**4. Evaluation of antioxidants in neonates**

Antioxidant defense in neonates can be evaluated by measuring enzymatic and non-enzymatic systems. Among enzymatic systems, glutathione reductase, peroxidase, transferase, the oxidized/reduced glutathione ratio, superoxide dismutase, as well as other antioxidants such as ceruloplasmin, transferring are the most frequently measured.

The non-enzymatic antioxidant systems that can be measured in newborns are vitamins A, E, and C. Vitamin A and E values measured in newborns are presented in many studies. Shah et al. describe a correlation between hepatic vitamin A reserves and gestational age, as well as between nutritional status and maternal vitamin A levels. Vitamin A has an important role in the development of visual acuity, and also in lung development and surfactant synthesis [1]. Vitamin A levels are significantly lower in preterm compared to term neonates. Antenatal corticoid administration has a beneficial effect on vitamin A levels in premature babies. Thus, in preterm newborns benefiting from antenatal corticosteroids, the levels of these vitamins with antioxidant effect are higher than in preterm babies without antenatal treatment. The mechanism of corticosteroids in increasing vitamin synthesis is unknown. It seems that these act by increasing the plasma levels of retinol-binding proteins, stimulating the hepatic synthesis of these proteins [2].

Vitamin E, another important non-enzymatic antioxidant, with a role in stabilizing cell membranes, also has lower values in preterm compared to term neonates. Vitamin E has been used in many studies for its antioxidant effect in preventing retinopathy and bronchopulmonary dysplasia. However, in a 2003 Cochrane analysis, Brion et al. demonstrated that vitamin E plays a role in reducing the incidence of ROP and IVH, but increases the incidence of neonatal sepsis [3]. Allopurinol, melatonin, and acetylcysteine have been used in studies for their antioxidant effect, mainly as neuroprotective agents. Melatonin and acetylcysteine were used in the studies of Gitto, and subsequently Barceló, to reduce the incidence of NEC in premature neonates [4, 5]. However, there is no consensus regarding their use for the treatment of NEC in neonates or for the treatment of other conditions associated with hypoxia-ischemia. Nevertheless, it should be taken into consideration that exogenous antioxidant therapy with high doses of vitamin C and beta-carotene in particular will have a pro-oxidant effect.

For the evaluation of antioxidant defense in newborns, the levels of ceruloplasmin were measured in our service. This non-enzymatic antioxidant defense marker proved to be deficient in preterm compared to term neonates. Ceruloplasmin is a peroxyl radical scavenger. Free oxygen radical excess caused by certain oxidative stress-inducing situations will put a strain on the impaired defense mechanisms of the premature newborn. Antioxidant values will be lower compared to those of fullterm newborns. Ceruloplasmin determined by spectrophotometry had lower values in preterm neonates with respiratory distress. Also, ceruloplasmin levels decreased with the decrease in gestational age. Determinations evidenced lower ceruloplasmin values on the first day compared to the third day of life (**Table 1**).

Exposure to asphyxia at birth results in decreased ceruloplasmin levels. Under these oxidative stress-inducing conditions, the measurements performed evidenced lower ceruloplasmin values in preterm newborns with asphyxia compared to term newborns with asphyxia. Asphyxia is followed by a diminution of antioxidant levels and an increase in transferrin saturation. Current data confirm the fact that in


*FiO2-oxygen concentration; pH-value.*

*pCO2-CO2 partial pressure; pO2-oxygen partial pressure.*

*SaO2-oxygen saturation; CP-ceruloplasmin.*

*p-test significance; Z = test parameter.*

#### **Table 1.**

*Evolution of ceruloplasmin on 1st vs 3rd day of life (DOL).*

neonatal asphyxia and in the post-asphyxic period, ROS are generated particularly during the re-oxygenation phase after perinatal asphyxia [10]. The brain is the most susceptible to oxidative injury, for the following reasons:


Ceruloplasmin was measured by Lindeman [23], who evidenced the fact that in premature newborns, its levels are constantly low until the age of 3–6 weeks. Its deficiency in premature newborns increases the risk of oxidative stress under conditions of exposure to the oxidative attack of ROS.

Another marker of antioxidant defense is represented by hydrogen donors. Like total antioxidant activity, these assess several natural non-enzymatic antioxidants: cysteine, glutathione, ascorbic acid, tocopherol, polyphenols, aromatic amines, and sulfhydryl protein groups. In the case of measurements performed with 1,1-diphenyl-picrylhydrazyl in neonates admitted to our service, we found a correlation of hydrogen donor values with the severity of respiratory distress. The presence of respiratory distress was a triggering factor for hydrogen donors, stimulating their antioxidant activity in a group of patients with impaired enzymatic antioxidant defense (**Table 2**).

Hydrogen donor levels in healthy, late preterm newborns are higher compared to those of preterm newborns with oxidative stress-inducing conditions such as respiratory distress or asphyxia at birth. Non-enzymatic antioxidant defense assessed by hydrogen donor values improves with time; our determinations showed significantly higher values on the third day compared to the first day (p < 0.05) (**Table 3**).

The enzymatic antioxidants studied in neonates are: catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). These are endogenous antioxidants and have the following antioxidant action mechanisms: superoxide dismutase catalyzes superoxide radical dismutation, resulting in hydrogen peroxide.

**191**

**Figure 1.**

*Enzymatic antioxidant mechanism.*

*Antioxidants at Newborns*

*b*

*\**

**Table 2.**

**Table 3.**

*Hydrogen donors (HD) by groups.*

*as compared to control*

*DOI: http://dx.doi.org/10.5772/intechopen.85175*

Mild Stat (p-value)\*b 42.20 (39.80-45.30)

Moderate Stat (p-value)\*b 62.70 (59.40-64.20)

Severe Stat (p-value)\*b 48.30 (46.48-51.31)

*median (Q1-Q3), Q = quartile, Wilcoxon Matched Test*

**1**

−3.11 (0.0019)

1.55 (0.1218)

−1.02 (0.3082)

Control 13 54.38 ± 7.33 2.03

Control 13 54.38 ± 7.33 2.03

*Hydrogen donors by severity of respiratory distress and comparisons with the controls.*

**st day 3rd day Stat (p-value)**

46.65 (41.53-52.05) −2.09 (0.0369)

61.50 (60.43-64.35) 1.68 (0.0926)

49.60 (45.30-54.00) −0.91 (0.3650)

2.42 (0.0157)

0.00 (0.9999)

0.0 (0.9999)

Glutathione peroxidase and catalase catalyze hydrogen peroxide reduction to water and oxygen. Thus, they exert a protective effect against oxidative injury (**Figure 1**). The levels of these enzymes decrease with the decrease in gestational age. Another factor that influences enzymatic antioxidant mechanisms is neonatal development. In neonates with intrauterine growth restriction, the antioxidant enzymes SOD, CAT, and GSH-Px have lower values than in term AGA neonates [12, 13]. In our study, for the assessment of enzymatic antioxidant defense capacity, erythrocyte SOD was measured using the Winterbourn method. Hemoglobin concentration was determined in K3 EDTA samples by the Drabkin method.

HD 1 Case 24 45.82 ± 10.36 2.12 −2.64 (0.0124)

HD 2 Case 24 49.03 ± 11.97 2.44 −1.47 (0.1514)

**Group n Mean±Stdev Std. Error Mean t-value (p-value)**

#### *Antioxidants at Newborns DOI: http://dx.doi.org/10.5772/intechopen.85175*


*median (Q1-Q3), Q = quartile, Wilcoxon Matched Test \* as compared to control*

#### **Table 2.**

*Antioxidants*

**Table 1.**

neonatal asphyxia and in the post-asphyxic period, ROS are generated particularly during the re-oxygenation phase after perinatal asphyxia [10]. The brain is the most

FiO2—DOL1 & FiO2—DOL3 59 52,5000 6,149,591 0.000000 pH—DOL1 & pH—DOL3 60 175,5000 4,346,968 0.000014 pCO2—DOL1 & pCO2—DOL3 60 412,5000 3,429,860 0.000604 pO2—DOL1 & pO2—DOL3 60 573,0000 2,014,110 0.043999 SaO2—DOL1 & SaO2—DOL3 60 208,5000 3,761,957 0.000169 CP—DOL1 & CP—DOL3 60 492,0000 2,814,343 0.004888

**N T Z p-Level**

• Neuronal membranes are rich in polyunsaturated fatty acids, an important

• Some brain areas are rich in iron [10]. The increase in CP in mild and severe asphyxia cases can represent a form of adaptation of the organism to the action

Ceruloplasmin was measured by Lindeman [23], who evidenced the fact that in premature newborns, its levels are constantly low until the age of 3–6 weeks. Its deficiency in premature newborns increases the risk of oxidative stress under

Another marker of antioxidant defense is represented by hydrogen donors. Like total antioxidant activity, these assess several natural non-enzymatic antioxidants: cysteine, glutathione, ascorbic acid, tocopherol, polyphenols, aromatic amines, and sulfhydryl protein groups. In the case of measurements performed with 1,1-diphenyl-picrylhydrazyl in neonates admitted to our service, we found a correlation of hydrogen donor values with the severity of respiratory distress. The presence of respiratory distress was a triggering factor for hydrogen donors, stimulating their antioxidant activity in a group of patients with impaired enzymatic antioxidant

Hydrogen donor levels in healthy, late preterm newborns are higher compared to those of preterm newborns with oxidative stress-inducing conditions such as respiratory distress or asphyxia at birth. Non-enzymatic antioxidant defense assessed by hydrogen donor values improves with time; our determinations showed significantly higher values on the third day compared to the first day (p < 0.05) (**Table 3**). The enzymatic antioxidants studied in neonates are: catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). These are endogenous antioxidants and have the following antioxidant action mechanisms: superoxide dismutase catalyzes superoxide radical dismutation, resulting in hydrogen peroxide.

• The activity of antioxidant enzymes (catalase and SOD) is significantly

susceptible to oxidative injury, for the following reasons:

conditions of exposure to the oxidative attack of ROS.

source of free oxygen radicals.

diminished in the brain.

*FiO2-oxygen concentration; pH-value.*

*SaO2-oxygen saturation; CP-ceruloplasmin. p-test significance; Z = test parameter.*

*pCO2-CO2 partial pressure; pO2-oxygen partial pressure.*

*Evolution of ceruloplasmin on 1st vs 3rd day of life (DOL).*

of oxidative stress [15].

**190**

defense (**Table 2**).

*Hydrogen donors by severity of respiratory distress and comparisons with the controls.*

