Thiol/disulfide homeostasis as a new oxidative stress marker in patients with neonatal transient tachypnea

Abstract

AimTransient tachypnea of the newborn (TTN) is the most common cause of respiratory distress occurring in delayed clearance of lung fluid. This is the first report to examine thiol-disulfide homeostasis in patients with TTN.MethodsThirty TTN and 30 controls were included in the present study. The dynamic thiol-disulfide balance was determined by the new colorimetric method developed by Erel et al. TAS, TOS and OSI levels were evaluated using the previously described method developed by Erel.ResultsThiol levels were found to be significantly lower between patient and the control groups. However, disulfide levels were not significantly higher in the TTN group compared with the control groups. Disulfide/native thiol, disulfide/total thiol and native/total thiol levels were statistically significantly different between the TTN and control groups. Moreover, we found that TAS, TOS and OSI levels were also statistically significantly different between patient and control groups.ConclusionThis study indicates that the measurement of dynamic thiol-disulfide homeostasis may contribute to the pathophysiological mechanism, and follow-up of the disease in patients with TTN. In addition, increased TOS and decreased TAS levels may be related with increased oxidative stress and a functional reduction of antioxidant defense system.

Keywords

Introduction

The most common reason for hospitalization of an infant to the neonatal intensive care unit is respiratory distress (RD). RD in the newborn is considered one or more signs of increased work of breathing, such as tachypnea, nasal enlargement, chest tightness, or grunting.¹ Transient tachypnea of the newborn (TTN) is a common cause of respiratory distress in neonates and results from impaired fetal lung fluid clearance.² During fetal life, fluid is secreted into the alveoli to maintain normal growth and development, and fetal lung volume approaches the functional residual capacity that would be created when air breathing is initiated.³ Fetal alveolar fluid filling the alveoli during the intrauterine period has a positive effect on lung mechanics by stretching the lungs. In order to have effective gas exchange after birth, it is necessary to clean the existing liquid from the environment.⁴ TTN was first described in 1966 and is the most common cause of RD in term and late preterm infants.² TTN is usually characterized by a respiratory rate of over 60 breaths per minute and symptoms of respiratory distress.⁵ The estimated incidence of TTN is 4.0 - 5.7 per 1000 term births.^2,6 Common risk factors in TTN formation include precipitous delivery, fetal distress, maternal sedation, low birth weight and gestational diabetes.⁷ Although TTN is considered a benign condition in infants, it is known to increase respiratory and stress load.⁸
Oxidative stress (OS) occurs as a result of the deterioration of the balance between free radicals and antioxidants and is responsible for the pathogenesis of various types of diseases such as cancer, neurological and inflammatory disorders.^9-10-11-12 Newborn infants are very sensitive to oxidative damage due to their limited antioxidant capacity.¹³ Therefore, the evaluation of OS levels in newborns plays a very vital role in terms of preventing unfavorable consequences that may occur due to OS and applying appropriate treatment methods.
Thiol groups are the primary target of reactive oxygen species (ROS) produced in the cell as a result of OS. Thiols are compounds that contain a sulfhydryl functional group in their structure. Thiols are exposed to oxidation reactions by increased oxidant molecules as a result of OS and disulfide structures are formed.¹⁴ Disulfide bonds are reduced back to thiol groups by reducing agents, and thus a dynamic thiol-disulfide homeostasis is achieved.
To the best of our knowledge, this is the first report to evaluate dynamic thiol-disulfide levels in patients with TTN.

Materials and Methods

Study DesignThis study was performed at the 3rd level neonatal intensive care center of Aksaray University Training and Research Hospital Neonatal Intensive Care Unit, in order to determine the thiol-disulfide balance in patients with TTN.Patient And Control GroupsA total of 60 subjects, including 30 controls and 30 TTN, were included in this study. There was no statistically significant difference in terms of age and gender in the patient group and healthy controls. Demographic data of the study and control groups were collected from the hospital automation system. Infants born 37 weeks before or after 39 weeks, those with congenital pneumonia and RDS, those with persistent hypoglycemia, hypocalcemia and polycythemia, and infants with heart disease or meconium aspiration were extricated from the current study. Our study was conducted in accordance with the ethical standards of the World Medical Association Helsinki Declaration Principles. Written informed consent was obtained from all patients and control families who agreed to participate in the study. The present study was approved by the Hatay Mustafa Kemal University Clinical Research Ethics Committee (protocol number: 2020/08).Sample Collection Of The StudyAll working samples were taken into ethylenediaminetetraacetic acid (EDTA) vacuum tubes and centrifuged at 3600 × rpm for 10 minutes. The serum obtained was aliquoted into Eppendorf tubes and kept in a deep freezer at -80°C until analysis.Measurement Of TAS And TOS LevelsTotal oxidant status (TOS) and total antioxidant status (TAS) levels were measured spectrophotometrically based on the method developed by Erel.¹⁵ The measured data were expressed in µmol Trolox equivalent per liter for TAS and mmol H2O2 equivalent per liter for TOS. Then, oxidative stress index (OSI) (OSI (arbitrary unit) = TOS (µmol H2O2 Eq/L) / TAS (µmol Trolox Eq/L) × 100) was calculated.Determination Of Thiol LevelsThiol levels (native and total) were determined by the fully automatic spectrophotometric method previously described by Erel and Neselioglu.¹⁶ Concisely, disulfide bonds are reduced to thiol groups via sodium borohydride (NaBH4). In order to prevent reduction of DTNB (5,5’-dithiobis-(2-nitrobenzoic) acid), the remaining NaBH4 was consumed and removed with formaldehyde from the environment. Disulfide levels were determined by dividing the difference obtained by subtracting native thiols from total thiols by two.Ethical ApprovalEthics Committee approval for the study was obtained.Statistical AnalysisIBM SPSS statistics package program version 22 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The normal distribution of the data was determined by the Shapiro-Wilk test. The mean differences between the two independent groups were compared with Student’s t-test. A comparison of values that did not fit the normal distribution was carried out using the Mann-Whitney U test. The Spearman correlation test was used for correlation analysis. The p-value < 0.05 was accepted as the level of statistical significance.

Results

The present study consisted of 60 patients, 30 in the TTN group and 30 in the control group. There was no statistically significant difference between the study groups in terms of age and gender (Table 1).
In the TTN group, 14 (46%) of the patients were male and 16 (54%) were female. In the control group, 16 (54%) of the participants were male and 14 (46%) were female. The birth weight was 3339.6 (2850-3860) g. in the control group, it was 3160 (2650-3800) g. in the patient group (p<0.034). There was no statistically significant difference in laboratory parameters between the two groups (Table 2).
Native and total thiol levels were found to be significantly lower in patients with TTN compared with the control subjects (p<0.001). However, disulfide levels were not significantly higher in the TTN subjects compared with the control group (p<0.064). Disulfide/native, disulfide/total and native/total levels were statistically significantly different between the TTN and control groups (p<0.005). Moreover, TAS, TOS and OSI levels were also statistically significantly different between patient and control groups (p<0.001) (Table 3).

Discussion

We demonstrated that total and native thiol levels were significantly lower in patients with TTN than in control subjects. However, disulfide levels were significantly higher in patients with TTN compared to the control subjects. We also demonstrated that serum TAS levels were lower in patients with TTN group than in healthy controls. However, TOS and OSI levels were significantly higher in the patient group compared to the controls. In our study, we also evaluated the ratios of disulfide/native, disulfide/total and native/total thiol levels between the control and patient groups and a statistically significant difference was found. This is the first report investigating thiol-disulfide homeostasis as an oxidative marker in patients with TTN.
The excess need for oxygen immediately after birth leads to an increase in reactive oxygen species and a decrease in antioxidants. OS occurs as a result of the disruption of the balance between free radicals and antioxidants and is responsible for the pathogenesis of several neonatal diseases such as respiratory distress syndrome, bronchopulmonary dysplasia, and retinopathy of prematurity.^17-18-19 Thiols are organic compounds containing a sulfhydryl group in their structure and are very sensitive to oxidation reactions.²⁰ After oxidation reactions, thiol groups convert into disulfide bonds as a result of their interaction with free radicals. After that, they are reduced back to thiol groups by antioxidants and thus a dynamic thiol-disulfide balance is achieved.
In the study of Unal et al. in low-birth-weight preterms, they showed that both thiols and disulfide values were high and disulfide values were decreased in the first week measurements, but thiols increased compared to the first week as a result of the third-week measurement.²¹ They concluded that since breast milk contains a high amount of glutathione, the serum glutathione levels of breastfed preterms may have increased and this may cause an augmentation in thiol levels. They also reported that increased disulfide levels are due to the increase in oxidative damage by phototherapy and antibiotics, which are common treatments in the first week of preterm infants.
In another study by Oktem et al. they applied antibiotherapy to newborn patients with urinary tract infections. They indicated that native and total thiol levels of the pretreatment group were lower compared to both post-treatment and control groups.²² However, they showed that disulfide levels were higher in the pre-treatment group. They concluded that the increase in proinflammatory cytokines due to the increase in infection may cause an increase in ROS, resulting in a decrease in native and total thiol levels and an increase in disulfide levels. In the study by Aydogan et al. it was indicated that native and total thiol levels were lower in newborns with neonatal sepsis compared to controls.²³ They hypothesize that this was due to the increase in OS. In the same line with the previous studies, we found that native and total thiol levels were lower, while disulfide levels higher in the TTN group with respect to the control subjects. One of the reasons for the decrease in serum thiol levels may be the depletion of sulfhydryl-containing antioxidant molecules, especially glutathione, to remove ROS as previously suggested.²⁴
In another study, Bulut et al. demonstrated that TOS and TAS levels were significantly higher in newborn infants with hyperbilirubinemia compared to the controls.²⁵ However, the OSI level did not change significantly in the patient group compared to the control group. They hypothesized that TAS levels were higher due to the increased bilirubin levels. They also concluded that increase in TOS levels may be associated with elevated OS levels due to increased bilirubin levels. In the present study, we found that TOS and OSI levels were higher in patients with TTN than controls. However, TAS levels were lower in patients with TTN compared to the controls. We concluded that increased OS may be related to a decrease in the antioxidant defense system.
The limitation of the present study is the small sample size. Studies with larger sample size are needed for further prospective studies.

Limitations

Our study was conducted in a single center due to sample conditions and patient population. Therefore, the sample size of our study is small. Large sample size studies are needed to evaluate the results.

Conclusion

In conclusion, we found that TOS and OSI levels were high, while TAS levels were low in patients with TTN. These findings show us that the oxidant- antioxidant balance is impaired. We also found decreased native and total thiol levels and increased disulfide in patients with TTN groups. This result might indicate that increased OS lead to formation of disulfide bonds resulting in decreased antioxidant capacity in patients with TTN. Measurement of dynamic thiol-disulfide levels can contribute to follow-up of the disease and the application of appropriate treatment.

References

1. Reuter S, Moser C, Baack M. Respiratory distress in the newborn. Pediatr Rev. 2014;35(10):417-428. doi:10.1542/pir.35-10-417
   Article · PubMed · Google Scholar
2. Alhassen Z, Vali P, Guglani L, Lakshminrusimha S, Ryan RM. Recent advances in pathophysiology and management of transient tachypnea of the newborn. J Perinatol. 2021;41(1):6-16. doi:10.1038/s41372-020-0757-3
   Article · PubMed · Google Scholar
3. Guglani L, Lakshminrusimha S, Ryan RM. Transient tachypnea of the newborn. Pediatr Rev. 2008;29(11). doi:10.1542/pir.29.11.e59
   Article · PubMed · Google Scholar
4. Katsumata M, Fujisawa T, Kamiya Y, et al. Effects of long-acting muscarinic antagonists on promoting ciliary function in airway epithelium. BMC Pulm Med. 2022;22(1):186. doi:10.1186/s12890-022-01983-3
   Article · PubMed · Google Scholar
5. Moresco L, Romantsik O, Calevo MG, Bruschettini M. Non-invasive respiratory support for the management of transient tachypnea of the newborn. Cochrane Database Syst Rev. 2020;4(4). doi:10.1002/14651858.cd013231.pub2
   Article · PubMed · Google Scholar
6. Gutvirtz G, Wainstock T, Sheiner E, Pariente G. Prematurity and long-term respiratory morbidity: what is the critical gestational age threshold? J Clin Med. 2022;11(3):751. doi:10.3390/jcm11030751
   Article · PubMed · Google Scholar
7. Edwards MO, Kotecha SJ, Kotecha S. Respiratory distress of the term newborn infant. Paediatr Respir Rev. 2013;14(1):29-36. doi:10.1016/j.prrv.2012.02.002
   Article · PubMed · Google Scholar
8. Liem JJ, Huq SI, Ekuma O, Becker AB, Kozyrskyj AL. Transient tachypnea of the newborn may be an early clinical manifestation of wheezing symptoms. J Pediatr. 2007;151(1):29-33. doi:10.1016/j.jpeds.2007.02.021
   Article · PubMed · Google Scholar
9. Özcan O, Erdal H, Çakırca G, Yönden Z. Oxidative stress and its impacts on intracellular lipids, proteins and DNA. J Clin Exp Invest. 2015;6(3):331-336. doi:10.5799/ahinjs.01.2015.03.0545
   Article · PubMed · Google Scholar
10. Cakirca G, Celik MM, Erdal H, Neselioglu S, Erel O, Basarali MK, et al. Investigation of thiol/disulfide homeostasis in familial Mediterranean fever patients. Clin Anal Med. 2018;9(3):231-234.
    PubMed · Google Scholar
11. Sezgin B, Kinci MF, Pirincci F, et al. Thiol-disulfide status of patients with cervical cancer. J Obstet Gynaecol Res. 2020;46(11):2423-2429. doi:10.1111/jog.14480
    Article · PubMed · Google Scholar
12. Erdal H, Ciftciler R, Tuncer SC, Ozcan O. Evaluation of dynamic thiol-disulfide homeostasis and ischemia-modified albumin levels in patients with chronic lymphocytic leukemia. J Investig Med. 2022.
    PubMed · Google Scholar
13. Saugstad OD. Update on oxygen radical disease in neonatology. Curr Opin Obstet Gynecol. 2001;13(2):147-153. doi:10.1097/00001703-200104000-00009
    Article · PubMed · Google Scholar
14. Ozcan O, Erdal H, Ilhan G, et al. Plasma ischemia-modified albumin levels and dynamic thiol/disulfide balance in sickle cell disease: a case-control study. Turk J Haematol. 2018;35(4):265-270.
    PubMed · Google Scholar
15. Erel O. A new automated colorimetric method for measuring total oxidant status. Clin Biochem. 2005;38(12):1103-1111. doi:10.1016/j.clinbiochem.2005.08.008
    Article · PubMed · Google Scholar
16. Erel O, Neselioglu S. A novel and automated assay for thiol/disulphide homeostasis. Clin Biochem. 2014;47(18):326-332. doi:10.1016/j.clinbiochem.2014.09.026
    Article · PubMed · Google Scholar
17. Hamid ERA, Ali WH, Azmy A, Ahmed HH, Sherif SH, Saleh TM. Oxidative stress and anti-oxidant markers in premature infants with respiratory distress syndrome. Open Access Maced J Med Sci. 2019;7(17):2858-2863.
    PubMed · Google Scholar
18. Thébaud B, Goss KN, Laughon M, et al. Bronchopulmonary dysplasia. Nat Rev Dis Primers. 2019;5(1):78. doi:10.1038/s41572-019-0127-7
    Article · PubMed · Google Scholar
19. Kim SJ, Port AD, Swan R, Campbell JP, Chan RVP, Chiang MF. Retinopathy of prematurity: a review of risk factors and their clinical significance. Surv Ophthalmol. 2018;63(5):618-637. doi:10.1016/j.survophthal.2018.04.002
    Article · PubMed · Google Scholar
20. Erel O, Erdogan S. Thiol-disulfide homeostasis: an integrated approach with biochemical and clinical aspects. Turk J Med Sci. 2020;50:1728-1738. doi:10.3906/sag-2003-64
    Article · PubMed · Google Scholar
21. Unal S, Isik DU, Bas AY, et al. Evaluation of dynamic thiol-disulfide homeostasis in very low-birth-weight preterms. J Matern Fetal Neonatal Med. 2019;32(7):1111-1116. doi:10.1080/14767058.2017.1400007
    Article · PubMed · Google Scholar
22. Oktem A, Zenciroglu A, Dilli D, et al. Thiol-disulfide homeostasis in neonatal patients with urinary tract infection. Am J Perinatol. 2022;39(13):1460-1464.
    PubMed · Google Scholar
23. Aydogan S, Akduman H, Dilli D, et al. The role of thiol-disulfide homeostasis in neonatal sepsis. J Matern Fetal Neonatal Med. 2021;34(10):1522-1528. doi:10.1080/14767058.2019.1638904
    Article · PubMed · Google Scholar
24. Arner ES, Holmgren A. The thioredoxin system in cancer. Semin Cancer Biol. 2006;16(6):420-426.
    PubMed · Google Scholar
25. Bulut O, Erek A, Duruyen S. Effects of hyperbilirubinemia on markers of genotoxicity and total oxidant and antioxidant status in newborns. Drug Chem Toxicol. 2022;45(1):451-455. doi:10.1080/01480545.2019.1710182
    Article · PubMed · Google Scholar

Tables

Additional Information

Publisher’s Note
Bayrakol MP remains neutral with regard to jurisdictional and institutional claims.

Rights and Permissions

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). To view a copy of the license, visit https://creativecommons.org/licenses/by-nc/4.0/

View full license details →