Comparative assessment of oxidative stress and enzymatic antioxidants in primigravidae pregnant and non-pregnant women

Abstract               Background: The generation of reactive oxygen species and the activation of antioxidant defence mechanisms are important components of female reproductive physiology. It's impairment alters cellular signalling and can damage cellular macromolecules. Consequently, oxidative stress has emerged as a likely promoter of several pregnancy-related disorders, such as spontaneous abortions, embryopathies, preeclampsia, foetal growth restriction, preterm labour and low birth weight. Aims: The present study aimed to investigate the status of malondialdehyde (MDA), superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-P) with age matched pregnant & nonpregnant women. Methods & Materials: Blood samples were collected during the ante partum period of primi gravidae pregnant women and in non - pregnant women. Study subjects were matched with respect to maternal and gestational age in all the three trimesters. Results: MDA levels were significantly increased whereas the levels of enzymatic antioxidant's i.e. SOD, CATALASE and glutathione peroxidase were significantly decreased in pregnant women as compared to non –pregnant women. Conclusion: These results suggest that an equilibrium maintained between the production of free radicals and antioxidant production during pregnancy ensures healthy maternal and foetal growth.

Key Word: Malondialdehyde, Superoxide dismutase, Reactive oxygen species, Lipid peroxidation, Glutathione peroxidase, Oxidative stress.

INTRODUCTION

Pregnancy is a stressful condition in which many metabolic and physiological functions are altered to a considerable extent¹. It is a physiological development characterized by the drastic increase in energetic and oxygen demands for adequate foetal development and growth. Thus, both mother and foetus are likely to experience oxidative stress, at the time of pregnancy². In late pregnancy, negative energy equilibrium may be the reason for the development of oxidative stress; increased lipid per oxidation and decreased antioxidant activity which are also contributing factors for the development of complications in pregnancy³. Oxidative stress can be defined as a state of disrupted balance between reactive oxygen species and the mechanisms of detoxification and repair. These reactive oxygen species (ROS) are generated as by-products of aerobic respiration⁴. Excessive production of these reactive oxygen species leads to “oxidative” stress that play a vital role in many pathological conditions i.e. neoplastic diseases, neurodegenerative disorders, or illnesses with a viral, toxic, or inflammatory etiology⁵. Pro-neoplastic activity of ROSs results mainly from DNA damage, proteins, and lipids and modifying these molecules may increase the risk of permanent genetic mutation⁶. Oxidative stress forms an imbalance between pro-oxidants and antioxidant capacity which has been implicated in sub optimal reproductive performance from the earliest stage of foetal development to labour till delivery⁷. From the biochemical point of view, oxidative stress is associated with increased production of oxidizing species or a significant decrease in the effectiveness of anti-oxidant defence. Oxidative degradation of lipids is referred to as lipid peroxidation. Lipid peroxidation is a process in which free radicals remove the electrons from membrane lipid ensuing in cell membrane damage. Markers of lipid peroxidation have been confirmed in many diseases such as ischemic heart disease, diabetes and neurodegenerative disease⁹. Oxidative stress is also involved in the aetiology of defective embryo development¹⁰.In recent years the role of decreasing antioxidants and increasing oxidative stress is gaining momentum as they are threat for the normal pregnancy. Certain biochemical indices are useful in assessing the progression of pregnancy. Hence the present study was under taken to assess the role of antioxidants, lipid peroxidation and superoxide generation in normal pregnancy¹¹. A common reliable marker of lipid peroxidation is malondialdehyde (MDA). The antioxidant defence mechanisms can be categorized in to two types- free radical scavenging and chain breaking antioxidants. The free radical scavenging mechanisms include antioxidant enzymes like Superoxide dismutase (SOD), Glutathione peroxidase (GSH-P), Glutathione reductase (GSH-R) and Catalase, which limit the cellular concentration of free radicals and prevent excessive oxidative damage¹². Lipid peroxidation is a well-established mechanism of cellular injury in both plants and animals, and is used as an indicator of oxidative stress in cells and tissues. Lipid peroxides, derived from polyunsaturated fatty acids, are unstable and go off to form a complex series of compounds. These include reactive carbonyl compounds, in which the most abundant is malondialdehyde. Therefore, measurement of malondialdehyde is widely used as an indicator of lipid peroxidation¹³. Based on this, a drop in total antioxidant status during pregnancy was concluded following significant decrease in plasma and whole blood selenium and selenium-dependent glutathione peroxidase activities during gestation¹⁴. Superoxide dismutase and glutathione peroxidase are the most prominent natural antioxidant enzymes that can eliminate ROS.¹⁵ Some of these antioxidant enzymes are activated by the presence of metal ions, while in some metal ions have structural roles. On the other hand, low levels of antioxidants have been reported to cause many adverse pregnancy outcomes and even fetal and childhood slow or abnormal development¹⁶. According to a study carried out in pregnant ewes, Catalase activity in maternal blood decreases during pregnancy. The decreased activity of Catalase could be due to decrease in lipid peroxidation in the third trimester of pregnancy¹⁷.

MATERIALS AND METHODS

This study was conducted in the Department of Biochemistry, ESIC Hospital, Okhla. It comprised of total 200 women in the age group of 18-40 years, of which 50 each were in all the three trimesters of pregnancy and are primigravida and 50 were non-pregnant women. The subjects with Diabetes mellitus under medication and untreated diabetes, any acute and chronic disease, severely anaemic (<6.0gm% of Hb) and those suffering from any other systemic disorder were excluded from the study. The cases and controls were studied with informed written consent. A detailed clinical history including age, sex, occupation, socio-economic status, duration of illness and any associated risk factor contributing for the illness was elicited from the subjects. With all aseptic precautions blood samples (5 ml) were drawn by venipuncture and collected in heparinized tubes to measure MDA and the antioxidant enzymes. The method of thiobarbituric acid, which measured MDA-reactive products¹⁸ there is formation of pink colour which gives the measure of thiobarbituric acid reactive substance (TBARS) and was read at 532 nm using spectrometer. SOD activity was measured as described by Das et al¹⁹ The method involves generation of superoxide radical by photoreduction of riboflavin and its detection by nitrite formation from hydroxylamine hydrochloride at 543 nm. Catalase²⁰ and GSH-P ²¹ activity were measured as described by the authors.

Statistical Analysis: The result are presented in mean ± SD. The MDA, SOD, GSH-P, and catalase levels were compared by using Unpaired t- test between cases and control. The Pearson’s correlation coefficient were calculated among the study parameters. The p- value <0.05 was considered significant. All the analysis was carried out by using Statistical Package for Social Sciences (SPSS) version 22.

OBSERVATION

Table 1: Comparison of MDA, Sod, Catalase and Gutathione Peroxidase Between Cases and Control

Malondialdehyde and enzymatic antioxidant levels in the non-pregnant and normal pregnant women (Ist, IInd and IIIrd trimester). N – Number of cases, p – Probability (exact level of significance)

RESULT

In table 1, the levels of MDA, SOD, Catalase and glutathione peroxidase were observed in cases and in controls and the difference was statistically significant. A significant increase (p<0.0001) was found in the levels of Malondialdehyde in pregnant women when compared to Non-Pregnant women and a significant decrease (p<0. 0001) was found in the levels of catalase, SOD, GSH-P in pregnant women when compared to non– pregnant women. The level of MDA was observed to be higher in III^rd trimester 5.25±1.31 as compared to I^st and II^nd trimester among cases 2.27±0.27 and 2.71±1.29 respectively and MDA level was found lower compared with controls. The level of SOD was found to be lower in I^st, II^nd and III^rd trimester among cases 0.59±0.33 ,0.50±0.24 and 0.46±0.17 respectively when compared with controls. The level of catalase was found to be lower in I^st, II^nd and III^rd trimester among cases 1.52±0.85, 1.31±0.72 and 1.28±0.70 compared with controls. The level of glutathione peroxidase was found lower in I^st, II^nd and III^rd trimester among cases 2.27±0.20, 1.79±0.14, 1.64±0.14 compared with controls.

DISCUSSION

In normal pregnancy there is increase in oxidative stress because of high demand of metabolic and increase requirement of tissue oxygen. MDA is a stable end product of free radicals which is produced by lipid peroxidation and it is a reliable marker for the assessment of free radical induced damage to tissue. In this study we have assessed the serum levels of MDA, SOD, Catalase And glutathione peroxidase level. The values of these markers were compared between Pregnant and Non- Pregnant women. There is a balance between the production of ROS and antioxidant status in the predelivery period ²².Lipid peroxides are important because their uncontrolled production can result in oxidative stress, with significant damage to cell²³. MDA, a metabolite of lipid peroxides, is detectable in plasma and is used as an indicator of lipid peroxidation.CAT, SOD, and GSH-P are important components of the antioxidant defence system. They control the level of free radicals in cells. GSH-P activity was higher in the second and third trimesters than in the first trimester. Placenta plays a major role in the lipid peroxide production²⁴. NADPH oxidase is localised in the placental synctial microvillous membrane generates increased amount of the superoxide radicals²⁵. As stated in previous study lipid peroxide levels in non-pregnant and normal pregnant women show remarkable increased levels of lipoperoxides in II^nd and III^rd trimesters of pregnancy as compared to non-pregnant women²⁶. A decrease in antioxidant enzymes, where they argued that it probably could be due to a response to the increased lipid peroxide load in some forms of pathologic conditions (preeclamptic and eclamptic patients)²⁷. SOD, CAT and GSH-P activities compared to the non-pregnant controls were also observed. The level of these enzymes suggested increased turn-over of these enzymes due to oxidative burden arising from increased production of lipid peroxides. As stated in previous studies, Calcium is a strong inducer of uterine contraction that potentially lead to preterm labour. Thus, suppression of oxidative stress by antioxidant system, primarily by catalase, play very important role in preventing preterm labor²⁸. Decreased antioxidant activity, is indicative of the disturbance in the antioxidant system, which results due to diminished individual antioxidants. Overall it was observed that there was reduction in the individual antioxidant levels in normal pregnany²⁹. In a prospective, randomized controlled study of 251 primigravida women, it was found that oral supplementation with antioxidant lycopene reduces the development of pre‐eclampsia and intrauterine growth retardation in primigravida women³⁰. It is the dynamic equilibrium between various antioxidants. So even though individual antioxidants rise during pregnancy, net result may be a lower anti oxidative capacity. In our study decrease in catalase, SOD and GSH-P and increase of MDA were statistically significance in pregnant women.

CONCLUSION

Hence, in conclusion, it is observed that there exist an correlation between the generation of reactive oxygen species and the antioxidant defense mechanisms in our body. The antioxidant enzyme activities could be influenced by various physiological factors i.e. diet, hormones and presence of systemic illness as well. There is still dearth of data about other enzyme and free radical scavenging mechanisms operating in physiological and pathological pregnancy states. The effect of oxidative stress in the postpartum phase and its effect on lactation needs to be studied.

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