PECULIARITIES OF METABOLISM (BY THE LEVEL OF NITRATES, NITRITES, MALONDIALDEHYDE AND SIALIC ACIDS) IN 6-9 MONTHS OLD INFANTS BORN FROM MOTHERS WITH METABOLIC SYNDROME, WHO HAD HYPOXIC- ISCHEMIC ENCEPHALOPATHY IN THE EARLY NEONATAL PERIOD
Authors: Kovalova O. , Pokhylko V. , Cherniavska Y. , Tsvirenko S. , Rossokha Z. , Davydenko A. 
Published: December 2024

According to the World Health Organization, asphyxia and hypoxic-ischemic encephalopathy remain the leading neurological causes of disability and mortality in newborns. The degree of brain damage and subsequent neurological complications is determined by a number of factors, including duration of hypoxia/ischemia, cerebral circulation, neonatal central nervous system immaturity, and maternal somatic status.
The aim of the study was to determine the metabolic characteristics (by nitrate, nitrite, malondialdehyde and sialic acid levels) of infants aged 6-9 months born to mothers with metabolic syndrome who had hypoxic-ischemic encephalopathy in the early neonatal period, and the effect of eNOS (G894T) and IL-1b (C3953T) gene variants on the metabolism of these metabolites.
Materials and methods. To this end, a prospective case-control study was conducted in 30 children aged 6-9 months. The main study group consisted of 16 children born to mothers with metabolic syndrome who had an Apgar score of less than 7 points at 5 minutes and diagnostic clinical and neurosonographic signs of hypoxic-ischemic encephalopathy, as determined during a consultation with a pediatric neurologist at 6-9 months of age. The comparison group consisted of 14 randomly selected relatively healthy children aged 6-9 months, born to mothers without metabolic syndrome and without manifestations of hypoxic-ischemic encephalopathy in the early neonatal period.
The concentration of nitrites was determined by determining the diazo compounds formed in the reaction with sulfanilic acid, and then the reaction was carried out with α-naphthylamine (Grisse-Ilosway reagent). The concentration of free malondialdehyde was determined by the spectrophotometric method. The concentration of sialic acids in urine was determined by the reaction with acetic-sulfuric acid reagent (Hess reaction). Molecular genetic analysis was carried out using buccal epithelium as biological material. Determination of IL1B 3953C>T (rs1143634) and eNOS G894T (rs1799983) polymorphic variants was carried out by polymerase chain reaction using the commercial kit "DreamTaq Green PCR Master Mix" (Thermo Scientific, USA) and specific oligonucleotide sequences (Metabion, Germany).
Statistical analysis. Traditional methods of parametric and non-parametric statistics were used to process quantitative values; non-parametric methods were used for the analysis of qualitative characteristics expressed mainly in percentages. Under the normal distribution of data, the main statistical characteristics were used, namely: the average value (M) to determine the central tendency; the standard error of the mean (m) for the precision of the estimate of the mean, the confidence interval (CI) for determining the 95% interval of the mean. Hypotheses regarding the equality of general means were tested using a two-tailed Student's t-test. Comparison of relative or percentage values was performed using Fisher's exact two-sided test. Relationships between count variables were determined using Poisson multiple regression analysis. Statistical analysis was performed using the STATA 14.0 software package.
The study was conducted in compliance with the ‘Rules for Ethical Principles for Scientific Medical Research Involving Human Subjects’ approved by the Declaration of Helsinki (1964-2013), ICH GCP (1996), EEC Directive 609 (24.11.1986), Order of the Ministry of Health of Ukraine No. 690 of 23.09.2009 and confirmed by the conclusion of the Biomedical Ethics Commission of Poltava State Medical University (Protocol No. 233 of 21.11.2024), which approved the study. Written consent was obtained from the parents of the newborns.
The study was conducted within the framework of the joint budget research work of the Department of Pediatrics №1 with Neonatology of the Poltava State Medical University on the topic «Development of clinical and laboratory criteria, methods for predicting and preventing metabolic disorders in children of early age», state registration number 0120U102856, term of execution 2020-2024.
Research results. A study of the indicators of nitric oxide exchange in the group of examined children showed that the concentration of nitrites was significantly higher in children with hypoxic ischemic encephalopathy in the period of 6-9 months of life, 2.69 nmol/l compared to healthy children, 1.39 nmol/l (p<0.001). Similar differences were obtained in the concentration of nitrates (5.41 vs 2.62; p<0.001). We did not obtain significant differences in the levels of nitrates, nitrites and malondialdehyde depending on the genotypes of the IL1B gene (C3953T, rs1143634), but we found a higher (at the level of p <0.1) concentration of sialic acids in children with the CC genotype than in children with СT or TT genotype. When studying the eNOS gene (G894T, rs1799983), we did not find significant differences in the levels of nitrates, nitrites, sialic acids and malondialdehyde in children with different genotypes. The study revealed a reliable direct relationship (at the level of p<0.1) between the level of nitrates in the urine of children of the examined groups and the concentration of sialic acids (Coef. 0.753, p=0.062).
Conclusion. The results of the study indicate an increase in the levels of nitrates (5.41 vs. 2.62; p<0.001) and nitrites (2.69 vs. 1.39; p<0.001) in children of mothers with metabolic syndrome in the recovery period of hypoxic ischemic encephalopathy, which can indicate the progression of pathological processes caused by hypoxic damage or testify to the adaptive mechanisms of recovery after brain tissue damage. A reliable direct relationship was also found between the level of nitrates in urine in children of the examined groups and the concentration of sialic acids (Coef. 0.753, p=0.062). At the same time, these changes were not associated with polymorphic variants of the eNOS (G894T, rs1799983) and IL1B (C3953T, rs1143634) genes. Thus, determining the concentration of nitrates and nitrites can be implemented in clinical practice for early diagnosis and prediction of possible complications in children with hypoxic ischemic encephalopathy, because the increased level of nitrites, nitrates and sialic acids can serve as a marker of distant consequences of hypoxic damage. This allows us to consider them as promising predictors of a child's suboptimal neuropsychological development.