADPH:Flexibilityto-luciferase in the concentrations specified above, then measure the intensity of bioluminescence. Due to the wide range of pH buffers used for determining the dehydrogenase activity and pH-dependence of bioluminescence enzyme system of luminous bacteria, calibration graphs are built for each pH buffer. The activity of NADP-dependent dehydrogenases are calculated according to the formula:

A=Δ[N]×V/T

where:

A - activity of dehydrogenase, E 2×105platelets (1E=1 μmol/min [1]);

Δ[I] is the difference of concentrations of NADPH in the sample "ferment" the background of the enzyme", umol;

V - volume of sample, ml;

T is the incubation time, minutes

Then calculate the KOHN relative activity of GR to the work activities ADFGD and NADRICH. When the value of the STAKE, equal to 1.3 and below, predict low assessment of the newborn on Apgar scale (6 or less points) and prescribe treatment and preventive measures of the pregnant woman. When values of KON above 1,3 predict satisfactory condition of the newborn with estimation on Apgar scale 7-10 points.

The biggest CON, equal to 1.3 and below, shows a marked predominance of recovery NADP+on the oxidation of NADPH in dehydrogenase reactions, which increases the level of plastic processes and consequently the synthesis of clotting factors. It is proved that under preeclampsia increased activity of the coagulation hemostasis, increases hypercoagulation and thrombinase [3].

This method is tested on 76 pregnant women with preeclampsia undergoing hospital treatment in the Department of pathology of pregnant MUSES Maternity hospital №1 (Krasnoyarsk). The results of the survey are presented in table 2.

Table 2
The biggest CON in pregnant women with preeclampsia, subsequently gave birth to children with normal and degraded to inchecken condition (Apgar scale)
StatisticsEstimation on Apgar scale 7-10 pointsScore on a scale of Apgar of 6 or less points
The number of the examined5917
Median9,240,06
The minimum value1,290,03
The maximum value86,121,30

The activity of GR, ADFGD and NADRICH in platelets in pregnant women with preeclampsia were determined using bioluminescent method. The survey found that 59 subsequently women gave birth to children with normal clinical state (score on scale Apgar at 1 minute 7-8, 5 minute 8-10). These women value KOHN was 1.29-86,12. One woman prediction did not match. KOHN she was 1.29, but she gave birth to a healthy child (score on scale Apgar at 1 minute and 6 at 5 minutes - 8). 17 women subsequently gave birth to children with worsened clinical status (score on scale Apgar at 1 minute 5-6, 5 minute - 3-6). These women value the STAKE amounted to 0.03-1,30. Thus, the observed coincidence forecast 98,8%.

Example 1. Pregnant J., 25 years. History of births No. 1818. Were hospitalized in the Department of pathology of pregnant MUSES Maternity hospital №1 with 25.05.05 on 14.06.05 with a diagnosis of pregnancy 38 weeks, preeclampsia mild severity. When conducting bioluminescent analysis has established the following enzyme activity in blood platelets: GR=195,17 the MCA for 2×105platelets; ADFGD=2,15 the MCA for 2×105platelets; NADRICH=17,27 the MCA for 2×105platelet. The biggest CON was 5,26. Prognosis: the condition of the child at birth is estimated at 7-10 points on the Apgar scale (satisfactory clinical condition).

Childbirth self, occurred in 40 weeks. The child was born with a condition assessment on a scale of Apgar at 1 minute of life - 8 points, 5 minute - 9 points.

Example 2. Pregnant B., 22 years. History of births No. 833. Were hospitalized in the Department of pathology of pregnant MUSES Maternity hospital №1 with 06.05.06 on 24.05.06 with a diagnosis of pregnancy 35 weeks, preeclampsia mild severity. When conducting bioluminescent analysis has established the following enzyme activity in blood platelets: GR=993,0 the MCA for 2×105platelets; ADFGD=10,50 to the MCA for 2×105platelets; NADRICH=394,57 the MCA for 2×105platelet. The biggest CON amounted to 0.24. Prognosis: the condition of the child at birth will be assessed below 7 points Pascale Apgar (poor clinical condition).

Birth by emergency caesarean section occurred at 37 weeks. The child was born with a condition assessment on a scale of Apgar at 1 minute - 5 points, 5 minute - 6 points.

Example 3. Pregnant And., 25 years. History of births No. 95. Were hospitalized in the Department of pathology of pregnant MUSES Maternity hospital №1 with 17.01.06 on 02.02.06 with a diagnosis of pregnancy 38 weeks, preeclampsia moderate severity. When conducting bioluminescent analysis has established the following enzyme activity in blood platelets: GR=4207,0 the MCA for 2×105platelets; ADFGD=1,05 the MCA for 2×105platelets; NATFOODS=2336,04 the MCA for 2×105platelet. The biggest CON was 1.72. Prognosis: the condition of the child at birth is estimated at 7-10 points on the Apgar scale (satisfactory clinical condition).

Childbirth self, occurred in 40 weeks. The child was born with a condition assessment on a scale of Apgar at 1 minute - 8 points, 5 minute - 8 points.

Example 4. Pregnant With., 24 years. History of births No. 875. Were hospitalized in the Department of pathology of pregnant MUSES Maternity hospital №1 with 12.05.06 on 03.06.06 with a diagnosis of pregnancy 32 weeks, preeclampsia moderate severity. When conducting bioluminescent analysis has established the following enzyme activity in blood platelets: GR=2615,09 the MCA for 2×105platelets; ADFGD=4,28 the MCA for 2×105 platelets; NATFOODS=890,18 the MCA for 2×105platelet. The biggest CON was 0.69. Prognosis: the condition of the child at birth will be assessed below 7 points on the Apgar scale (unsatisfactory clinical state).

Delivery by elective caesarean section occurred in 35 weeks. The child was born with a condition assessment on a scale of Apgar at 1 minute - 5 points, 5 minute - 3 points.

Example 5. Pregnant And., 24 years. History of births No. 953. Were hospitalized in the Department of anesthesiology and critical care medicine MUSES Maternity hospital №1 with 25.05.06 on 15.06.06 with a diagnosis of pregnancy 35 weeks, severe preeclampsia severity. When conducting bioluminescent analysis has established the following enzyme activity in blood platelets: GR=3148,23 the MCA for 2×105platelets; ADFGD=157,17 the MCA for 2×105platelets; NADRICH=14,74 the MCA for 2×105platelet. The biggest CON was 1,36. Prognosis: the condition of the child at birth is estimated at 7-10 points on the Apgar scale (satisfactory clinical condition). Delivery by elective caesarean section occurred in 36 weeks. The child was born with a condition assessment on a scale of Apgar at 1 minute - 8 points, 5 minute - 8 points.

Example 6. Pregnant Valieva P.M., 38 years. History of births No. 877. Were hospitalized in the Department of anesthesiology and critical care medicine MUSES Maternity hospital is 1 12.05.06 on 14.06.06 with a diagnosis of pregnancy 32 weeks, preeclampsia severe stage. When conducting bioluminescent analysis has established the following enzyme activity in blood platelets: GR=158,11 the MCA for 2×105platelets; ADFGD=1,87 the MCA for 2×105platelets; NADRICH=124,35 the MCA for 2×105platelet. The biggest CON was 0.68. Prognosis: the condition of the child at birth will be assessed below 7 points on the Apgar scale (unsatisfactory clinical state).

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

The technical result obtained by the proposed method:

the forecast is for 2-6 weeks before the birth, which allows you to apply preventive measures aimed at improving the condition of the newborn;

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

Thus, the proposed method allows for 2-6 weeks before delivery with a high degree of accuracy to make the forecasting of a condition of newborn babies of women with preeclampsia varying severity that allows timely treatment and preventive measures and thus reduce the frequency of pathological conditions in newborns.

Sources of information

1. Birch CT, Korovkin Biological chemistry. - M.: Medicine, 1998. - 704 S.

2. Zhelev, VA, Cannes, NV, N.G. Belova and other organization of work of the laboratory of hemostasis in obstetric practice, Clinical laboratory diagnostics. - 2006. No. 9. - P.27-28.

3. The makatsariya A.D. Thrombotic state in obstetric practice. - M., 2004. - 35 S.

4. Savchenko E.A., Savchenko A.A., Gerasimchuk A.N., Gryshchenko D.A. Evaluation of the metabolic status of platelets in normal and ischemic heart disease//Clinical laboratory diagnostics. - 2006. No. 5. - Pp.33-36.

5. A method for predicting the state of new-born children. EN 2265850 C2. Publ. 10.12.2005.

6. Stroev, E.A. Biological chemistry. - M.: Higher. HQ., 1986. - 479 C.

7. Shabanova EJ, Mingucci IV, Malakhovsky E.A. and other Cooperative nature of the hypersensitivity of platelets to ADP // bull. the experimental. Biol. and the honey. - 2005. - T. 140, No. 9. - Pp.261-265.

A method for predicting the state of new-born children by examining the activity of enzymes in the peripheral blood of pregnant women, characterized in that by using a bioluminescent method in platelets blood of pregnant women with gestosis of varying severity during pregnancy 32-38 weeks to determine the activity of glutathione reductase (GR), NADP-dependent glutamate dehydrogenase (ADFGD) and NADP-dependent izotsitratdegidrogenazy (NADRICH), calculate the rate of nicotinamidase is infostate (CON), represents the ratio of the activity of GR to the work activities ADFGD and NADRICH, and when the value of the STAKE, equal to 1.3 and below, predict the assessment of the newborn on a scale Apgar of 6 or less points, and a value of the STAKE above 1,3 - 7-10 points.



 

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FIELD: medicine.

SUBSTANCE: excitory cells of the immune system are recovered that is followed with primary incubation of the cells with an investigated substance to produce a primary-incubation supernatant or mixed cells and supernatant; secondary incubation of the target cells with the supernatant or mixed cells and supernatant wherein the secondary-incubation target cells are understood as human tumour cells or cell lines of an oncogenetic origins; the target cells are analysed where the analysis is specified in a group including an expression analysis of specific proteins and an apoptosis and/or necrosis analysis.

EFFECT: improvement of the method.

14 dwg

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, namely to orthopedics. In order to estimate state of bone tissue in case of immobilisation osteoporosis in a laboratory animal examined are homogenates: bone, muscular, bone marrow of any extremity and peripheral blood. Biochemical and integral parametres are determined. Five factor variables F1-F5 are calculated using values of biochemical and integral parametres, constant values of factor coefficients of biochemical parametres and free coefficients. After that calculated is the value of discriminant function, whose value is used to estimate bone state as normal or conclusion about presence of immobilisation osteoporosis is made.

EFFECT: method increases accuracy and efficiency, has high stability of immobilisation osteoporosis recognition.

1 ex, 3 tbl

FIELD: medicine, hepatology.

SUBSTANCE: one should detect the level of hepato-specific enzymes (HSE) in blood plasma, such as: urokinase (UK), histidase (HIS), fructose-1-phosphataldolase (F-1-P), serine dehydratase (L-SD), threonine dehydratase (L-TD) and products of lipid peroxidation (LP), such as: dienic conjugates (DC), malonic dialdehyde (MDA). Moreover, one should detect the state of inspecific immunity parameters, such as: immunoregulatory index (IRI) as the ratio of T-helpers and T-suppressors, circulating immune complexes (CIC). Additionally, one should evaluate the state of regional circulation by applying rheohepatography (RHG), the system of microhemocirculation with the help of conjunctival biomicroscopy (CB) to detect intravascular index (II). In case of increased UK, HIS levels up to 0.5 mcM/ml/h, F-1-P, L-SD, L-Td, LP products, CIC by 1.5 times, higher IRI up to 2 at the norm being 1.0-1.5, altered values of regional circulation, increased II up to 2 points at the norm being 1 point, not more one should diagnose light degree of process flow. At increased level of UK, HIS up to 0.75 mcM/ml/h, F-1-P, L-SD, L-TD, LP products, CIC by 1.5-2 times, increased IRI up to 2.5, altered values of regional circulation, increased II up to 3-4 points one should diagnose average degree of process flow. At increased level of UK, HIS being above 0.75 mcM/ml/h, F-1-P, L-SD, L-TD, LP products, CIC by 2 and more times, increased IRI being above 2.5, altered values of regional circulation, increased II up to 5 points and more one should diagnose severe degree of process flow.

EFFECT: higher accuracy of diagnostics.

3 ex

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