OXIDANT AND ANTIOXIDANT STATUS IN NEONATES WITH SPECIFIC INTRAUTERINE INFECTIONS

Objective : To evaluate lipid peroxidation (LPO) and antioxidant defense (AOD) system in full-term and preterm neonates with specific intrauterine infections (IUI). Methods : Eighty full-term and preterm newborns with specific IUI were examined; 48 of them (60%) with a severe course of IUI, and 32 (40%) with an extremely severe IUI course. The control group included 30 relatively healthy newborns, including 22 full-term and 8 late premature (born at 34-37 weeks of gestation) neonates. The state of LPO and AOD was assessed by the levels of malondialdehyde (MDA), superoxide dismutase (SOD), ascorbic (AA), and sialic (SA) acids. Enzyme-linked immunosorbent assay (ELISA) of blood serum of newborns with IUI and their mothers was carried out in paired sera, with IgG, IgM, and avidity level (%) of IUI pathogens determined. Results : Analysis of epidemiological data on TORCH infection in the examined neonates revealed diagnostic titers of cytomegalovirus infection (91.3%), herpes (70.4%), toxoplasmosis (50.1%), and chlamydia (43.4%). Comparative analysis of MDA level in the first and control groups showed a statistically significant difference (p<0.05); while its comparison between the 2 nd and control group showed even higher level difference (p<0.05). The levels of SOD, AA and SA in the 1 st and 2 nd groups were highly significantly different from the control group (p<0.001). All these tests showed significant differences between the 1 st and the 2 nd group (p<0.05), except for the levels of sialic acid (p>0.05). Conclusion : In neonates with specific IUI, statistically significant changes in LPO and AOD parameters were obtained compared with the control group. Enzymatic and non-enzymatic antioxidant parameters can be diagnostically significant for early prediction of infectious processes in the body of a newborn. The revealed changes in the LPO and AOD indicators in the neonates with IUI, dictate the need for timely and adequate antioxidant therapy along with etiotropic treatment.


Introduction
According to WHO data, every year more than 7 million children in the first years of life get sick on the background of intrauterine infection, and more than 600,000 of them die in the neonatal period and infancy. These figures are significant in the context of infant mortality in the world among children under 5 years of age [1][2][3].
Modern clinical and fundamental studies in the field of cellular physiology and pathophysiology confirm a significant role of free radical oxidation in cellular metabolism. It activates protein, lipid, and nucleic acids metabolism, and enhances the synthesis of prostaglandins, which regulate the permeability of the vascular endothelium and cell membranes [3,4].
Dyslipidemia of neonates combined with minimal impact of pathogenic microorganisms leads to activation of LPO and the formation of reactive oxygen species (ROS), which is an important link in the development of a disadaptation syndrome in the early neonatal period [5].
In newborns with bacterial pneumonia, MDA plays a significant role, changing the ionic parameters of cell membranes and affecting their function [6]. Along with this, on the background of hypoxia and anaerobic glycolysis, activation of LPO results in major metabolic disorders which may serve as prognostic indicators of the severity of the hypoxia-associated pathological processes [7,8].
Pronounced cellular hypoxia of mixed genesis in the perinatal period causes an acid-base and ionic disbalance, metabolic dissociation, and the formation of free radicals, leading to oxidative stress in cells [8]. LPO and AOD in neonates with IUI have not been subjected in detail, which justifies the expediency of this research.

Purpose of the study
To assess the level of LPO and AOD in full-term and preterm neonates with the specific IUI.

Methods
The study included 80 newborns with specific IUI, hospitalized in the neonatal pathology department of the National Medical Center of the Republic of Tajikistan "Shifobakhsh" from 2019 to 2021. The control group consisted of 30 relatively healthy neonates, including 22 full-term and 8 late preterm (born at 34-37 weeks of gestation) age-and gender-matched children.
The general condition of the patients and the severity of their disease were assessed using the modified NEOMOD scale and according to the results of clinical and laboratory investigations. According to this scale, the children were divided into two groups (Fig. 1).
The diagnosis was established on the basis of an obstetric anamnesis, an anamnesis of the child's life, clinical and laboratory data, and was confirmed by ELISA with the detection of specific antibodies against certain IUI pathogens in the blood serum of neonates and their mothers.
In addition to routine examination methods (study of anamnestic, clinical and laboratory data, physical examination results, assessment of physical development, results of microbiological blood tests, determination of markers of TORCH infections, biochemical parameters of blood serum, electrolyte levels in the blood, cultures of blood and feces for microflora) a study of the oxidant and antioxidant systems of the neonates was carried out.
The material for the study was venous blood serum. The study of LPO and AOD indicators was carried out on the basis of the Department of Biochemistry of the Avicenna Tajik State Medical University. The level MDA was assessed using the reaction with 2-thiobarbituric acid according to the method of Stalnaya ID and Garishvili TG [9]. The activity of the SOD was determined by the method of Fried R (1975), with an assessment of its inhibition of the aerobic reduction of nitroblue tetrazolium to formazan [9,10]. The level of non-enzymatic antioxidant -AA -was determined by the method of Sokolovsky VV. The level of SA was determined by reaction with thiobarbituric acid or by the Hess colorimetric method.
The study was approved by the Ethics Committee of the Avicenna Tajik State Medical University (protocol No. 10 of October 8, 2019).
Statistical analysis of the obtained results was performed using the Statistica for Windows 8.0 software package (StatSoft Inc., USA, 2008) and an online calculator for statistical criteria (http://medstatistic.ru/calculators.html). Quantitative indicators were presented as M±σ, where M is the mean value, and σ is the standard deviation. Nonparametric Mann-Whitney tests were used to compare data between two independent groups. Multiple comparisons for dependent samples were carried out using the Kruskal-Wallis H-test. Differences in indicators were considered statistically significant at p<0.05.
An analysis of the obstetric anamnesis of women showed a close relationship between infectious and inflammatory diseases and gestational hypertensive disorders, which significantly affected the fetus and were considered as a specific link in the development of chronic intrauterine hypoxia. In accordance with studies by a number of authors, pregnant women with infectious and inflammatory diseases exhibit activation of Th1 cells, an increase in the number of pro-inflammatory substances, an elevation of the level of anti-angiogenic proteins, an increase in the intensity of gene expression of the vascular endothelial growth factor (VEGF) with the VEGF-R1 receptor, and vascular endothelial dysfunction, which are the basis for the formation of fetal-placental disorders and serve as a trigger for the development of intrauterine fetal hypoxia [11].
Analysis of epidemiological data on TORCH infections in mothers of observed newborns revealed diagnostic titers of cytomegalovirus infection (97.3%), herpes (89.5%), chlamydia (54.3%), and toxoplasmosis (61.5%). Epidemiological data presented by some researchers confirm the widespread prevalence of cytomegalovirus and herpetic infections ranging from 40% to 100% among women of reproductive age [2,3]. Our results on these pathogens prevalence in observed women were comparable with data from international epidemiological studies. However, it should be noted that in most cases the women enrolled in our study had a complicated course of the infectious process and a combination of infectious agents, which was most likely due to the unsatisfactory quality of medical monitoring in the antenatal period, the traditional ritual dependence of young mothers on the husband's family and husband's health. It is important to emphasize that in most cases (86.5%) the fathers of the observed neonates were labor migrants, and the results of the survey of mothers confirmed the unfavorable living conditions of husbands during labor migration. In addition, almost 40% of the observed women confirmed that their husbands received inpatient and outpatient treatment for infectious diseases of the genitourinary system.
Among the newborns under our observation, 32 (40%) were from the 1 st pregnancy, 28 (35%) from the 2 nd , and 12 (15%) from the 3 rd and subsequent pregnancies. At the same time, all the children we observed were born with asphyxia of varying severity. According to the Apgar scale, 42 (52.5%) were rated at 6-7 points, 30 (37.5%) -4-5 points, and 8 (10%) neonates -1-3 points. It is known that intrauterine hypoxia and asphyxia during childbirth are the leading mechanisms for the development of the disadaptation syndrome and act as the main cause of death of newborns in the early and late neonatal periods [12].

Fig. 2 ELISA indicators for TORCH infection in neonates
Data on the detected diagnostically significant titers of antibodies to viral and bacterial pathogens of IUI in ELISA tests are shown in Fig. 2.
Oxidative stress is a trigger in the development of cellular hypoxia, however, on the background of the above clinical symptoms, the formation of free radicals is accelerated, which contributes to the intensification of LPO, as a result of which the lipid layer of cell membranes is damaged [13,14]. Polyene lipids of biomembranes are targets for a number of endotoxic substances, such as oxygen radicals and lysosomal hydrolytic enzymes, under the action of which lipids and highly active enzyme-like substances are formed, attacking the biopolymer molecules. During this process, not only the primary LPO products (diene conjugates) are important, but also intermediate products of free radical lipid oxidation, such as MDA, play a significant role.
Our study showed that LPO in neonates with IUI differed between the two groups of patients. MDA level was significantly higher in the neonates of the 1 st group compared to the control group (p<0.05). Even higher difference in MDA levels (p<0.05) was observed between the 2 nd clinical and control groups (Fig.3).
The above changes confirm the aggressiveness of oxidative stress and cell metabolism disorders that developed on the background of chronic cellular hypoxia. The intensity of free radical formation was observed in both groups of children with IUI, while a more pronounced acceleration of LPO was noted in premature neonates with an extremely severe course of IUI. With a high probability, this indicates the limitations of the adaptive resources of the body in these groups of newborns and the rapid depletion of the compensatory mechanisms on the background of hypoxia.
Our data are in agreement with a study on the levels of MDA in the blood serum of neonates residing in a zone of increased radiation [15]. This similarity indicates the dependence of the MDA level not only on the cause of the LPO intensification, but also on the duration of oxidative stress during the perinatal period.
The Table presents pro-and antioxidative indicators in the surveyed group of children.
As follows from the Table, for all the studied indicators, a statistically significant difference was obtained when comparing

ВЕСТНИК АВИЦЕННЫ
Том 25 * № 2 * 2023 both groups of children with IUI and the control group (p 1 <0.001). When comparing the levels of these indicators between the first and second groups, a statistically significant difference (p 2 <0.05) was also found, except for the levels of SA (p 2 >0.05).
It is known that SOD is an enzyme protecting the cells from the damaging effect of ROS. In addition, this enzyme is a predictor of organic damage to cells and tissues [8,14,15]. According to some authors, in response to the activation of infectious agents and exposure to their metabolic products, the level of free radical compounds released from activated immunocompetent cells in the mother's body and from the placenta increases [5,11,16]. Long-term exposure to free radicals, oxidative stress, and the continuous accumulation of under-oxidized products in the body of the fetus and neonates with IUI lead to depletion of the body's AOD, hypoxic damage to the lipid layer of erythrocyte biomembranes, the development of hemic hypoxia that occurs on the background of the fetal hemoglobin breakdown and destruction of other membrane structures of the newborns. Initially, during prenatal development, the above changes may be accompanied by a temporary increase in the level of SOD, which will gradually decrease on the background of a prolonged IUI later in the perinatal period. This indicates the depletion of adaptive resources of the AOD system of neonates with IUI [16].
Along with enzymatic antioxidants, there are a number of non-enzymatic antioxidant substrates in the body, such as AA and SA, which are actively involved in the process of ROS deactivation and intensively work in favor of the body's AOD. In oxidative stress and hypoxia developed as a result of certain pathological conditions, the level of these substrates in the blood serum is unstable [15].
Due L et al (2022) studied sialo-containing substances in the placenta of pregnant women and confirmed the significance of determination of the non-enzymatic antioxidant (SA) for the early diagnosis of an infectious and inflammatory process in the fetus организма у этих групп НД и быстром истощении компенсаторных механизмов организма на фоне гипоксических явлений.
Our study of LPO and AOD showed that the process of free radical peroxidation in IUI begins in the prenatal period when the mother's body reacts aggressively to the reactivation of an infectious agent. After birth, on the background of chronic hypoxia and protracted infection, the body of premature and full-term neonates with specific IUI undergoes even more pronounced hypoxia and hypoxemia. Changes in MDA, SOD, AA, and SA levels indicate a disruption of cellular metabolism, intensification of free radical oxidation in cells, and disturbance of the ion exchange in cell membranes, which are clinically manifested by dysfunction of organs and systems affected by the viral and bacterial pathogens.

Conclusion
In neonates with specific IUI, statistically significant changes in LPO and AOD indicators were obtained compared with newborns in the control group. Enzymatic and non-enzymatic antioxidants may be diagnostically significant markers for the early prediction of infectious processes in the body of neonates. The revealed disturbances in the LPO and AOD in newborns with IUI underlie the importance of timely and adequate antioxidant therapy along with etiotropic treatment.