Method for prediction of cerebral affections in newborns

FIELD: medicine.

SUBSTANCE: five-percent placental tissue extract is analysed for the concentration of arginine and histidine to be related, and if the relation is lower than 0.86, development of cerebral affection in a newborn is predicted.

EFFECT: more precise and specific prediction of perinatal CNS affection in the newborn and enabled well-timed pathogenetic therapy.

3 dwg, 1 tbl

 

The invention relates to medicine, namely to obstetrics and Perinatology, and can be used to predict lesions of the Central nervous system of the newborn.

Perinatal brain damage takes the leading place in the structure of morbidity and mortality, due to a progressive increase in the frequency of cerebral disorders in newborns, leading to their role in the formation of a disability since childhood. In this regard, this problem is beyond the scope of biomedical and acquires social value (Beresnev, Y.M. Hypoxic encephalopathy: the hypothesis of the pathogenesis of cerebral disorders and the search for methods of drug therapy //Ross. Bulletin of perinat. and pediat. 2002. No. 1. - P.6-13).

Prenatal factors leading to the defeat of the brain of the newborn can be different: diseases transferred by the woman during pregnancy, perinatal infection, immunological imbalance, metabolic disorders in the mother's body. However, the leading role among them plays intrauterine hypoxia that accompanies many of the complications of pregnancy. Despite these successes remain some difficulties in accurate and objective diagnosis and especially the prediction of perinatal brain damage.

Thus, there is a need develo the TCI of new methods for the prediction of cerebral damage the newborn with the purpose of timely adequate pathogenetic therapy, to reduce the frequency of manifestation and severity of cerebral pathology.

Among well-known in the medical-scientific and patent literature methods of diagnosis of brain damage can be noted electroencephalography of the fetus at birth, but the accuracy of this diagnosis is very low due to functional immaturity of brain structures and the large variability of the bioelectrical activity of the brain of the fetus (Beresnev SCI, Bubnov NI, Sorokina SH and other Perinatal brain pathology: the margin of safety, and short and long-term prognosis // ROS. news. perinatol. and the pediatrician. - 1998. No. 4. - P.6-12). Data on the diagnostic value of determining the level of α-fetoprotein in the amniotic fluid for the specified purpose relate mainly malformations (neural tube Whirlow et al. Amniotic fluid. - 1998. - S). Fairly accurate, however, very traumatic, is a method of determining neurospecific peptide - homocarnosine in the amniotic fluid taken during pregnancy by amniocentesis (Madhu M Homocarnosine amniotic fluid and its value in the diagnosis of brain damage in the fetus. - Abstract. Diss. Kida. the honey. Sciences. 1990. - 19 C.). Traumatic is the diagnosis and perinatal CNS damage by determining the intensity of processes of lipid peroxidation in the Kyrgyz Republic the wee baby at 3 and 6 days of life (Lagumina NN. Early diagnosis and preventive therapy of perinatal CNS lesions in children. Rostov-on-don. - Abstract. Diss. Kida. the honey. Sciences. 1998. - 22 S.).

There is a method of predicting health disorders children with perinatal CNS damage in the first year of life on the study of umbilical cord blood, wherein determine the relative content teilinstitut cells in their content of 9% and over predict the preservation of neurological symptoms by the end of the first year of life (patent RF №2121682, 1998). The disadvantages of this method include nespecificnomu - change T-link the immune system takes place and other pathological conditions.

In the patent of Russian Federation №2148262 (1999) proposed a method for the diagnosis of hypoxic lesions of the Central nervous system in newborns using the definition of the MDA content in the cerebrospinal fluid of a child in the 2nd and 6th day of life. With the increase of this indicator on the 2nd day over to 0.72 µmol/l and on the 6th day in 2 times in comparison with the norm diagnose the progression of the pathological process in the brain. This method is traumatic because it required taking liquor from a child in the neonatal period, which may lead to serious consequences.

There is a method of forecasting encephalopathy in newborns (RF patent No. 2157534, 1999), which in cord blood define an is iheni loci And, In, DR HLA system, and if one of the antigens A32, B8, B27, DR3 or combinations thereof predict perinatal hypoxic-ischemic encephalopathy. The method is tedious and not sufficiently specific - different loci HLA-system observed in various diseases, including systemic.

In the patent of Russian Federation №2254573 (2005) proposed a method for predicting newborn encephalopathy by determining the activity of NADPH-oxidase in placental homogenates. Increased activity of this enzyme to 13.5 units of optical density and above allows us to predict the development of encephalopathy in the newborn. The disadvantage of this method is its lack of specificity. Proposed in this method, the enzyme activity is determined for diagnostic purposes, firstly, does not reflect the state of the placental blood flow and hemodynamics, the violation of which is the primary cause of hypoxic-ischemic brain injury fetus and newborn. Secondly, disorders of energy metabolism that can lead to serious modification of biochemical processes in the brain, primarily associated with the reactions of oxidative phosphorylation and metabolism of makroergov, and then the ratio of NADPH and NADP (Lubchenco A.M., Lukyanova L.D. Trigger role of energy metabolism in the cascade of functional-metabolic the definition of violations during hypoxia. //Problems of hypoxia: molecular, physiological and medical aspects. - M. 2004. - P.51-84).

Another method for early diagnosis of perinatal CNS lesions in newborns is the concentration of MDA in the amniotic fluid (RF patent No. 2289136, 2005), and increased more 4,50 µmol/l diagnosed with perinatal CNS damage. However, this method is non-specific, because the increase in MDA level in the amniotic fluid can lead not only to the defeat of the Central nervous system, but also to cardiopathy newborn and thyroid gland (Vierow et al. Amniotic fluid.-1998. - 215 C.). Therefore, this method can only be used in screening tests.

Similar disadvantages are characteristic of the diagnostic method of the consequences of perinatal CNS damage is mild on the patent of Russian Federation №2312351 (2006), where the diagnostic test is proposed total level of antibodies in the serum of children. This indicator is also not specific in relation to cerebral damage and can, in particular, increase in autoimmune diseases in children (Menshchikova E.B., Zenkov NICHOLAS, Shergin S.M. Biochemistry of oxidative stress. Oxidants and antioxidants. Novosibirsk. - 1994. - 203 C.).

There is a method of diagnosing consequences of perinatal CNS lesions, mild (pathé is t No. 2339043, 2007) by estimating the level of IL-1β in the serum. In this way not predicted the development of CNS lesions in the newborn, and already diagnosed with the aftermath, and therefore cannot timely pathogenetic therapy before irreversible complications.

Thus, the main disadvantages of the above methods of prediction and diagnosis of cerebral disorders in newborns are: in some cases the trauma, in the other - nespecificnomu or complexity, in some cases the need to use expensive equipment to perform the analysis.

The prototype of the claimed invention, the selected method for predicting CNS pathology in newborns (patent No. 2317043, 2008).

The essence of the prototype method is that in cord blood of a newborn determine the level of endothelin, and on the sixth day spend dopplerometrical the study of the middle cerebral artery, and if the level of gli is 8,14 PCG/ml or more, the resistance index is equal to 0.73 and more, predict the development of neurological symptoms in this child in the first 3 months of life.

This method has several disadvantages:

1. The impossibility of predicting neurological disorders in the newborn immediately after birth (Doppler is only about 6 hours).

2. The method is not sufficiently specific - changes in the level of endothelin in the umbilical cord blood reflects only one component of a complex process of regulation of the tone of blood vessels. The effect of the vasoconstrictor may be reduced due to the opposite effect extremely active vasodilators and primarily of nitric oxide (Reutov V.P. Cycle of nitric oxide in mammals // Success Biol. chemistry. - 1995. - T.35. - P.189-228).

3. The method is time-consuming, its implementation requires expensive equipment and import sets of reagents for immunoassay analysis, which hinders its widespread use in clinical practice.

4. Some degree of inaccuracy in the results diplomaticheskii changes can make extraordinary lability vascular reactions of the newborn, and severe stages of flow hypoxic lesions of the Central nervous system (change vasospasm and vasodilation) in this period.

These drawbacks are proposed to be eliminated in the present method. The objective of the proposed method is to develop to enable specific way with high accuracy to predict cerebral damage in the newborn.

The problem is solved in the following way: after delivery of placenta (at the final stage of labor) take a portion of its tissue, prepare a 5%extract, which definition shall have the content of arginine and histidine, calculate the ratio of the first to the second, and when the magnitude of the relationship below 0,86 predict the development of cerebral lesions in infants.

The technical result is obtained during use of the invention is to improve the accuracy of predicting the development of cerebral lesions in the newborn, which allows you to assign in the early neonatal period adequate pathogenetic therapy.

The novelty of the proposed method lies in the fact that for the first time proposed to predict cerebral lesions in the newborn in the early neonatal period, and in the case of a delayed their development on a single definition in the placenta ratio of the content of arginine and histidine.

Biochemical and, as a consequence, the functional changes in the placenta (placental insufficiency) are the main trigger in the development of the violations of her hemodynamics and fetal-maternal circulation, accompanied by chronic intrauterine hypoxia (Savelyeva G.M. et al., Placental insufficiency M.: "Medicine". - 1991. - 276 C.). The latter, in turn, can lead to the development of cerebral damage the fetus and later a newborn baby.

It is now established that Autonomous control of placental blood flow is competing influence of the vases is active compounds - signal molecules produced locally in the placenta. These include nitric oxide (NO), which has an extremely wide spectrum of action. He is not only a potent vasodilator, but neurotransmiters (Snyder S.H. //Science. - 1992. - vol. 257. P.494-496), controls the aggregation of platelets to the endothelium, regulates oxygen transport processes (Markov oil on canvas //Advances in physiological Sciences. - 1996. - So 27. No. 4, P.30-43). However, NO is a short - lived molecule in different tissues exists from a fraction to several seconds, and its metabolites present an incomplete picture of the possible effects of nitric oxide. As you know, the main source of NO synthesis is the amino acid arginine (E.S. Severin, Biochemistry. M: GEOTAR-MED. 2003, S-188), the contents of which allows to judge about the dynamics NO. In addition, arginine is involved in the regulation of many physiological processes.

Arginine is used in the tissues as a building material and energy, as well as operates as an independent signaling molecule. He is involved in communication between nerve cells (Dmitrienko EXAMPLE, the gut THUS, Chandarana YEAR, Ukr. her oncologist. log. No. 1. - 2008. - P.137-140).

An essential amino acid synthesized in the placenta and also performs numerous functions, is histidine, imidazole ring which expands its participation in biochemical reacts is Yah. Education high energy derivatives of imidazole may affect tissue energy pool. The product of the decarboxylation of histidine - histamine exerts opposite nitric oxide vasoconstrictor effect, affecting the tone of the placental vessels and, consequently, the intensity of circulation. Simultaneous determination of placental amino acids with multidirectional influence on the blood vessels endothelial component allow you to more objectively judge the state of the placental blood flow and fetal as oxygen and plastic substances necessary for proper development of the fetal brain and then the brain of the newborn.

We installed a prognostic parameter for the development of perinatal brain damage, by definition, 5%placenta extract ratio arginine/histidine, when the value is below 0,86 predict cerebral lesions of the newborn.

The method is as follows.

Before conducting biochemical research areas of the Central part of the placenta is washed from the blood chilled saline solution, weighed 1 g of tissue and the extraction is carried out amino acid 10% solution of trichloroacetic acid (in the ratio 1:5). To precipitate proteins, the mixture is centrifuged at 3000 rpm for 10 minutes. The supernatant in an amount of 0.1 ml of UNOSAT on a strip of chromatographic paper (density 60-80), pre-moistened with acetate-pyridine buffer solution. Electrophoresis of amino acids is carried out in the same buffer at a pH of 5.2 to 5.4 in the instrument for electrophoresis (Institute of physiology. Bohomolets, Ukraine). The duration of electrophoresis for 2 hours at 120 V/cm and a current of 0.8 mA/see At the end of electrophoresis pregrammy fixed in a drying Cabinet at 90° C for 10 min and stained with 0.5% ninhydrin solution in acetone. The color intensity is measured on the spectrophotometer SF-26 (Russia) at a wavelength of 530 nm. The location of the arginine and histidine on ForageMax identify coincidentally mobility spots - the standard of the corresponding amino acids with mobility spots placenta extract. Calculations of the content of arginine and histidine carried out on the calibration graphs prepared using standard solutions of amino acids (ICN, Belgium).

The performance of the proposed method is confirmed by the following clinical examples.

Example 1. Pregnant I-VA, 25 years. East. No. 906. The patient was in the dispensary in the antenatal clinic with 10 weeks of pregnancy. The first pregnancy. Entered the maternity ward 7.10.2008, Diagnosis: pregnancy 39 weeks, fetoplacental insufficiency. In the second trimester was installed anemia, on this occasion, has taken the necessary treatment. The first period is on delivery. 8.10.2008, was born full-term baby, the baby's weight at birth 3200 g, height 51 cm, estimation on Apgar scale 8/8 points.

Study 5%placenta extract has allowed to establish that the contents of arginine and histidine 0.85, indicating that opportunities for the development of CNS pathology in the newborn. The child was discharged from the hospital on the 6th day in a satisfactory condition. However, at the age of 2 months when viewed by the child neurologist identified General motor restlessness, tremor of extremities, muscle dystonia, reflexes quickly fade away. The clinical picture was rated as perinatal hypoxic-ischemic lesion of the Central nervous system.

Thus, the prediction made by the present method, were confirmed.

Example 2. Pregnant C-WA, 29 years. East. No. 793. Was at the dispensary in the antenatal clinic with 9 weeks of pregnancy. This first pregnancy. Entered the maternity ward 4.08.2008, Diagnosis: pregnancy 39-40 weeks, late preeclampsia, placental insufficiency. The first stage of labor. 5.08.2008 occurred this term spontaneous labour. The weight of the baby at birth 3300 g, height 52 cm, estimation on Apgar scale 7/8 points. In the study of 5% placenta extract the contents of arginine and histidine amounted to 0.80, which allowed to predict the development of perinatal porazeni the Central nervous system. From the first days of life the newborn was noted anxiety, tremor of extremities, muscle hypertonicity, the weakening of the sucking recovery of reflexes. Diagnosed with hypoxic-ischemic CNS damage the child was transferred to the neonatal care unit.

Thus, the prediction made by the present method, were confirmed.

Example 3. Pregnant E, 28 years. East. No. 849. The first pregnancy. The patient was in the dispensary in the antenatal clinic with 9 weeks of pregnancy. Entered the maternity ward 24.09.2008 was diagnosed with pregnancy 39-40 weeks, early preeclampsia, pyelonephritis. 25.09.2008, was born full-term baby, the baby's weight at birth 3250 g, height 52 cm, estimation on Apgar scale 8/8 points. Condition at birth satisfactory. In the study of 5%placenta extract the value of the ratio of the content of arginine and histidine was 0.86, indicating that the absence of risk of the development of the Central nervous system in the newborn. The child was discharged on the 6th day in a satisfactory condition. Subsequent examinations neurologist in 2 months and 6 months revealed no neurological pathology.

Thus, the prediction by the present method were confirmed.

By the present method, we have examined 102 women with a 5%placenta extract the content of arginine and histidine and the calculated is an ratio of these amino acids. Of the surveyed mothers in 39 the ratio of arginine to histidine was below 0,86, and 63 is 0.86 and above. In 38 children, in the placenta of mothers which studied the figure below 0,86 found the CNS, and only one child in this group cerebral CNS lesions within 6 months of follow-up was lacking. All babies born to women with placental ratio of arginine to histidine 0,86 and above, had no neurological symptoms either at birth or in later periods of postnatal development (up to 6 months). These studies are presented in the table.

Table
IndexThe number of the examined
Total female102
True-positive result38
False positive result1
True-negative result63
False-negative result0

From the presented data it follows that the accuracy and specificity is 99%.

The above confirms prognosticheskoe value of the claimed method.

Thus, the claimed method is more effective in comparison with the known and has a number of advantages:

1. High accuracy - 99%, specificity 99%.

2. Noninvasive.

3. Normally requires a single study.

4. The availability of way into widespread clinical practice.

5. The possibility of prediction of perinatal brain lesions of the newborn and timely preventive treatment of this pathology.

The inventive method for predicting cerebral damage in newborns allows to carry out preventive therapy to reduce the risk of irreversible complications and can be widely used in clinical practice.

A method for predicting cerebral lesions in newborns, including the study of biological material, characterized in that the five percent extract of placenta tissue determine the content of arginine and histidine, compute their value and when the value is less than 0,86 predict the development of the newborn cerebral lesions.



 

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