Oxidative and Antioxidative Biomarker Profiles in Neonatal Hypoxic-Ischemic Encephalopathy: Insights for Pathophysiology and Treatment Strategies

Introduction

Neonatal hypoxic-ischemic encephalopathy (HIE) is a significant cause of perinatal and postnatal morbidity and mortality worldwide [1]. No specific marker of neonatal hypoxia with strong predictive capabilities has been identified [2]. Therapeutic hypothermia has emerged as the established treatment protocol aimed at enhancing neurological recovery in infants affected by HIE [3]. The cerebral impairment and subsequent long-term neurological consequences commonly associated with this condition are frequently cited as significant factors affecting term and preterm infants during the perinatal phase [4].

Oxidative stress, glutaminergic excitotoxicity, failure of mitochondrial energy production, and apoptosis are involved in the pathophysiology of HIE [1]. Oxidative stress is defined as a dysregulation between the production of reactive oxygen species (ROS) and endogenous antioxidant defense mechanisms, referred to as a “redox state”. When ROS are present in low concentrations, they play a critical role in cell homeostasis. However, excessive ROS are neurotoxic and cause cellular dysfunction, protein and lipid peroxidation, and DNA damage and ultimately lead to irreversible cell damage and death [5].

There are numerous markers of oxidative stress, including the oxidation products of lipids, DNA, proteins, and carbohydrates, which are formed by the reactions of free radicals [1]. Malondialdehyde (MDA), one of these markers, is formed when free radicals attack membrane lipids containing carbon-carbon double bonds, resulting in lipid peroxidation and oxidation of polyunsaturated fatty acids in the membrane structure [6]. MDA, which is cytotoxic and mutagenic, is in different isoforms according to the ambient potential of hydrogen (pH), is found in the free enolate form at physiologic pH, and shows low reactivity against amino groups. However, its reactivity increases at low pH and ultimately adversely affects proteins [7]. Many studies have shown that MDA, whose concentration increases in plasma as a result of disruption of oxidant/antioxidant balance, is effective in the pathogenesis of cancer, Alzheimer disease, Parkinson disease, and many other chronic diseases by binding to proteins, phospholipids, and nucleic acids and causing toxic effects. Regarding oxidant products, the lipid peroxidation level is evaluated by MDA [6]. Ischemia-modified albumin (IMA) is a novel biomarker for the identification of myocardial ischemia of myocardial necrosis and can also be increased in liver, brain, and intestinal ischemia. ROS produced during ischemia/reperfusion, which are the main steps in perinatal asphyxia, modify albumin with highly reactive hydroxyl radicals and convert it into IMA. Therefore, IMA can be valuable in the prediction and diagnosis of perinatal asphyxia [8].

In vivo metabolism, antioxidant enzymes utilize their specific substrates to prevent cellular damage from oxidative stress, reduce oxidants, or convert them into other molecules [9]. Catalase (CAT) is the most crucial hydrogen peroxide-scavenging enzyme in the body, and CAT activity detection is used to detect levels of inflammation and oxidative stress [10]. That enzyme can dissociate millions of hydrogen peroxide (H2O2) molecules every second into molecular oxygen and water through reaction steps, without producing radicals [11]. In the neonatal period, glutathione (GSH) is the most critical non-enzymatic endogenous antioxidant [7]. Glutathione performs its primary function in antioxidant defense by inhibiting ROS and protecting the cell by scavenging free radicals and reducing H2O2. This function of GSH occurs in 2 stages. In the first stage, the enzyme glutathione peroxidase (GSH-Px) breaks down the H2O2 formed in cells into water, while reduced GSH is converted into oxidized glutathione (GSSG). In the second step, GSSG is converted back into GSH by the activity of the enzyme GSH reductase [12]. In the brain, which is one of the tissues highly affected by metabolic changes, endogenously produced GSH with low molecular weight and catalase as an enzyme play a role as antioxidants [13].

The aim of this manuscript is to provide a comprehensive understanding of the oxidant-antioxidant balance and its implications for cellular mechanisms by elucidating the intricate pathways and interplay between oxidant and antioxidant status in neonates with hypoxia.

Material and Methods

ETHICS APPROVAL AND INFORMED CONSENT:

Before conducting the study, approval was secured from the local ethics committee (Yuzuncu Yil University Clinical Research Ethics Committee date-number: 20231115-05), following the guidelines outlined in the Declaration of Helsinki. The study ensured that the legal guardians of patients and healthy infants were adequately informed about the research, and their consent was obtained through the completion of an informed consent form.

PATIENTS, STUDY DESIGN, AND DATA COLLECTION:

The research involved 59 infants with a diagnosis of HIE and a gestational age of 37 weeks or more. These infants were admitted to the Neonatal Intensive Care Unit (NICU) at Van Yuzuncu Yil University Hospital between 2023 and 2024. The diagnosis of perinatal hypoxia was based on the presence of an acute intrapartum/peripartum event with pH <7.00 or base deficit >12 mmol/L in the first hour after birth or blood gas from cord blood, APGAR score <5 at 5 to 10 min, or the need for prolonged resuscitation [14]. The causes of the HIE cases were cord prolapse, uterine rupture, problems with blood flow to the placenta, placenta previa, placental abruption, and shoulder dystonia. Additionally, 32 healthy infants born in the same hospital were included as the control group and received follow-up care solely from their mothers. Healthy infants in the control group had a gestational age of 37 weeks or more and were free from congenital anomalies, congenital heart defects, genetic diseases, cerebral anomalies, and postnatal complications. Gestational age of control and hypoxic newborns was determined by the mother’s last menstrual period and confirmed through early antenatal ultrasound and Ballard scoring in first 24 h. Infants with gestational age less than 37 weeks, cerebral or congenital anomalies, congenital heart defects, genetic diseases, sepsis, or lacking parental consent were excluded from the study. Our hospital is a reference center for hypothermia treatment, and not only patients born in our hospital but also patients with hypoxia from other centers are frequently admitted. It was not possible to obtain cord blood from every patient. Since hypothermia treatment should be started in the first 6 h, blood samples were taken together with the first blood tests to see the condition before hypothermia. The first blood samples were taken at a median of 75 min (min–max: 60–90 min). In the control group, blood samples were taken for routine blood glucose and blood group analyses in the first hours of life, close to the time when blood samples were taken from the patient group, and blood samples were also taken for the study at a median of 75 min (min–max: 65–85 min). No extra invasive procedure was performed to the neonates for these test samples.

The Sarnat staging system classifies HIE with stages I, II, and III, denoting mild, moderate, and severe HIE, respectively [13]. The HIE group received standard NICU support as per NICU protocol guidelines. Infants in the control group remained with their mothers and did not undergo any NICU interventions.

SPECTROPHOTOMETRIC MEASUREMENTS:

A total of 3 mL of venous blood was drawn from each participant in the patient and control groups, then the blood was placed into a biochemistry tube. Half (1.5 mL) of the 3 mL of blood taken from the patient and control groups was used for routine tests, and half (1.5 mL) was used for CAT, GSH, MDA, and IMA measurements. After 20 min was allowed for clotting, the tubes were centrifuged at 4000 rpm for 15 min. The resulting sera for the 4 measurements were carefully collected and stored separately in 4 different Eppendorf tubes after centrifugation at a temperature of −80°C until the day of the study.

CAT ACTIVITY: Serum CAT activity was determined using the method described by Aebi et al in 1984 [14], in which H2O2 was used as a substrate to determine the activity. According to the Aeibi method, 1400 μL of 30 mM H2O2 was placed in a blank tube and 100 μL of phosphate buffer was added. Then, 100 μL serum and 1400 μL of 30 mM H2O2 was added to the sample tube and mixed using a vortex mixer. Absorbances were recorded at 240 nm twice at 30-s intervals, due to the disappearance of H2O2. Enzyme activity was expressed as enzyme units per liter of serum (U/L) at 25°C. Activity=(2.3/Δx)×(log A1/log A2), with activity calculated in U/L; Δx=30 s; and 2.3=1 μmol optical density of H2O2 in 1cm light path.

REDUCED GSH LEVEL: The GSH level was determined using the method described by Beutler et al in 1963 [15]. GSH was measured by the reaction of sulfhydryl groups in the blood with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) resulting in the formation of yellow color. According to the method, 800μL of phosphate buffer was added to 200μL of serum. The first absorbance (OD1) at 412nm was recorded. Then, 100μL of Ellman reagent was added to the same tube, and the second absorbance was read. OD2 was recorded.

Activity (mg/mL)=([OD2-OD1]/13600×E1 1.25)×1000, where OD1=first absorbance before the addition of DTNB at 412nm; OD2=second absorbance after the addition of DTNB at 412n; E1=1 in the calculations; 13600=the molar extinction coefficient of the yellow color formed during the interaction of GSH with DTNB. GSH level was expressed as millimoles per milligram protein per deciliter (mmol/mg).

MDA LEVEL: According to the method described by Gutteridge in 1995 [16], the serum MDA level was measured spectrophotometrically by UV-VIS spectrophotometer by the thiobarbituric acid method. The method was performed as follows: 200μL of serum samples were taken into each tube. Then, 800μL phosphate buffer, 25μL Butyl hydroxy toluene solution, and 500μL 30% trichloro acetic acid were added. The tubes were mixed in the vortex, capped, and kept in an ice bath for 2 h. The tubes were readied to room temperature. The tubes were then capped and centrifuged at 2000rpm for 15 min. Then, 1mL of the supernatant obtained from the centrifuge was transferred to other tubes, and 75μL ethylenediamine tetra acetic acid and 25μL thiobarbituric acid was added to 1mL of the filtrates. The tubes were mixed in the vortex and kept in a hot water bath for 15 min (70°C). Then, they were brought to room temperature, and their absorbance was read on a UV/Vis spectrophotometer at 532nm. The MDA level was calculated as millimoles per liter (mmol/L), where C=concentration, F=dilution factor, and A=absorbance:

IMA LEVEL: The so-called albumin cobalt binding test is based on the binding of cobalt, a transition metal, with serum albumin. The method developed by Bar-Or et al was used for IMA determination [17]. For serum IMA measurement, 50 μL of cobalt chloride hexahydrate (0.1%) (CoCl2.6H20, 1 g/L) was added over 200μL of serum, followed by incubation for 10min. Next, 50 μL dithiothreitol (DTT) (1.5 g/L H20) was placed to the measurement cuvette and mixed. After 2min, 1mL of saline 0.9% NaCl (9 g/L) was added. Color change with DTT was read against a serum-cobalt blank without DTT, at 470 nm, in a UV/Vis spectrophotometer. Reading against the blank was taken as absorbance activity. IMA level was expressed as absorbent units per milliliter (U/mL).

STATISTICAL ANALYSIS:

The mean and standard error of the oxidant (IMA, MDA) and antioxidant (CAT, GSH) parameters were calculated in the healthy and HIE newborn groups. Normal distribution of variables was assessed visually (histogram and probability plots) and using an analytical method (Shapiro-Wilk test). Categorical variables are presented as numbers and percentages. The independent t test and Mann-Whitney U test were used for comparisons between categorical variables and continuous variables, respectively. Results were considered statistically significant at a P value of 0.05. SPSS version 26 (IBM SPSS Statistics for Windows, IBM Corp, Armonk, NY, USA) was used for the calculations.

Results

Fifty-nine newborns older than 37 weeks of gestation and with hypoxia, 64.4% (n=38) of whom were born by vaginal delivery (32 male, 27 female), were included in our research during the specified period. Thirty-two healthy newborns (16 male, 16 female), who were born in the same hospital, stayed with their mothers and did not receive any NICU care. Healthy infants in the control group had a gestational age of 37 weeks or more, as stated for the HIE group. The vaginal delivery rate in the control group was 62.5% (n=20). The birth weight was 3019.2±58.72 g in the HIE group and 3006.2±97.39 g in healthy newborn group. In the HIE group, 39 (66.1%) newborns were born in the university hospital, and 20 (33.8%) were referred from other health centers. All the newborns with HIE had therapeutic hypothermia treatment in the first 6 h of life.

Demographic data and clinical features are reported in Table 1. Maternal age, birth weight, sex distribution, type of delivery, and gestational age were not different between the groups. APGAR scores at 1, 5, and 10 min were significantly lower in the HIE group (Table 1). Initial blood gas values and biochemical test results of the newborns in the HIE and healthy groups are reported in detail in Table 2. Serum blood urea nitrogen, creatinine, creatine kinase, lactate dehydrogenase, alanine aminotransferase, and uric acid measurements were statistically significantly higher in newborns in the HIE group.

Magnetic resonance imaging (MRI) results were available for all 59 patients in the HIE group and was performed after hypothermia treatment, at a median age of 5 days. MRI scan results showed diffuse white matter injury, watershed infarcts, and basal ganglia injuries. Three HIE newborns had basal ganglia injuries. Four (6.77%) newborns with severe HIE died and had extensive diffusion restriction in cerebral MRI. Twenty-eight (47.4%) newborns with HIE had a normal cranial MRI (Figure 1). After evaluating the relationship of early diffusion MRI findings with oxidant and antioxidant levels of patients with different findings on MRI, no significant relationship was found.

Compared with healthy newborns (2694.12±442.54, n=32), CAT was statistically significantly lower in the HIE group (782.12±135.37, n=59; P=0.0001), while MDA serum level was significantly higher in the HIE group (1.08±0.0083, n=59) than in healthy newborns (1.04±0.009, n=32; P=0.01). There was no significant difference between newborns with hypoxia (0.0036±0.0004, n=59) and healthy newborns (0.0038±0.0009, n=32) in GSH measurements (P=0.054). No significant difference was found in IMA measurements (P=0.19) between newborns with hypoxia (0.39±0.007, n=59) and healthy newborns (0.40±0.01, n=32; Figure 2).

No statistically significant difference was found between female and male HIE newborns in terms of CAT (P=0.37), GSH (P=0.58), MDA (P=0.59), and IMA levels (P=0.12; Table 3).

While MDA measurements were significantly higher in infants born by cesarean delivery in the HIE group (1.09±0.01, 1.06±0.01, respectively, P=0.039), no significant difference was found in the control group in terms of delivery type (P=0.12). On the other hand, GSH, IMA, and CAT measurements did not differ between the type of delivery in newborns in both the HIE (P=0.34, P=0.65, P=0.48 respectively) and healthy groups (P=0.64, P=0.45, P=0.68, respectively).

Of the 59 patients who had hypothermia treatment, there were 37 (62.7%) patients in stage II and 22 patients in stage III (37.2%), according to Sarnat staging. No significant difference was found in CAT, GSH, MDA, and IMA levels between stages (P>0.05).

The post-discharge follow-up of the patients continues at the time of this writing. Fourteen (23.7%) of 59 patients were discharged with anticonvulsant treatment, and 2 (3.38%) of them continued to receive NICU care after tracheostomy. One (1.7%) of them needed ventriculoperitoneal shunt, due to hydrocephalus after intraventricular hemorrhage.

Discussion

In the results of antioxidant and oxidant analysis of newborns with HIE and healthy newborns older than 37 weeks of gestation, CAT was significantly lower and MDA was significantly higher in the HIE group, but no difference was found in GSH and IMA levels. Many studies have shown how hypoxia affects living organisms in human and experimental studies, which factors modify these effects, and how they respond to the effects of hypoxia [1,2,4,10].

The pathophysiology of HIE involves several different events that are tightly linked to each other. The main events triggered by severe anoxic brain injury are mitochondrial dysfunction, excitotoxicity, calcium surge, ROS accumulation, and inflammation. They are the starting point for a multitude of complex pathways [1]. In the first phase, primary neuronal death is observed as primary energy deficiency and oxidative metabolism failure, cytotoxic edema, and excitotoxin accumulation [13,20]. With the restoration of cerebral circulation, the second phase begins, and apoptosis, microglia activation neuronal cell membrane damage, and excitotoxicity follow this phase [13,21].

There are many studies in the literature showing that the female sex is more protected from hypoxia, while the male sex is more affected [22,23]. Sexual dimorphism-related hypoxia can be ascribed hormone-related events, such as estrogen and testosterone secretion, gene-linked conditions, and apoptotic cascade [24]. After HIE, females display a greater reliance on the caspase-dependent pathway, whereas males rely more on the caspase-independent pathway. In contrast, previous studies have shown a difference in the amount of caspases activated following HIE injury. More caspases tend to be activated in males, which is associated with a more extensive apoptotic event and can lead to a greater susceptibility in males and, thus, early brain damage. [25]. Talat et al demonstrated that there is no significant association between sex and brain lesions [26]. We did not find differences between sexes in terms of CAT, GSH, MDA, and IMA levels in newborns with HIE (P=0.37, P=0.58, P=0.59, P=0.12, respectively).

At 5 min of life, an APGAR score 0 to 3 was defined as severe, APGAR score 4 to 5 as moderate, and APGAR score 6 to 7 as mild [27]. While APGAR scores are still used as the first way to evaluate newborns, the 5-min APGAR score is an important factor related to mortality for HIE [28]. In different studies, APGAR scores have been found to vary significantly between the stages of hypoxia [26,28] when compared with control groups [29], while the 5-min APGAR score was found to be significantly lower in patients with severe hypoxia than in those with mild and moderate hypoxia [28]. Oncel et al studied urinary markers in newborns with perinatal asphyxia, and the rate of those with an APGAR score <5 at 5 min was 73%, while this rate was zero in healthy newborns [29]. Our babies with hypoxia had statistically significantly lower APGAR scores than healthy babies at 1, 5, and 10 min after birth.

When we look at the mortality rates in our study, 4 of 59 patients with hypoxia died (6.7%), while a study from Egypt in 2020, which aimed to assess the value of IMA levels as a diagnostic marker for neonatal hypoxic-ischemic encephalopathy, found a mortality rate of 20%. Mortality percentages in different stages of HIE are 0%, 13.3%, 67.7% in mild, moderate, and severe, respectively [26]. In studies in which different blood parameters were studied in neonatal hypoxia, mortality varied between 10% [29] and 18% [28]. The variability of patients presenting at different stages and time of hypothermia initiation and the unequal distribution of the patients included in the study for each stage may cause the different reporting of mortality rates.

When the effects of mode of birth were analyzed in regards to MDA measurements in HIE newborns in the literature, MDA levels were found to be more elevated in the cord blood of newborns born by cesarean delivery in small for gestational age newborns than in appropriate for gestational age newborns [30]. Although not statistically significant, mean cord blood IMA levels were slightly higher in cesarean deliveries than in vaginal deliveries in HIE and control group babies [26]. Prematurity and cesarean delivery has also been observed to cause antioxidant deficiency in human babies [31]. In our study, CAT, IMA, and GSH levels were not found to be statistically significant for cesarean and vaginal delivery according to the type of delivery in both the HIE and control group newborns. However, MDA measurements were statistically higher in neonates born by cesarean section in the HIE group.

Several factors may have contributed to free radical formation in the cesarean delivery group. Inhaled oxygen caused an increase in oxygen free radical activity in maternal blood during anesthesia, and the surgery itself possibly participated in lipid peroxidation [32]. Numerous studies have shown that oxidative stress is a contributing factor to various diseases and conditions, including atherosclerosis, certain cancers, and aging. Additionally, oxidative stress has been linked to inflammatory, ischemic, traumatic, and neurological disorders, as well as to conditions such as emphysema, hemochromatosis, acquired immunodeficiency syndrome, hypertension, preeclampsia, and gastric ulcers, and to diseases related to alcohol and tobacco use [9,33–35].

There are various oxidative stress markers that contain oxidation products of lipids, DNA, proteins, and carbohydrates formed by reactions with free radicals. MDA is one of these markers and is formed due to lipid peroxidation caused by free radical attacks on membrane lipids containing carbon-carbon double bonds and oxidation of polyunsaturated fatty acids in the membrane structure. MDA, which is cytotoxic and mutagenic, exists in different isoforms depending on the pH of the environment. At a physiological pH, it is present in the free enolate form and exhibits low reactivity toward amino groups. The increased concentration of MDA in plasma due to the disruption of the oxidant/antioxidant balance can cause toxic effects by binding to proteins, phospholipids, and nucleic acids. However, its reactivity increases at a low pH, ultimately leading to negative effects on proteins [6]. Studies have demonstrated the involvement of MDA in the pathogenesis of cancer, lung, and liver diseases, Alzheimer disease, Parkinson disease, diabetes [6], cirrhosis and liver transplantation [36], allergy-related diseases [37], and spinal injuries [35]. Due to its easy reaction with thiobarbituric acid, MDA has been widely used for many years as a suitable biomarker for lipid peroxidation of omega-3 and omega-6 fatty acids [18]. Increased plasma and cerebrospinal fluid MDA levels are associated with perinatal asphyxia, according to Kumar et al [38]. When the lipid oxidation level in the brain tissue was determined using an MDA kit, Zheng et al showed that the MDA level in the brain tissue of the sham operation group was significantly lower than the mean MDA level in the brain tissue of the hypoxia-ischemia group [39]. In an experimental study, plasma urea, creatinine, and MDA levels were increased in rats exposed to hypoxia. The increase in blood uremia profile, determinants of toxicity and lipid peroxidation enzymes, showed that hypoxia induced renal failure. Histological structures of the kidneys of hypoxic animals demonstrated severe disorganization of the glomeruli and enlargement of the renal tubules. These data suggest that nephrotoxicity or acute renal failure can occur in hypobaric hypoxia and that it influences the gut microbial population [40]. Our study also showed that MDA was statistically significantly higher in blood levels obtained in the first 6 h in infants with hypoxia.

IMA is another biomarker of oxidative stress, which occurs due to changes in the structure of albumin under ischemic and hypoxic conditions caused by ROS. Therefore, IMA is considered a biomarker for many diseases that combine ischemia and oxidative stress [41]. In a study involving 60 HIE newborns in which IMA was measured from cord blood, it was found that IMA levels were statistically significantly higher in newborns who died due to asphyxia than in newborns with asphyxia and living newborns, and when IMA measurements were compared in newborns with mild, moderate, and severe HIE, it was observed that IMA levels gradually increased as the severity of the disease increased [26]. Newborns with perinatal asphyxia had significantly higher cord blood IMA levels than newborns without asphyxia [42]. IMA might be beneficial in addressing certain perinatal complications, such as intrauterine growth restriction. Recent studies have shown increased levels of maternal serum IMA levels during early normal pregnancies, suggesting that proper trophoblast development occurs in a hypoxic intrauterine environment [43]. Additionally, excessive intrauterine hypoxia and the resulting reperfusion oxidative damage could lead to impaired trophoblast development. Therefore, IMA levels in the first trimester could serve as a potential biomarker for identifying abnormal placental development [44]. IMA seems to be produced in patients with stroke, end-stage renal disease, stroke, pulmonary embolism, skeletal muscle ischemia, acute mesenteric ischemia, and intrauterine ischemia [8]. Although this elevation has been shown in cord blood samples or tissue samples in different studies, the IMA elevation may not have been shown in blood samples after birth before the onset of hypothermia in our study. To the best of our knowledge, there is no study that makes measurements before hypothermia treatment immediately after birth.

Antioxidant enzymes in living metabolism prevent cellular damage from oxidative stress by using specific substrates to reduce oxidants or convert them into other molecules [9]. The antioxidant defense system works by removing initiator reactive derivatives, reducing oxygen concentration, and removing catalytic metal ions to prevent ROS formation [45]. In our study, conducted to observe the effects of hypoxia on oxidant markers and antioxidant enzymes in neonatal hypoxia, we found that CAT and MDA were significantly altered.

CAT is particularly important, as it scavenges H2O2, decomposing millions of H2O2 (approximately 107 M/sec) molecules every second into molecular oxygen and water without producing free radicals [11,46]. It can protect brain tissues against ROS cytotoxicity by catalysis, maintaining a neutral balance [34]. CATs reduce neuroinflammation, mitochondrial damage, astrogliosis, and cell death induced by hypoxia [10]. The hypoxia-ischemia lesion induces mitochondrial impairment, leading to increased release of ROS, often associated with changes in the activity of the antioxidant enzyme CAT [47]. In a biochemical hypoxia model, cobalt chloride (CoCl2) was shown to decrease CAT, superoxide dismutase activity, and gene expression in the mouse brain cortex [48]. While CAT and superoxide dismutase levels were significantly decreased in hypoxia-exposed rats in comparison with controls, plasma urea, creatinine, electrolytes, and malonaldehyde levels were increased [40]. Similar to many studies in the literature, in our study, CAT levels were found to be significantly lower, and blood urea nitrogen, creatinine, ALT levels were higher in newborns with hypoxia than in healthy babies.

GSH is another important component of antioxidant defense. It is a tripeptide composed of γ-L-glutamyl-L-cysteinyl-glycine amino acids found in high concentration in the liver and in all mammalian tissues. To protect the cell from oxidative damage, it is important that the ratio of GSH (thiol-reduced) to GSSG (disulfide-oxidized) is high and the GSSG content is less than 1% of the GSH content. Glutathione serves as the primary defense against oxidative stress by preventing ROS and reducing H2O2 through scavenging free radicals [12]. Analysis of GSH redox status and ROS products demonstrated lower concentrations of GSSG, higher concentrations of reduced GSH, and higher GSH/GSSG ratios during hypoxia [49]. In several investigations of hypoxia and reoxygenation in mice, high GSH/GSSG ratios were measured when reoxygenation was performed at low oxygen concentrations in the neonatal period. In addition, GSH is the major non-enzymatic endogenous antioxidant. The GSH/GSSG ratio is regarded as a robust indicator of redox homeostasis in a number of preclinical and clinical investigations [2]. Plasma GSH-Px, CAT, and superoxide dismutase were significantly higher in neonates with perinatal asphyxia and showed a progressive increase with the severity of hypoxic-ischemic encephalopathy. Higher plasma and cerebrospinal fluid levels of MDA and CAT have been documented in newborns who died of HIE than in survivors, but no such difference was found in plasma levels of GSH-Px and superoxide dismutase [38]. We found no statistically significant difference in GSH measurements in the first blood samples taken after birth between hypoxic and healthy newborns.

This study has some limitations because an equal number of patients at different stages of HIE could not be obtained, comparisons could not be made according to the stages, and cord blood could not be obtained from each patient. In future studies, we plan to evaluate the results of patients with hypoxia at different stages and with additional parameters in more comprehensive studies.

Conclusions

The rapidly growing nature of neonatal tissues makes them particularly vulnerable to oxidative damage. Conditions associated with oxidative stress are extremely common in the early neonatal period and contribute to an increase in mortality and morbidity that can have lifelong consequences. To guide interventions and monitor outcomes, the search for new biomarkers to serve as diagnostic tools has become increasingly important, using accurate analytical methods on matrices that can be obtained without causing pain or damage to vulnerable newborns. Assessing bedside oxidative stress using microsamples and providing rapid results will allow earlier diagnosis, improve our understanding of the consequences of our interventions, prevent potential complications, and evaluate the success of the therapies applied. Elucidating the mechanisms linking the balance between oxidants and antioxidants to cell death is crucial for understanding the pathophysiology of HIE. In the future, new studies should more extensively investigate the diagnostic and therapeutic value of the different oxidative stress biomarkers and antioxidants to diminish oxidative tissue injury in the developing newborn.