Журнал «Здоровье ребенка» 7 (50) 2013

During the first days of life "oxidative stress" stimulates the formation of reactive oxygen species (ROS), which cause the increase of lipid peroxidation (LPO) and the corresponding activation of the antioxidant defense system (ADS). Under the influence of  hypoxia, use of high oxygen concentrations during resuscitation and subsequent treatment of respiratory failure lipid peroxidation activates, but this is not followed by the adequate activation of ADS in newborns, increasing the risk of a variety of pathological conditions in this group of patients [T. K. Znamenska et al., 2008; D.O. Dobrjanskuj, 2000]. Newborns are at risk for the development of ROS-induced diseases: bronchopulmonary dysplasia, necrotizing enterocolitis, retinopathy of prematurity and periventricular leukomalacia [Jennifer W. Lee, 2011; N.V. Nagornaja, 2010].

LPO is one of the most studied form of free radical damage. It is known that spontaneous activity of lipid peroxidation increases with the decrease of gestational age. Antioxidant function of vitamin A - protection of biological membranes from damage by reactive oxygen species. Vitamin E reacts with the hydroxylic radical, inactivates superoxide radicals and inhibits lipid radicals. The activity of vitamin E increases with gestational age [D.O. Dobrjanskuj, 2000].

Exploring indicators of oxidative stress resulting from an imbalance between LPO and ADS makes it possible to reveal the pathogenesis of pathological processes, to evaluate the risk of their occurrence and to predict the characteristics of the disease. Removal of oxidative stress  increases the effectiveness of treatment and rehabilitation measures.

The aim of research was to examine the state of prooxidant-antioxidant system in neonates with respiratory disorders (pneumonia, respiratory distress syndrome (RDS), transient tachypnea, asphyxia) on ventilation mode SIMV/PS (synchronized intermittent ventilation with pressure support) and nCPAP (spontaneous breathing with continuous positive airway pressure via nasal cannula), to evaluate its diagnostic and prognostic value.

Materials and methods                 

The levels of  LPO indicators (TBA-active products, lipid hydroperoxides, diene conjugates) and ADS indicators (superoxide dismutase, glutathione peroxidase, vitamin A, vitamin E) were measured in the blood of 40 newborns treated at the neonatal intensive care unit of Lviv City Children's Hospital. All infants had symptoms of respiratory distress that appeared on the first day after birth. Clinical diagnosis was made basing on the evaluation of data history, results of objective, instrumental (chest X-ray; neurosonography; electro- and echocardiography) and laboratory (general blood and urine tests; biochemical parameters of blood serum; standard bacteriological tests) surveys. All children received respiratory therapy (mechanical ventilation, nCPAP) standard infusion therapy, antibiotics, etc. According to the applied method of respiratory therapy infants were divided into 2 groups: 1st - 27 children who were on mechanical ventilation mode SIMV /PS, 2nd - 13 children on nCPAP. The average gestational age of children in the first group – 35.5 ± 0.82 weeks, mean birth weight – 2556.7 ± 212.97 g. In newborns of the second group – 35.9 ± 1.0 weeks and 2801.5 ± 269.58 g respectively. Boys predominated in both groups (74.1% - 1st group, 53.8% -  2nd group).

Patients received respiratory support with the help of machines: VIP "BIRD", VIP "Newport", VIP "Inspiration", and nCPAP - Infant Flow system. The respiratory therapy was started on the 1st day of life in both groups. In 14 newborns  of the first group (51.9%) mechanical ventilation was started during the first 6 hours of life, in 2 infants (7.4%) - from 7 to 12 hours of life and in 11 (40.7%) - from13 to 24 hours. In 2 children (15.4%) of the 2nd  group nCPAP was started during the first 6 hours of life, in 4 (30.8%) – from 7 to 12 hours and in 7 infants (53.8%) - from13 to 24 hours. The average duration of ventilation (SD) in children of the first group - 6.4 (4.5) days, in neonates from the second - 3.6 (1.5) days.

Considering the heterogeneity of lung diseases in both groups of newborns the index of the pulmonary injury severity (IPIS) was determined by the method of M. Palta et al. According to the received scores 10 infants on mechanical ventilation mode SIMV/PS had the hardest pulmonary injury (IPIS> 50 scores), 6 - heavy (IPIS> 20 scores), 3 - light pulmonary injury (IPIS <20 scores). For newborns on nCPAP, one child had light pulmonary injury (IPIS <20 scoress), one neonate −  moderate lung injury (IPIS = 20 scores), and 11 children - heavy lung injury.

 The status of LPO was determined by the following methods: TBA-active products − method  of Korobejnikov; diene conjugates (DC) − method of Stalna; lipid hydroperoxides (HPL) - method of Myronchyk. The activity of antioxidant enzymes was measured in the red blood cells: superoxide dismutase (SOD) - the method of Dubinin and glutation peroxidase (GP) – method of Moin. The levels of vitamins A and E in serum were determined with the liquid chromatography apparatus "Milihrom-4".

Statistical analysis of the data was performed using  program STATISTICA 10 (Statsoft, USA). Values are presented as arithmetic mean (M), standard error of the mean (SE), n-sample size. The difference between groups was established using Student's test (t-test) under the normal distribution. If the distribution was different from the normal we used a nonparametric Mann-Whitney test (U-test). Differences in nominal performance was evaluated using ^{ᵪ 2} criteria or Fisher's exact test. All results were considered reliable at p <0.05. In order to determine the relationship between the studied parameters we defined Spearman's correlation coefficient.

Results and discussion

Analysis of data history revealed that 17 children (62.96%) of the first group were born from pregnancies with complicated course (threatened miscarriage, hydramnion, hypamnion, preeclampsia, gestosis of the second half of pregnancy, episodes of acute infectious diseases). In 18 cases (66.7%) labour were with complications (premature discharge of amniotic fluid, fetal distress, weakness of labour, tight loop of cord around the neck, placental presentation, uterine bleeding). In addition, in 18 cases (66.7%) delivery were conducted by Cesarean section, 6 of them – repeatedly. 7 babies (25.9%) were born with Apgar scores on the 1st minute less than 4 points, 15 infants (55.6%) − with Apgar scores from 4 to 6 points. 17 newborns (62.96%) required resuscitation procedure in the delivery room.

Concerning newborns from the 2-nd group, 4 children (14.8%) were born from pregnancies with complicated course (threatened miscarriage, hydramnion, episodes of acute infectious diseases). In 4 cases (14.8%) labour were with complications (bleeding due to placental presentation, tight loop of cord around the neck, abruption of placenta). In 6 cases (46.2%) Cesarean section was performed, 5 of them – repeatedly. One child was born with Apgar score on the 1st minute less than 4 points, 7 infants (53.8 %) − 4-6 points. 4 infants (14.8%) required of resuscitation in the delivery room.

There were no significant differences found between the groups in terms of maternal infectious morbidity (all relevant data were subjected to analysis, but not shown).

In the structure of primary diseases, which manifested in newborns with respiratory distress no significant difference was found between the groups in RDS (44.4% vs. 46.2%, p = 0.92), congenital pneumonia (25.9% vs. 30, 8%, p = 0.75), asphyxia (29.6% vs. 15.4%, p = 0,33). In addition, one neonate in the second group had transient tachypnea. We also did not reveal significant difference between groups concerning the age of neonates at the beginning of respiratory support (66.7% vs. 46.2%, p = 0,43).

The results show a more pronounced activation of LPO in infants on mechanical ventilation mode SIMV/PS compared with neonates on nCPAP (TBA-active products 2.57 ± 0.11 nmol / ml vs 1.84 ± 0.17 nmol / ml, p <0.01; diene conjugates 21.30 ± 0.48 mmol / l vs 14.77 ± 0.46 mmol / l, p<0.01; lipid hydroperoxides 2.27 ± 0.15 Od480/ml vs 1.7 ± 0.09 Od480/ml, p ≤ 0.01). The levels of TBA-active products, lipid hydroperoxides, diene conjugates exceed the corresponding rates in children on nCPAP 1.4 times. According to literature, the accumulation of lipid hydroperoxides show the active course of the initial stages of LPO. In turn, the increase of TBA-active products is the evidence of prolonged pathological process that does not lose its sharpness [S.K. Sokodajeva, 2008] We also established a number of significant correlations: between gestational age and levels of diene conjugates and lipid hydroperoxides (r = -0.59, p <0.01 ; r = -0.35, p <0.05 respectively) between the start of respiratory therapy and TBA-active products (r = -0.47, p <0.05), diene conjugates (r = -0.47, p <0.05), lipid hydroperoxides (r = -0.38, p <0.05); between IPIS and TBA-active products (r = 0.78, p <0.001), diene conjugates (r = 0.62, p <0.001), lipid hydroperoxides (r = 0.45, p <0.01).

In addition to activation of LPO in neonates on mechanical ventilation mode SIMV/PS a decrease in activity of ADS was found in comparison with children on nCPAP (SOD 2.6 ± 0.13 IU/mg Hb vs 3.3 ± 0.23 IU/mg Hb, p <0.01; GP 0.15 ± 0.02 mkmol/min. * mg Hb  vs 0.25 ± 0.02 mkmol/min. * mg Hb, p <0.01; Vitamin A 0.78 ± 0.12 mmol/l  vs 2.17 ± 0.11 mmol/l , p <0.01; Vitamin E 7.71 ± 0.62 mmol/l vs 16.68 ± 0.71 mmol/l, p <0.01). Thus, the level of SOD was 1.3 lower, GP - 1.7 times, vitamin A - 2.8 times, vitamin E - 2.2 times lower. Reduction of antioxidant enzymes was probably related to depletion of ADS in response to increased activity of LPO. Vitamins deficiency may also be associated with neonatal hypoxia (due to respiratory failure), which disrupts the synthesis of coenzyme forms of vitamins. Besides persistent inflammation in the airways with increased activity of LPO, which dramatically increases the catabolism of vitamins. Reliable inverse correlation was found between IPIS and levels of vitamins A and E (r = -0.43, p <0.05; r = -0.44, p <0.05, respectively).

 We also conducted an analysis of correlations between the initial settings for mechanical ventilation in newborns on ventilation mode SIMV/PS and indices of LPO. We revealed that high concentrations of oxygen (> 50%, r1), high peak inspiratory pressure (r2) and high average pressure in the airways (r3) are associated with activation of LPO: TBA-active products (r1 = 0.49, p <0.01; r2 = 0.81, p <0.01; r3 = 0.75, p <0.01), diene conjugates (r2 = 0.53, p <0.01), lipid hydroperoxides (r1 = 0.40, p <0.01; r2 = 0.43, p <0.01; r3 = 0.41, p <0.05). It was found that the levels of TBA-active products, diene conjugates, lipid hydroperoxides directly correlated with duration of respiratory therapy (for TBA-active products r = 0.53, p <0.01; for diene conjugates r = 0.46, p < 0.01, for lipid hydroperoxides r = 0.48, p <0.01). On the other hand we found inverse correlations between levels of vitamins A, E and duration of respiratory therapy (for vit.A r = -0.42, p <0.01; for vit. E r = -0.44, p <0.01).

Conclusions

1. In infants on mechanical ventilation mode SIMV/PS, along with activation of LPO a decrease of AOS was observed, which  may increase the risk of various pathological conditions in this group of patients.

2. High concentrations of oxygen (> 50%), high peak inspiratory pressure and high average pressure in the airways  are associated with activation of lipid peroxidation.

3. The disparity between the potential of LPO and activity of AOS associate with increased duration of respiratory therapy in newborns.