MEASUREMENT OF OXIDANT- ANTIOXIDANT MARKERS IN PREMATURE NEWBORN WITH RESPIRATORY DISTRESS SYNDROME

Ali K Al-Zahrani 1, 2 ,Nader M Mohamed 1, 3 and Mona G Amer 4, 5 . 1. Department of Pediatrics, college of Medicine, Taif University, Taif, Saudi Arabia. 2. Department of Neonatology, King Abdel Aziz Specialist Hospital (KAASH),Taif, Saudi Arabia. 3. Department of Pediatrics & Neonatology, AlAhrar teaching Hospital, Zagazig, Egypt. 4. Department of Anatomy & Histology, College of Medicine, Taif University, Taif, Saudi Arabia. 5. Department of Histology & Cell Biology, Faculty of Medicine, Zagazig University, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

1288 peripheral airways and immaturity of cells important for lung maturation are the major causes of poor surfactant production by type II cells and inadequate antioxidant responses to increased ambient oxygen (Gleason et al., 2012). In addition to prematurity, premature neonates could be complicated by diminished antioxidant stores and antioxidant enzymes production. Oxidative stress is oxidant/antioxidant imbalances that resulting in production of free radicals. Adequately mature and healthy infants are able to tolerate this drasticchange in the oxygen concentration. A problem occurs when theintrauterine development is incomplete or abnormal. Preterm  Oxidative stress in infantsis implicated in the pathogenesis of the major complications of prematurityincluding respiratory distress syndrome (RDS), necrotizing enterocolitis, chronic lung disease, retinopathy of prematurity andintraventricular hemorrhage (Mutinati et al., 2014).
Newborns and especially pre-term infants are probably more prone to oxidative stress than are children and young adults as they are exposed to high oxygen concentrations, have infections or inflammation, have reduced antioxidant defense, and have free iron which enhances the Fenton reaction leading to production of highly toxic hydroxyl radicals (Saugstad, 2003; For instance, the superoxide dismutase (SOD) activity appears in the developing lungs concomitantly with the surfactant synthesis by type II pneumocytes. So, the current study investigated the alterations in the biochemical parameters of oxidant/antioxidant by quantification of levels of malondialdehyde (MDA),TRBAS along with total antioxidant capacity in cord blood of healthy premature newborns compared with those with RDS.Umbilical cord blood provides valuable information regarding the status of the infant at birth thatgives early predictors of some of the metabolic disorders in future adult life.

Patients and methods:-
A total of 90 neonates were included in this case-control study. They were recruited from Neonatology department of King Abdul Aziz specialist Hospital (KAASH) Taif, Saudi Arabia, between August 2015 and september 2016. This study was approved by the Institutional Ethics Committee, and written informed consent for participation was obtained from the parents.
Cord blood samples were collected at the time of the delivery. The study group consisted of 40 preterm born < 33 weeks gestational age or of low birth weight < 1500 g with diagnosis of RDS. The control group was composed of 50 preterm low birth weight newborns. Exclusion criteria of the study were infection, congenital malformations, metabolic disorder and newborns with history of difficult delivery, genetic disorder, severe intraventricular hemorrhages, congenital hemolytic diseases caused by blood-type incompatible pregnancies, diseases requiring surgical treatment, and a gestational age of 22 wk. RDS was diagnosed on the presence of typical clinical and radiological signs of the disease in the preterm infants. Newborns were considered to have RDS if they have tachypnea; grunting and cyanosis with several hours of birth required mechanical ventilation and typical radiographic findings on the chest X-ray. The diagnosis was established from the clinical symptoms and needed for oxygen treatment.RDS neonates were classified into mild, moderate and severe categories based on severity of oxygenation compromise.
Maternal age, date of last menstrual period, medical history and reproductive history were obtained from the hospital record.
Four mL of heparinized venous blood sample was collected from the umbilical cord just after delivery of the studied neonates and processed for the isolation of erythrocytes and plasma. The red cells were stored at 4°C and all serum 1289 samples at -20°C until analysis. The red cell samples were processed within 12 hours. For reduced glutathione determination erythrocyte mass was used, superoxide dismutase activity was determined in erythrocyte lysate (add 3 ml 0.9 % NaCl to 500 µl of blood and centrifuge 660 g/10 min at 4 °C, remove supernatant and repeat 4 times; after final removal of supernatant add 1.5 ml of chilled distilled water, leave for 20 min and freeze).
The following oxidant/antioxidant markers were measured Serum malondialdehyde (MDA) marker of lipid peroxidationwas measured using the colorimetric method described by Satoh et al. (1978), based upon the reaction of thiobarbituric acid (TBA) with MDA, one of the aldehyde products of lipid peroxidation. The absorbance of the MDA-TBA adduct thus produced was measured at 532 nm wavelength using a spectrophotometer. serum superoxide dismutase (SOD) in erythrocytes was determined by the method described by Marklund and Marklund (1974) as modified by Nandi and Chatterjee (1988), which is based on the inhibition of pyrogallol autoxidation brought about by SOD.
Reduced glutathione (GSH) was estimated by a method based on the development of yellow color with 5, 5' dithiobis-2-nitrobenzoic acid (DTB), which was measured at 432 um using a spectrophotometer (Beutler et al., 1963).

Statistical analysis:-
Descriptive analysis was performed for demographic and clinical characteristics of the patients. MDA, TBRAS, reduced glutathione and superoxide dismutase levels were expressed as mean ± SD. The levels were compared between the Study and Control groups by student's t-test or Mann 2 Whitney U test was used for comparison of numeric variables between two groups. Comparison between RDS group was determined by one-way analysis of variance (ANOVA) followed by Tukey post hoc test for multiple comparisons. Probability values (P) less than 0.05 were considered to be statistically significant.

Characteristics of the studied groups:
The study included 40 premature neonates with RDS and 50 healthy premature neonates. 75% of RDS group were delivered by cesarean and 57.5% of them were infant of diabetic mothers. 65% of RDS group and 42% of control group were male table (1). 1290 Difference in oxidative markers between RDS and control groups: High significant changes in mean ± SD of oxidative markers between both groups were observed. Significant increase in oxidant markers (MDA, TBRAS)was detected in RD group when compared with control group and significant decrease in antioxidant markers (GHS, SOD) table (2). Data are presented as means ± SD; n = number of cases in each group. Statistical analysis was done using student's t-test. *, P < 0.05 versus control

Difference in oxidative stress markers according to severity of RDS:
Mean ± SD levels of oxidant markers MDA, and TBRAS were significantly increased in moderate and sever cases of RDS when compared with mild cases. Also, the antioxidant markers GSH, and SOD were significantly decreased in sever/moderate cases when compared with mild cases p<0.05.

Discussion:-
The imbalance between reactive oxygen species (ROS) and enzymes needed for removal of free radicals is called oxidative stress. This process involved in the activation of a complexarray of genes involved in inflammation,coagulation, fibrinolysis, cell cycle andsignal transduction resulting in consequence on fetal structure.The rapid transition of the fetus from a relatively hypoxic to a relatively hyperoxic environment at birth gives rise to oxidative stress. However, oxidant defense mechanisms are induced late in gestation.Management of oxidative stress is a greatchallenge for both researchers and clinicians (Frank, 1991

; Dizdar et al., 2011).
In the present study, we detected evidence of oxidative stress in cord blood of premature neonates suffering from RDS that was prominent with severe cases. Most of RDS cases in the present work were delivered by Caesarean section and some of them were infants of diabetic mothers. Substantial increased levels of oxidant markers MDA and TBARS in our study were accompanied with significant decreased levels of GHS and SOD.
Many authors have demonstrated that there is the same degree of fetal oxidative stress in vaginal delivery as that resulting from Caesarean section (Fogel et al., 2005; Hracsko et al., 2007). It is unclear whether oxidative stress is related to delivery itself or whether it reflects a pre-existing fetal level of oxidative status. Respiratory distress syndrome is accompanied by inflammatory processes with free radical generation and oxidative stress (Nemeth, 1994, Krediet, 2006. The imbalance between the oxidative forces and the antioxidant defense systems was suggested to predispose the lungs to the development of RDS (Lang et al., 2002). Many studies have shown increased oxidant stress markers and/or reduced antioxidant defense in preterm infants with RDS (Ogihara  et al., 1996, Miller et al., 1993).
Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acids peroxidation. The present study showed increased concentration of MDA in neonates with respiratory disorders than that of control in consonance with the reported study (Negi et al., 2015 ).In addition, preterm infants appear to have deficient quantities of enzymes responsible for scavenging ROS, including superoxide dismutase. Frank, (1987). ROS potentiate tissue damage by lipid peroxidation (Saugstad, 2005). A common site of damage is the basement membrane and other elements of the lung matrix. One consequence of this damage is increased microvascular permeability and vascular leakage, resulting in oedema formation containing protein rich fluid (Groneck et al., 1994).This protein rich fluid is consideredas an ideal target for oxygen reactive species. In order to initiate the oxidative attack, ROScauses an imbalance in the lung protease-antiprotease system through inactivation of alpha-1 protease The present study observed reduction in total antioxidant status in newborns with RDS compared to healthy newborns. One study also reported decreased level of total antioxidant activity in newborn with RDS (Dizdar et al.,  2011). Oxidant-antioxidant balance shifts in favor of the oxidative damage in premature newborns with RDS due to diminished antioxidant activity. Antioxidant defenses of the immature lung will be prepared neither for the hyperoxic environment nor the inflammation found in association with respiratory distress (Davis and Auten, 2010). The lung depends on a delicate balance between oxidant and antioxidant systems to maintain normal cellular function. The lungs of prematurely born infants suffering from respiratory distress syndrome may be ill-adapted for protection against ROS. Antioxidants clearly have an important role in the defense against free radical induced lung injury in newborns with RDS (Negi eta ., 2015).
The present study showed that the elevated level ofMDA and TBRAS might be risk factor; reduced level of total antioxidant status (reduced GHS and SOD) might lead to the RDS in newborn and control its severity. Total antioxidant status might serve as prognostic marker in newborns with RDS and might help distinguish high risk infants.

Conclusion:-
The result of this study indicates that newborns with RDS were manifested by oxidative stress accompanied by reduced antioxidant defenses which could play a role in the pathogenesis of RDS and affect its degree of severity.