|Year : 2015 | Volume
| Issue : 2 | Page : 109-114
Assessment of methemoglobin concentration, serum nitrate, and nitrite levels and their interrelationships with antioxidant status in the cord blood of neonates born via normal delivery versus neonates delivered by cesarean section in an Indian population
Manali Sinharay1, Indranil Chakraborty2, Partha Sarathi Chakraborty3
1 Department of Biochemistry, Calcutta National Medical College and Hospital, Malda, West Bengal, India
2 Department of Biochemistry, Malda Medical College and Hospital, Malda, West Bengal, India
3 Department of Gynaecology and Obstetrics, IPGMER and SSKM Hospital, Kolkata, West Bengal, India
|Date of Web Publication||6-Apr-2015|
Dr. Manali Sinharay
Department of Biochemistry, Calcutta National Medical College and Hospital, 32, Gorachand Road, Kolkata - 700 014, West Bengal
Source of Support: None, Conflict of Interest: None
Background: Oxidative and nitrosative stress are known to be associated with the transition from fetal to neonatal life at birth, when newborns having a compromised antioxidant defense system are suddenly exposed to a normoxic environment (from a hypoxic state). The current study explores the levels of antioxidants, markers of oxidative and nitrosative stress, in the cord blood samples of neonates born by cesarean section (CS) and normal delivery (ND), to assess the effect that the mode of delivery has on neonates, as far as oxidative and nitrosative stress are concerned. Aims: The main purpose of this study was to compare the levels of oxidative stress, nitrosative stress, and the antioxidant status in the cord blood of neonates delivered by ND, versus neonates delivered by CS. Materials and Methods: Cord blood was collected from 80 full-term, mature neonates of both sexes; 40 born via ND and 40 delivered by elective CS under regional (spinal) anesthesia. The samples were tested for the percent of methemoglobin as a marker of oxidative stress, as well as the antioxidants red blood cell (RBC) glucose-6-phosphate dehydrogenase (G6PD), RBC reduced glutathione (GSH), and serum nitrate and nitrite as markers of nitrosative stress. The statistical evaluation of the data obtained was done using SPSS 17 software, taking the significance level as P < 0.05. Results: The comparison of the analytes between the two groups revealed that the percent of methemoglobin (P < 0.001) was significantly higher, and both RBC G6PD (P < 0.001) and RBC reduced GSH (P = 0.003) were significantly lower in CS neonates than in ND neonates. There were no significant differences in the serum nitrate (P = 0.273) and serum nitrite (P = 0.060) between the two groups of neonates. Conclusion: The CS neonates were deficient in antioxidants and had higher levels of oxidative stress than the ND neonates, but no significant difference existed in the nitrosative stress between the groups.
Keywords: Antioxidant, nitrosative stress, oxidative stress
|How to cite this article:|
Sinharay M, Chakraborty I, Chakraborty PS. Assessment of methemoglobin concentration, serum nitrate, and nitrite levels and their interrelationships with antioxidant status in the cord blood of neonates born via normal delivery versus neonates delivered by cesarean section in an Indian population. J Clin Neonatol 2015;4:109-14
|How to cite this URL:|
Sinharay M, Chakraborty I, Chakraborty PS. Assessment of methemoglobin concentration, serum nitrate, and nitrite levels and their interrelationships with antioxidant status in the cord blood of neonates born via normal delivery versus neonates delivered by cesarean section in an Indian population. J Clin Neonatol [serial online] 2015 [cited 2020 Apr 5];4:109-14. Available from: http://www.jcnonweb.com/text.asp?2015/4/2/109/154105
| Introduction|| |
The living organism is constantly exposed to oxygen. Even in the mother's womb, the fetus is surrounded by enemies like the highly reactive free radicals, reactive oxygen species (ROS) as well as reactive nitrogen species (RNS).  The transition from fetal to neonatal life at birth involves fetal exposure from a hypoxic to normoxic environment, which induces an elevated production of ROS and RNS.  Newborns, and especially preterm infants, have reduced antioxidant defenses and free iron, which enhances the Fenton reaction and infections or inflammation, making them more prone to oxidative stress than children and young adults. ,
The mode of delivery has been reported to be associated with the oxidative stress of the newborn. Since the mode of delivery is almost always dependent on maternal or fetal indications, there is still no agreement as to which method is more suitable for avoiding unfavorable consequences for the baby. Some authors claim that cesarean section (CS) is advantageous in order to avoid oxidative stress, since during normal delivery (ND) mothers and their newborns are exposed to greater oxidative stress. , However, some studies suggest that newborns born by CS are subjected to significantly more oxidative stress than those delivered by ND, , since CS may cause a deficiency in antioxidant defenses in human newborns. , Conversely, some authors have concluded that oxidative stress in the fetal circulation does not depend on the mode of delivery. 
Based on the abovementioned information, we set out to study the differences in oxidative stress (methemoglobin concentration), nitrosative stress (serum nitrate and nitrite levels), and the efficiency of the antioxidant systems (reduced glutathione [GSH] and glucose-6-phosphate dehydrogenase [G6PD] levels) in the newborns delivered by CS versus those delivered by ND.
| Materials and Methods|| |
Umbilical cord blood samples (venous) were obtained from the Department of Obstetrics and Gynecology of the Medical College, Kolkata, West Bengal, India. The Institutional Ethics Committee of the Medical College, Kolkata approved the study protocol.
The two branches of the study were randomly selected neonates delivered by elective CS under regional (spinal) anesthesia, and those neonates delivered by a normal vaginal route in the medical college. Neonates of mothers diagnosed as apparently healthy by history, physical examination, and diagnostic investigation were included, after obtaining written informed consent, as per the local language.
The study period was from January, 2011 to January, 2012.
Samples were collected randomly. Eighty full-term mature neonates of both sexes, born between weeks 37 and 40 of gestation, were included in the study; 40 were born by ND, and 40 were delivered by elective CS. The birth weight was noted, and low birth weight cases were excluded; additionally, the Apgar scores after 1 min and 5 min were recorded.
All mothers chosen were free from any obstetrical complications, including pregnancy-induced hypertension, obstetrical hemorrhage, preterm birth, postterm pregnancy, fetal growth disorder, multifetal gestation, reproductive tract abnormalities, prolonged rupture of membranes, and chorioamnionitis (excluded clinically). Their vitals were normal, and their BMIs indicated that they were nonobese. The mothers received complete pregnancy care, with iron, folic acid, routine blood work, urine testing and tetanus toxoid vaccinations. No special medications, including any other vitamins, were prescribed to the participants, and oxygen supplementation was given to the CS mothers only. All subjects were singleton pregnancies selected from the general obstetrics population. Patients with a history of renal disease, diabetes, or significant perinatal complications were excluded, and there were no incidences of perinatal asphyxia, congenital anomalies, or special morbidities.
This was a hospital based noninterventional cross-sectional study.
Sample collection and storage
Cord blood (venous) was collected from the newborns from the labor room as well as from the operating theatre of the Department of Gynecology and Obstetrics, Medical College and Hospital, Kolkata, after taking a proper history, checking the vitals, and other antenatal routine investigations. Cord blood which was collected was divided into two aliquots: One with ethylenediaminetetraacetic acid (EDTA) for the estimation of the G6PD activity in the red blood cells (RBCs), reduced GSH in the RBCs, methemoglobin level and hemoglobin (Hb) level; the other aliquots were preserved without any anticoagulant for the assessment of the serum nitrate and serum nitrite. After the collection of the samples and carrying them to the Department of Biochemistry, the clotted blood was centrifuged at 1500 rpm for 5 min and the serum was harvested. The estimation of the methemoglobin was conducted within 1 h of collection, with the other assays being completed on the following day. The EDTA blood sample was stored at 2-8°C in the door of the refrigerator, and the serum was stored in the freezer compartment (-20°C) of the refrigerator. The estimation of the Hb) was done using the cyanmethemoglobin method with the EDTA treated blood.
Reduced glutathione in red blood cells
The GSH was measured using the method of Beutler, Duron, and Kelly,  with 5,5'-dithiobis 2-nitrobenzoic acid (Ellman's reagent, DTNB), which produces a yellow colored compound with a sulfhydryl group containing GSH. Estimation of G6PD: The G6PD was measured using the G-SIX KIT (Kinetic method) , from Crest Biosystems.
The Hi estimation was done using Evelyn and Malloy's , method in which, after the lysis of the EDTA blood, the lysates were divided into two parts. In one part, when cyanide was added, the absorption band at 630 nm disappeared, and the resulting change in absorbance was directly proportional to the concentration of Hi. In another part, the total Hb (THb) in the sample was measured after complete conversion to HiCN by the addition of a ferricyanide-cyanide reagent. The methemoglobin was expressed as a percentage of the THb.
Serum nitrate, nitrite
The serum nitrate and nitrite levels were measured by the cadmium reduction method, in accordance with Cortas and Wakid. 
The data obtained from the above tests were analyzed for differences between the medians of the five analytes studied between Group 1 (neonates delivered by CS) and Group 2 (neonates delivered by ND).
Kolmogorov and Smirnov method
The Kolmogorov and Smirnov method was used to check whether the data were normally distributed.
In order to study the significance in the differences between the two groups, the Mann-Whitney U-test was performed for all parameters, since the data did not pass the normality test.
Spearman's correlations were done between all analytes.
All of the tests were completed with a P <0.05 as the significance level, and all statistical analyses were carried out using the SPSS 17 software (SPSS Inc. Chicago, USA).
| Results|| |
The comparison of the analytes between the cord blood of the neonates delivered by CS and that of the neonates delivered via ND as reported in [Table 1] revealed that the median methemoglobin% (P < 0.001) was significantly higher in the CS neonates than in the ND neonates [Figure 1]. Both the medians of the RBC reduced GSH (P = 0.003), and RBC G6PD (P < 0.001) were significantly lower in the CS neonates than in the ND neonates [Figure 2]. The median serum nitrate and nitrite concentrations were higher in the CS neonates, but the difference was not statistically significant [Figure 3] and [Figure 4].
|Figure 1: Box plot showing statistically significant differences in the medians of the methemoglobin% (MHB) in normal delivery neonates (ND) versus cesarean section neonates (CS)|
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|Figure 2: Box plot showing statistically significant differences in the medians of red blood cell (RBC) glucose-6-phosphate dehydrogenase (G6PD)-U/g of hemoglobin (Hb), and RBC reduced glutathione (GSH)- μmol/g of Hb, in normal delivery neonates (ND) versus cesarean section neonates (CS)|
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|Figure 3: Box plot showing no statistically significant difference in the medians of serum nitrate (NO3)-μmol/L, in normal delivery neonates (ND) versus cesarean section neonates (CS)|
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|Figure 4: Box plot showing no statistically significant difference in the medians of serum nitrite (NO2)-μmol/L, in normal delivery neonates (ND) versus cesarean section neonates (CS)|
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|Table 1: Comparison of medians of analytes between cord blood of neonates delivered by CS and ND|
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The correlation between the analytes [Table 2] showed a strong negative correlation in both the methemoglobin% and RBC G6PD (ρ = −0.894 in ND group, ρ = −0.864 in CS group), as well as between the methemoglobin% and RBC reduced GSH (ρ = −0.732 in ND group, ρ = −0.862 in CS group) in the neonates of each group. The RBC G6PD and RBC reduced GSH (ρ = +0.843 in ND group, ρ = +0.996 in CS group) were strongly correlated positively. The correlation between the methemoglobin% and serum nitrate/serum nitrite was positive, but not at all significant; additionally, the level of significance was different for the two groups.
These analyses established that the CS neonates were significantly deficient in antioxidants, both the RBC reduced GSH and RBC G6PD, versus the ND neonates. The higher methemoglobin% reflected higher oxidative stress in the CS neonates than in the ND neonates. Moreover, according to the correlation study, the deficiency in antioxidants might have caused higher oxidative stress in the CS neonates, since there existed a strong negative correlation between the antioxidants and the methemoglobin%.
| Discussion|| |
Free radicals, being highly reactive molecules containing one or more unpaired electrons, have a wide range of biologically toxic effects. Oxygen-derived reactants are called ROS and RNS when produced in excess, and lead to the oxidation of lipids, proteins, and polysaccharides, and to DNA damage (fragmentation, base modifications, and strand breaks).
Term labor may induce periods of both hypoxia and oxidative stress for the newborn, where oxidative stress plays an important role in the initiation of labor. The superoxide anions generated may contribute to matrix metalloproteinase activation, leading to the degradation of the extracellular matrix, causing effacement and dilation of the uterine cervix or rupture of the membranes.  Term labor has been demonstrated to cause an up-regulation of the antioxidant reserve in the fetal compartment,  which is beneficial for the newborns of the ND group.
Oxygen supplementation is commonly given to parturients undergoing CS under regional anesthesia, with the aim of improving fetal oxygenation, but inspiring a high oxygen fraction (FiO 2 ) can also increase free radical activity and lipid peroxidation in both the mother and baby, as concluded by Khaw et al.  in a prospective, randomized, double-blind study. Argüelles et al.  found a good correlation between the oxidative status of the mother and of the neonate.
The methemoglobin level is maintained by an HMP shunt within the erythrocytes and two enzyme systems: Diaphorase I or cytochrome b5 reductase, which plays a major role utilizing NADH; and diaphorase II or nicotinamide adenine dinucleotide phosphate-dependent methemoglobin reductase, which has a minor role in adults, but is more important in newborns who have an underdeveloped diaphorase I or cytochrome b5 reductase activity. , Thus, RBC G6PD and RBC reduced GSH play pivotal roles as antioxidants in lowering the formation of oxygen-derived free radicals; thus checking methemoglobin formation.
In one study, Raijmakers et al.  found the median (5 th -95 th percentile) GSH levels in venous and arterial umbilical samples were higher after ND, as compared to CS. Similar results were seen in studies conducted by Paamoni-Keren et al.  and Nabhan et al.  Compagnoni et al.  also concluded that ND neonates have higher levels of coenzyme Q10, an antioxidant. Deficiencies in these antioxidants in CS neonates have caused significantly higher methemoglobin levels than in ND neonates. Moreover, a significantly strongly negative correlation exists between the antioxidants and methemoglobin levels in both the groups in the current study.
Nitric oxide is a precursor to RNS, but it cannot be measured directly. Nitrates and nitrites, which are metabolites of NO, are measured as biomarkers of RNS. Oxidative stress increases both ROS and RNS. Therefore, CS neonates are expected to have higher nitrosative stress than ND neonates, but the difference is not significant in our study. The higher asymmetric dimethylarginine level, the endogenous inhibitor of NO synthase, after cesarean birth compared with vaginal birth, may contribute to decreased nitric oxide production, as established in a study by Vida et al.  This may have caused an insignificant difference in the nitrate and nitrite concentrations between the neonates of the two modes of delivery. The correlation between oxidative stress represented by the methemoglobin level and nitrosative stress was also insignificant and weak.
In the current study, ND has been concluded to be favorable for neonates, based on the assessment of oxidative stress, a marker of fetal well-being, in the two modes of delivery in the Indian population. Based on maternal and fetal outcomes, the consequences of a drastic change in trend from ND to CS have been assessed by the WHO. Their guidelines (in 1985, revised in 1994) concluded that the proportion of cesarean births should range between 5% and 15% and no additional benefits accrue to the perinates or the mothers when the rate exceeded the level.  The third phase of the WHO global survey in Africa and Latin America in 2004-2005, and in Asia in 2007-2008,  established that the overall rate of CS was 27.3% and of operative vaginal delivery was 3.2%. The risk of maternal mortality, the morbidity index, and perinatal outcomes increased for operative vaginal delivery and all types of CSs. The Indian scenario is no different. A study by the Indian Council of Medical Research in 33 tertiary care institutions noted that the average CS rate increased from 21.8% in 1993-1994 to 25.4% in 1998-1999,  and a 5-year audit from a large teaching hospital in Kolkata showed a CS rate of 49.9% (Pahari et al. 1997).
The current study was conducted because there was a scarcity of studies on this aspect of the Indian population. Moreover, consensus regarding the effects of the two modes of delivery on the oxidative stress of newborns varied from study to study. There were some limitations of the present study: One being the sample size and the other being that the study population was a local one. A potential limitation was the absence of long-term clinical follow-up.
In the present study, neonates delivered by CS were found to be deficient in antioxidants in the RBCs, resulting in greater oxidative stress than in the neonates born by vaginal delivery. The WHO guidelines and this study have interpreted that, to improve maternal and perinatal outcomes, CSs should be done only when there is a medical indication. Thus, CSs, although indispensable in saving the lives of both mother and fetus, must be carefully considered when the mother could easily undergo ND, in order to improve the fetal outcome.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]