Proteomic analysis of blood serum - a new approach to the search for diagnostic markers of bronchial asthma in children.

Currently, bronchial asthma (BA) is one of the most pressing medical and social problems, the molecular aspects of the formation and development of BA are insufficiently studied and the diagnosis is not perfect. Carrying out proteomic analysis of BA will not only reveal new biomarkers specific to this disease, but also bring us closer to understanding its pathogenetic mechanisms. The purpose of the study: to study the proteomic profile of blood serum of children with BA to identify proteins associated with this disease A comprehensive clinical and laboratory examination of children suffering from BA and control group patients was performed. Proteomic analysis of depleted blood serum included high-resolution two-dimensional electrophoresis (1 direction: immobiline strips 17cm, pH 3-10, 2 direction: denaturing electrophoresis in 12.5% polyacrylamide gel), protein staining on gels with fluorescent dye Flamingo, protein identification by MALDI-TOF mass spectrometry using the search algorithm Mascot and the Swiss-Prot database. Comparison of the proteomic profile of BA serum and the control group patients serum allowed us to establish that the production of a number of proteins is reduced in this pathology. Among them, proteins in the molecular weight range of 16-33 kDa (p<0.05) were identified: glutathione peroxidase 3, transtyretin, complement components C4b and C3. Research shows that changes in the children's serum proteome occur in BA, affecting proteins that play an important role in immune responses, ligand transport, and antioxidant protection. Special attention should be paid to the differences identified in the course of this work (glutathione peroxidase, transtyretin, C3 and C4 fragments of the complement system) or their combinations. Studying the features of their expression will expand our understanding of the molecular mechanisms underlying chronic inflammation of this disease.

[Impairments in metabolism of amino acids - precursors of gasotransmitters - in the premature birth]. / Narushenie obmena aminokislot ­ predshestvennikov gazotransmitterov pri prezhdevremennykh rodakh.

The content of amino acids - sources of gasotransmitters and the activity of enzymes of their metabolism have been studied in the placenta and amniotic fluid in full-term and complicated by preterm birth (PB). Determination of amino acids was carried out using an automatic analyzer; specific spectrophotometric methods were used to assess the activity of enzymes. The development of PB is accompanied by changes in the amino acid level already in the second trimester of pregnancy. Correlation of differently directions was found between the level of amino acids and the activity of the corresponding enzymes. The imbalance of amino acids in the fetoplacental system in PB is accompanied by a change in the production of their low molecular weight derivatives gas transmitters (NO, CO, H2S), which play an important role in the regulation of numerous metabolic processes.

Violation of the balance of free metals and metal-containing proteins in amniotic fluid in placental insufficiency.

The content of metal ions and proteins containing or binding these metals in amniotic fluid during different periods of physiological pregnancy and placental insufficiency (PI) was studied. The content of zinc, copper, magnesium, iron, zinc-α-2-glycoprotein, ferritin, ceruloplasmin and the activity of Ca2+, Mg2+-ATPase were estimated using spectrophotometric methods, immunoturbometric and enzyme immunoassay methods. It was found that in PI in both trimesters there is a decrease in the content of zinc, copper, iron and an increase in the level of copper. The indices of ceruloplasmin, ferritin, Ca2+, Mg2+-ATPase in PI are lower, and zinc-α-2-glycoprotein is higher than in similar periods of physiological gestation. A close correlation of different directions has been established between the level of metals and the corresponding proteins. The revealed violations obviously play a certain pathogenetic role in the development of PI, and the indicators of the ratio between metals can serve as markers for predicting the state of newborns.

[Impairment of production and posttranslational changes of placental nuclear and membrane proteins with complicated pregnancy]. / Narushenie produktsii i posttransliatsionnye izmeneniia platsentarnykh iadernykh i membrannykh belkov pri oslozhnennoi beremennosti.

The content of nuclear and membrane proteins of the placenta, as well as posttranslational modification of these proteins in physiological pregnancy and placental insufficiency (PI) were studied. Differential centrifugation, electrophoresis in polyacrylamide gel, spectrophotometric methods were used. It was found that with PN there is a decrease in the degree of production of the studied proteins of varying degrees relative to control parameters. For chromatin proteins, a more pronounced decrease in the content of non-histone proteins was found in comparison with histones. Among histone fractions, the maximum decrease was detected in the H2A fraction. The degree of change in the amount of membrane proteins depends on the detergent used. Changes in posttranslational protein modifications disorders are characterized by a decrease in the content of amine and amide (especially difficult to hydrolyze) groups and an increase in carbonyl derivatives of proteins. The revealed changes in the composition and structure of the nuclear and membrane proteins of the placenta, performing numerous regulatory functions, can be triggering links in the chain of molecular damage in the placenta at PI.

[The imbalance of vasoactive components and arachidonic acid in the placenta and amniotic fluid in preeclampsia]. / Disbalans vazoaktivnykh komponentov i arakhidonovoi kisloty v platsente i okoloplodnykh vodakh pri preéklampsii.

The content of vasoactive compounds and arachidonic acid in the placenta and amniotic fluid was studied in full-term (39-40 weeks) physiological pregnancy and preeclampsia (PE). The content of metabolites of nitric oxide (NOx), endothelin-1, thromboxane B2 (TxB2), prostacycline (PGI2) and arachidonic acid was estimated using spectrophotometric, immunoenzyme methods and gas-liquid chromatography. It was found that in PE the content of vasoconstrictors, of endothelin and TxB2, increased in the placenta and amniotic fluid, while the content of vasodilators, PGI2 and NOx decreased. Despite the same directionality of changes in both studied objects, the degree of changes differed and was more pronounced in the placenta. A direct or inverse correlative relationship was found between various vasoactive components (depending on their effect on vascular tone). In the case of arachidonic acid changes in its content in PE correlated with the level of vasoactive compounds, the source of which it is. The revealed differences in the ratio of vasoactive components obviously play a pathogenetic role in the development of PE and its subsequent complications.

[The activity of enzymes of amino acid metabolism of the placenta in different terms of the physiological and complicated pregnancy.]

The activity of amino acid metabolism enzymes and the content of free amino acids in the placenta during physiological pregnancy and placental insufficiency (PI) were studied using spectrophotometric methods and ion-exchange chromatography. It was found that in PI placental activity of the studied enzymes: alanine-, cysteine-e, tyrosine-, glutamino- transferase, glutathione synthetase, glutamate dehydrogenase decreases at different periods of gestation. The opposite variations occur for aspartataminotranferase and glutaminase. Similar changes are detected for amino acids synthesized or used in the course of appropriate reactions: aspartic acid, glutamic acid, glutamine, alanine, cysteine, tyrosine, arginine. The correlation between enzyme activity and amino acid content was revealed. Different periods of pregnancy are characterized by varying degrees of change, especially expressed in the second trimester, characterized by the most intense growth and development of the fetus, and its increased needs for trophic material. The revealed changes obviously play a pathogenetic role in the formation and further development of PI.

[Features of redox processes in the amniotic fluid at placental insufficiency]. / Osobennosti okislitel'no-vosstanovitel'nykh protsessov v okoloplodnykh vodakh pri platsentarnoi nedostatochnosti.

Activity of prooxidant enzymes (NADPH-oxidase and xanthine oxidase), antioxidant enzymes (superoxide dismutase (SOD) and catalase), enzymes of the glutathione-dependent systems, as well as antioxidant vitamins (retinol and a-tocopherol), lipid peroxidation products (LPP) (conjugated dienes and Schiff bases), and peroxide chemiluminescence were studied in the amniotic fluid at different periods of physiological pregnancy and placental insufficiency (PI). It was found that at PI the activity of NADPH-oxidase, xanthine oxidase increased and the activity of SOD, catalase, glutathione peroxidase, glutathione reductase, glutathione transferase and the content of fat-soluble vitamins decreased. The direct and inverse correlation between the studied pro- and antioxidant parameters and the content of LPP products, was found ro be different in the II and III trimesters of gestation. The revealed differences obviously reflect metabolic impairments in the fetoplacental complex, and the activity and level of the paremeters of redox processes can be used as tests for pre- and postnatal disorders.

[Disbalance of redox processes in amniotic fluid at pre-eclampsia.]

The parameters of pro and antioxidant processes in the amniotic fluid in the dynamics of physiological pregnancy and preeclampsia were studied. Spectrophotometric and fluorimetric methods were used to evaluate the activity of NADPH-oxidase, xanthine-oxidase, superoxide dismutase (SOD), catalase, glutathione-dependent enzyme systems, the content of antioxidant vitamins - retinol and α-tocopherol, lipid peroxidation products (LPP) - diene conjugates and Schiff bases, and parameters of peroxide chemiluminescence. It was found that with preeclampsia there is an increase in the activity of prooxidant enzymes NADPH-oxidase and xanthine-oxidase, a decrease in the activity of antioxidant enzymes SOD, catalase, glutathione peroxidase, glutathione reductase, glutathione transferase, and the content of fat-soluble vitamins. Between pro- and antioxidant indices and the content of LPP, intensity of chemiluminescence a correlation is established, the degree of which is different in the II and III trimesters of pregnancy. The revealed changes obviously play a pathogenetic role in the formation and further development of preeclampsia, and the indicators of oxidation-reduction processes can serve as informative markers of pre- and postnatal pathology.

[Influence of amino acid imbalance in maternal and fetal organisms on the development of placental insufficiency and the course of the neonatal period.]

The content of amino acids in maternal serum and umbilical cord blood during physiological pregnancy and placental insufficiency (PI) was estimated using ion-exchange chromatography. It was found that that the content of arginine, serine, glutamine, alanine, cysteine, methionine, tryptophan, leucine, phenylalanine and proline, which are involved in many metabolic processes necessary to maintain the normal functioning of the mother-placenta-fetus system, was decreases in maternal serum at PI. Opposite deviations have dicarboxylic amino acids, lysine, histidine, glycine, tyrosine and threonine, the number of which increases, strengthening the nitrogen imbalance. In the blood serum of the umbilical cord for most amino acids, was a similar direction of changes, but the degree of it is more pronounced. The opposite direction of changes is found for glycine, threonine, tyrosine, the content of which is reduced. The results of the study make it possible to broaden the understanding of the mechanisms of PI development and to offer informative tests for predicting the state of newborns.

[Impairments of placental amino acid metabolism in fetal growth restriction]. / Narushenie platsentarnogo obmena aminokislot pri zaderzhke rosta ploda.

The content of the amino acids in the placenta during physiological pregnancy and fetal growth restriction (FGR) has been investigated my means of the method of ion-exchange chromatography. It has been found that in FGR the placental amino acid pool is characterized by a decreased content of arginine, proline, alanine, serine, cysteine, methionine, tryptophan, leucine, threonine, tyrosine, phenylalanine, glutamine and an increased content of dicarboxylic amino acids, lysine, histidine and glycine. These changes are accompanied by altered activity of some enzymes of amino acid metabolism, and the degree of these changes correlates with the level of corresponding amino acids.

Proteomic Profiling of the Blood Serum for Prediction of Premature Delivery.

Mass-spectrometric profiling of the serum in women at weeks 16-17 of gestation was carried out in order to detect proteomic predictors of preterm delivery. Changes in the production of 25 proteins (down-regulation for 13 proteins and up-regulation for 12 proteins) were detected in the sera of women whose pregnancies eventuated in premature deliveries. Among them, proteins with various regulatory functions were distinguished: antioxidant enzymes, chaperons, cytoskeleton proteins, cell adhesion molecules, and proteins involved in angiogenesis, proteolysis, transcription, inflammation processes, binding and transportation of various ligands. These results indicated the formation of proteomic imbalance as early as during trimester II, this eventually leading to premature delivery. The detected serum proteins were suggested as markers for early prediction of premature delivery.

[The imbalance of metal-containing proteins and free metal ions in the amniotic fluid during fetal growth].

The levels of zinc, copper, iron, and magnesium ions, and some of their binding proteins have been investigated in an amniotic fluid under the fetal growth retardation (FGR). FGR, developed under conditions of placental insufficiency, is characterized by a decrease in the content of zinc, iron, and magnesium ions and by an increase in the copper content in the amniotic fluid in the II and III trimesters of pregnancy. During these trimesters the levels of ceruloplasmin, ferritin, and Ca2+,Mg2+-ATPase were lower in FGR, while the level of zinc-a-2-glycoprotein was higher than during the same periods of normal pregnancy. Changes in the parameters studied in the amniotic fluid were associated with developmental disorders of the newborns. These changes obviously have a pathogenetic importance in the development of FGR, and the levels of metal ions and their ratio in the amniotic fluid can be used as markers of the pre- and postnatal pathology.

[Imbalance of system of glutamin - glutamic acid in the placenta and amniotic fluid at placental insufficiency].

Metabolism of glutamine and glutamic acid has been investigated in the placenta and amniotic fluid under conditions of placental insufficiency. The development of placental insufficiency is characterized by the increased content of glutamic acid and a decrease of glutamine in both placenta and amniotic fluid. These changes changes were accompanied by changes in the activity of enzymes involved in the metabolism of these amino acids. There was a decrease in glutamate dehydrogenase activity and an increase in glutaminase activity with the simultaneous decrease of glutamine synthetase activity. The compensatory decrease in the activity of glutamine keto acid aminotransferase did not prevent a decrease in the glutamine level. The impairments in the system glutamic acid-glutamine were more pronounced during the development of premature labor.

New approaches to molecular diagnostics of prenatal pathology.

Analysis of the spectrum of amniotic fluid proteins in physiological and abnormal pregnancy using proteomic analysis allowed detection of a number of difference proteins, that are absent or, alternatively, appear in gestosis. Among absent proteins, there were NADPH-dependent carbonyl reductase, epidermal fatty acid-binding protein, haptoglobin, calgranulins A and B. In contrast to proteomic spectrum of amniotic fluid in physiological pregnancy, 7 new proteins appear during gestosis, 3 of them were identified: C area of immunoglobulin K-chain, breast cancer metastasis suppressor-1, and protein-1 containing AIG2-like domain. Possible effects of revealed differences in proteomic spectrum on development of main disturbances during gestosis are discussed. Difference proteins detected in amniotic fluid may serve as gestosis markers.