Hypertens Pregnancy, 2015; 34(1): 125–135 ! Informa Healthcare USA, Inc. ISSN: 1064-1955 print / 1525-6065 online DOI: 10.3109/10641955.2014.988349
ORIGINAL ARTICLE
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Effect of socio-economic status and estimation of lipid peroxidation and antioxidant in preeclamptic pregnant women: a case–control study Md. Shahid Sarwar,1 Rakhesh Chandra Sarkar,2 Rumpa Bhowmick,2 Syed Masudur Rahman Dewan,2 Maizbha Uddin Ahmed,3 Abul Hasnat,3 Mamunur Rashid,1 and Mohammad Safiqul Islam2,4 1
Department of Pharmacy, Southeast University, Banani, Dhaka, Bangladesh, Department of Pharmacy, Noakhali Science and Technology University, Sonapur, Noakhali, Bangladesh, 3 Department of Clinical Pharmacy and Pharmacology, Faculty of Pharmacy, University of Dhaka, Dhaka, Bangladesh, and 4 Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Shimane, Japan 2
Objective: Increased extent of lipid peroxidation and decreased antioxidant defense has been postulated as a major contributor to the development of preeclampsia. The present study was conducted to determine the serum level of malondialdehyde (MDA) and vitamin C in these patients. Methods: This research was conducted as a case–control study to assess the extent of lipid peroxidation and antioxidant status in 150 preeclampsia patients and 174 healthy pregnant women. Lipid peroxidation was assessed by measuring serum MDA level, and antioxidant defense was evaluated for serum vitamin C by UV spectrophotometric method. Independent sample t test and Pearson’s correlation test were used for the statistical analysis with statistical software package SPSS, version 16.0 (SPSS, Inc., Chicago, IL). Results: Our study found significantly higher level of MDA (p50.05) and significantly lower level of vitamin C (p50.05) in preeclampsia patients in comparison to the control subjects. Pearson’s correlation analysis revealed that there was negative correlation between the serum level of MDA and vitamin C for both patient (r = 0.057, p = 0.669) and control groups (r = 0.128, p = 0.487). Analysis of socio-economic data revealed that preeclampsia was more prevalent among the rural pregnant than the urban pregnant women (OR 1.44, 95% CI 0.665–3.14, p = 0.352). It was also observed that primigravida pregnant were at higher risk for the development of preeclampsia than the multigravida pregnant women (OR 1.87, 95% CI 0.868–4.04, p = 0.108). Conclusions: Elevated serum MDA and depleted serum vitamin C may be associated with the development of preeclampsia. Keywords Malondialdehyde, Preeclampsia, Pregnant women, Socio-economic status, Vitamin C
Correspondence: Dr. Mohammad Safiqul Islam, Associate Professor, Department of Pharmacy, Noakhali Science and Technology University, Sonapur, Noakhali-3814, Bangladesh. Tel: +88-0321-71483. Fax: +88-0321-62788. E-mail: research_safiq @yahoo.com.
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INTRODUCTION Preeclampsia is a multisystem, multifactor disorder of pregnancy with elevated blood pressure (140/90 mmHg), proteinuria (300 mg/day) and pathological edema; usually occurring on or after 20 week of gestation (1). Its incidence is approximately 2–8% throughout the world which causes 16% of maternal death during pregnancy in Bangladesh (2). Preeclampsia is highly prevalent in first pregnancy, and its rate reduces extensively in subsequent pregnancies. It is the most common medical complication of pregnancy whose incidence has continued to increase worldwide and is associated with significant maternal morbidity and mortality, accounting for about 50 000 deaths per year throughout the world (3). Thus reducing maternal mortality by 75% between 1990 and 2015 has been considered as part of the millennium development goals of the World Health Organization (4). The prevalence of preeclampsia differs on the basis of geographic location but the most common contributing factors are maternal age below 20 or over 35 years, nulliparity, multiple gestation, twins, black race, low socio-economic status, hydatidiform mole, polyhydramnios, non-immune fetal hydrops obesity, diabetes, chronic hypertension and underlying renal disease (5). Although the exact pathophysiology of preeclampsia is unknown but it is suggested that increased extent of lipid peroxidation and decreased antioxidant profile serve as a contributing factor to preeclampsia (6). Several studies stated that placental perfusion triggers the activation and dysfunction of the maternal vascular endothelium, which leads to increased production of endothelin and thromboxane, and reduced production of vasodilators such as nitric oxide and prostacyclin (7,8). Generally, free radicals mediate lipid peroxidation within the cellular membrane which ultimately causes tissue damage (9). Free radicals generation leading to lipid peroxidation is a usual phenomenon in pregnancy which occurs with low intensity in normal pregnancy, because in normal pregnant women the extent of lipid peroxidation and the formation of antioxidants are well balanced. If this balance of oxidant and antioxidant system is interrupted, it may cause oxidative stress (1). Malondialdehyde (MDA) – the end product of lipid peroxidation – can be used as a marker of tissue damage resulting from oxidative stress (10). Antioxidant molecules act as body’s defense system to combat free radicalmediated oxidative damage. Several enzymatic (glutathione peroxidase, superoxide dismutase and catalase) and non-enzymatic (vitamin A, C, E and glutathione) antioxidants are available in human body which scavenge these free radicals, thus act as protection system against various diseases. Considering the aforementioned facts, the present study was designed to elucidate the possible relation of lipid peroxidation and antioxidant level with the pathogenesis of preeclampsia.
MATERIALS AND METHODS Materials The reagents used in the present study were collected from commercially available company. 2-Thiobarbituric acid (TBA), 2,4-dinitrophenylhydrazine (DNPH) and n-butyl alcohol and sulfuric acid (97%) were purchased from
Lipid peroxidation and antioxidant in preeclampsia
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Merck, Germany. Ascorbic acid was supplied by Globe Pharmaceuticals, Noakhali, Bangladesh, as a gift sample. Trichloroacetic acid (TCA) and hydrochloric acid (37%) were procured from Guangdong, China. The commercially available 1,1,3,3-tetraethoxypropane was purchased from SigmaAldrich, Germany, whereas metaphosphoric acid and thiourea were obtained from Loba Chemie, India. Methods Study Design and Blood Sample Collection This case–control study comprising 150 preeclamptic pregnant women as cases and 174 healthy pregnant women as controls was carried out in the Department of Obstetrics and Gynecology, Noakhali Medical College Hospital, Bangladesh. The study protocol was approved by the ethical review committee of the hospital. Preeclampsia was characterized by the presence of a maternal blood pressure above 140/90 mmHg after 20 weeks of gestation. A specialist obstetrician and gynecologist conducted the diagnosis of preeclampsia. Both the patients and controls were briefed about the objective of this study, and written consent was obtained from each of them. Prior to their inclusion in the study, detailed history was taken from patients and controls using a pre-designed questionnaire. Individuals with diabetes mellitus or other endocrine disorder, renal, liver or cardiovascular disease were excluded from the study. Patients who were unwilling to involve in the research and denied to share their clinical data were also excluded. Blood samples were collected from the antecubital vein of each patient and control in a metal-free sterile tube using a plastic syringe (JMI-Bangla Co. Ltd, disposable syringe, 5 mL) after overnight fasting condition. All blood samples were then kept at room temperature for about 30 min to clot and centrifuged at 2218 g for 15 min to extract the serum. The extracted serum was collected in Eppendorf tube and stored at 80 C for future analysis. Analysis of MDA and vitamin C were performed within few days after serum collection. Determination of Serum MDA Lipid peroxidation was assessed by measuring serum MDA level following the modified method of Satoh (11). According to this method, 0.5 mL of serum was mixed with 2.5 mL of 20 mg/dL trichloroacetic acid in a centrifuge tube and allowed to stand for 10 min at room temperature. After centrifugation of the mixture at 2218 g for 10 min, the supernatant was decanted, and the precipitate was washed with 0.05 M sulphuric acid. Then the precipitate was mixed with 2.5 mL of 0.05 M sulfuric acid and 3.0 ml of 0.2 mg/dL TBA (in 2 M sodium sulfate) which was heated in a boiling water bath for 30 min. This solution was cooled in ice cold water and extracted with 4.0 mL of n-butyl alcohol by vigorous shaking. The organic phase was separated by centrifugation at 2218 g for 10 min, and absorbance of the supernatant was measured spectrophotometrically (UV–VIS Spectrophotometer, Shimadzu, Japan) at 530 nm against a blank that contained all reagents except the serum sample. Serum MDA concentration was expressed as nmol/mL.
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Determination of Serum Vitamin C Serum vitamin C was determined by phenyl-hydrazine spectrophotometry method (12). The concentration of ascorbic acid in the serum was calculated as follows: mmol Absorbance of sample mg Concentration, C ¼ 56:78 Absorbance of standard dl L
Statistical Analysis All values were expressed as mean ± standard error mean (mean ± SEM). Statistical analysis was performed using the statistical software package SPSS, version 16.0 (SPSS Inc., Chicago, IL). Independent sample t test was done to determine the level of significance for various parameters between patient and control groups. Pearson’s correlation analysis was used to find the correlation among the study parameters.
RESULTS Socio-Demographic Profile Our study comprised a total of 324 samples with 150 preeclampsia patients and 174 healthy pregnant women. We observed that there was no significant difference for maternal age (p40.05), but statistically significant difference was found for gestational age between patients and control groups (p50.05) as represented in Figures 1 and 2, respectively. Both the systolic and diastolic blood pressure (SBP and DBP, respectively) were significantly higher in preeclampsia patients (p50.05) when compared with control group (Figure 3).
26
Maternal age (year)
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The 56.78 is conversion factor for ascorbic acid for standard international unit.
24
22
20 0 Patient
Control
Group Figure 1. Comparison of maternal age between patient and control groups.
Lipid peroxidation and antioxidant in preeclampsia
Pregnant women who were in their first pregnancy (primigravida) were of at particular risk for the development of preeclampsia (OR 1.87, 95% CI 0.868– 4.04, p = 0.108) than the multigravida pregnant women (Table 1). The comparative percentage of the primigravida and multigravida pregnancy was 62 and 38% versus 46.6 and 53.4% between patient and control groups, respectively. In this study, it was observed that pregnant women from the rural 40
* Gestational age (week)
30
25
20 0 Patient
Control
Group Figure 2. Comparison of gestational age between patient and control groups. Asterisk indicates statistically significant difference in comparison to control group (p50.05).
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Blood pressure (mm Hg)
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35
*
SBP
135
* DBP
90
45
0 Paent
Control Group
Figure 3. Comparison of SBP and DBP between patient and control groups. Asterisk indicates statistically significant difference in comparison to control group (p50.05).
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Variables Gravidity Primigravida Multigravida Area of residence Rural Urban Occupation Housewife Service Income level Low Medium High Educational status Illiterate Primary Secondary Higher secondary Higher studies
Preeclamptic pregnant
Normal pregnant
p Value
93 (62) 57 (38)
81 (46.6) 93 (53.4)
0.108 (OR 1.87)
96 (64) 54 (36)
96 (55.2) 78 (44.8)
0.352 (OR 1.44)
138 (92) 12 (8)
153 (87.9) 21 (12.1)
0.486 (OR 1.58)
102 (68) 42 (28) 6 (4)
102 (58.62) 54 (31.03) 18 (10.34)
48 54 33 6 9
(32) (36) (22) (4) (6)
60 45 42 12 15
(34.5) (25.90) (24.1) (6.9) (8.6)
–
–
OR: Odd ratio.
area had higher prevalence for preeclampsia than those from the urban area (OR 1.44, 95% CI 0.665–3.14, p = 0.352). The relative percentages of the pregnant women from the rural and urban area were 64 and 36% versus 55.2 and 44.8% in patient and control groups, respectively. It was found that majority of them were of lower (68 versus 10.25%) economic class women followed by medium (28 versus 58.62) and high (4 versus 31.03%) economic class. This study explored that most of the patients were housewives in both patient and control groups (92 versus 87.9%), and the remaining small populations were of service holder (8 versus 12.1%) (OR 1.58, 95% CI 0.434– 5.74, p = 0.486). Educational status studies of the preeclamptic pregnant women explored that 32% of them were illiterate, while 36, 22, 4, and 6% continues their study up to primary, secondary, higher secondary and graduate or post-graduate level, respectively, whereas in the control group these were 34.5, 25.90, 24.1, 6.9 and 8.6%, respectively.
Lipid Peroxidation and Antioxidant Status Serum level of MDA was found 6.66 ± 0.16 and 5.01 ± 0.21 nmol/mL, whereas the level of vitamin C was 17.75 ± 1.09 and 25.26 ± 1.65mmol/L in patient and control groups, respectively (Table 2). Statistical analysis reveals that the level of MDA was significantly higher in preeclamptic pregnant women (p50.05), whereas significantly lower level of vitamin C was found in this group (p50.05).
Lipid peroxidation and antioxidant in preeclampsia Table 2. Serum level of MDA and Vitamin C in the study population. Values (Mean ± SEM) Parameters MDA (nmol/mL) Vitamin C (mmol/L)
Preeclamptic pregnant
Normal pregnant
p Value
6.66 ± 0.16 17.75 ± 1.09
5.01 ± 0.21 25.26 ± 1.65
p50.05** p50.05**
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**p50.05 (significant difference between patient and control groups at 95% confidence interval).
Table 3. Correlation of socio-demographic characteristics with serum MDA and vitamin C in the patient and control groups. Preeclamptic pregnant Correlation parameters Maternal age and SBP Maternal age and DBP Maternal age and MDA Maternal age and vitamin C Gestational age and SBP Gestational age and DBP Gestational age and MDA Gestational age and vitamin C MDA and SBP MDA and DBP MDA and vitamin C Vitamin C and SBP Vitamin C and DBP
Normal pregnant
r
p
r
p
0.399** 0.151 0.062 0.169 0.127 0.042 0.170 0.182 0.010 0.144 0.057 0.055 0.023
0.004 0.296 0.667 0.240 0.380 0.773 0.237 0.206 0.943 0.320 0.669 0.707 0.874
0.309** 0.240 0.028 0.031 0.188 0.147 0.188 0.054 0.045 0.132 0.128 0.122 0.043
0.018 0.069 0.837 0.820 0.377 0.272 0.158 0.689 0.740 0.322 0.487 0.362 0.751
r correlation co-efficient; p significance. Values with negative sign indicate an inverse correlation. **p50.05; correlation is significant at 0.05 level (two-tailed).
Correlation Analysis In the present study, the research interest focused on the potential role of lipid peroxidation in the pathophysiology of preeclampsia. For this reason, a wide range of correlation was established to the effect of maternal age, gestational age, SBP and DBP on serum level of MDA and vitamin C in both patient and control groups (Table 3). This study found no significant correlation between serum MDA or vitamin C level with gestational age, SBP and DBP (p40.05), but statistically significant correlation was found between maternal age and SBP in both patient and control groups (p50.05). There was a negative correlation between the serum level of MDA and vitamin C in both patient group (r = 0.057) and control group (r = 0.128).
DISCUSSION Preeclampsia is considered as one of the three leading complications of pregnancy that may affect both mother and fetus. Circulating level of several
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chemicals may greatly contribute to the development of preeclampsia. Although the principle objective of this study was to determine several biochemical parameters that may have an association with the pathogenesis of preeclampsia, but we also analyze the socio-demographic features of the patients and controls to find out the relationship of these parameters with the disease process. All these data could help in improving the maternal and perinatal morbidity and mortality in preeclampsia. Our study found no significant difference for maternal age between the patient and control groups (p40.05), but the gestational age was significantly lower in patient group than the control group (p50.05), which confirms previous studies (13–15). The relationship between gravidity of pregnancy and its complications continues as an interesting research to the obstetricians. Gravidity has been used as a risk marker of preeclampsia with primigravida pregnant, and women who had previous preeclampsia history are identified as higher risk factor of preeclampsia. Primigravida pregnant, i.e. women who were pregnant for the first time are considered to be at risk of preeclampsia, whereas the multigravida pregnant are considered to be at risk for hemorrhage, malpresentation, anemia, uterine rupture and complications associated with chronic medical problems such as diabetes and hypertension (2). In this study, it was found that primigravida pregnancies were at higher risk of developing preeclampsia than women in subsequent pregnancies or multigravida pregnancies which was also reported by other studies (16,17). The results of this study indicate that women from the rural community have more toxemias in pregnancy than those from urban community which is consistent with previous study (18). This may be due to the lack of awareness regarding the antenatal care, insufficient healthcare facilities or lack of qualified physicians in the rural community of Bangladesh. But another study found higher incidence of preeclampsia among the pregnant women from urban area in another country (19). It was also observed that preeclampsia is more common among the lower socioeconomic class women possibly due to their poor life style and dietary habit. Nutrients deficiency may act as a contributing factor to preeclampsia development as these substances can modulate oxidative stress by increasing or decreasing free radicals and antioxidants and/or by supplying substrate for the production of free radicals (20,21). Jonas et al. (22) estimated the associations of socio-economic status (SES) with medical problems and pregnancy outcomes, and their results indicate that low SES is related to a higher risk profile for the development of preeclampsia. Interpretation of the extent of lipid peroxidation and antioxidant status is the relationship of cause and effect in preeclampsia. It is well established that free radicals have a tendency to cause lipid peroxidation. Markers of lipid peroxidation (MDA) are increased during several pregnancy complications. Generally, there is a physiological balance between the lipid peroxidation and antioxidative processes in normal pregnancies (23). Alteration of normal physiological balance in pregnancy causes modification of polyunsaturated fatty acids (PUFA) leading to altered function and structure of capillary endothelial cells (24). Thus, the endothelial cell injury or damage may be the initiator of the pathophysiological events of the preeclampsia. In the present study, it was observed that lipid peroxidation product (MDA) was significantly
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Lipid peroxidation and antioxidant in preeclampsia
increased in preeclampsia when compared with control (p50.05) which signifies the excessive ongoing lipid peroxidation in preeclampsia. This result correlates with numerous studies undertaken by other researchers (25,26). Suhail et al. (27) reported that preeclamptic placenta contains higher MDA than those from normal pregnancies which may contribute to the development of preeclampsia. The increase in extent of lipid peroxidation might not be so important if there is a compensatory increase in antioxidant protection. Several exogenic and endogenic antioxidants act as defense system against the free radicalmediated oxidative damage. Normally, they prevent lipid peroxidation by inactivating free radicals and quenching activated singlet oxygen molecules and superoxide radicals (28). This study showed that the level of antioxidant vitamin C was significantly depleted in preeclampsia patients when compared with the controls (p50.05). The outcome of increasing or decreasing concentration of a single component in human body is not limited to that component only, but the level of other biological components may be affected. Thus, correlation analysis may provide the information whether the level of one parameter affect the level of other parameter either positively or negatively. In this study, Pearson’s correlation analysis confirmed that there was a negative correlation between the serum level of MDA and vitamin C in both patient and control groups. This may be due to the fact that large extent of vitamin C may be utilized to counteract lipid peroxidation of cellular components, hence resulting in the depletion of this antioxidant level (29). Moreover, endothelial dysfunction resulting from high-lipid peroxidation state leads to enhanced endothelial damage in preeclampsia patients, whereas antioxidant vitamins try to neutralize the negative impact of MDA on endothelial cell which subsequently results in depletion of antioxidant vitamins. The findings of the present study reveal that socioeconomic factors have considerable effect on the pathogenesis of preeclampsia possibly due to their life style and dietary habit. Primigravida pregnant had higher risk for the development of preeclampsia than the multigravida pregnant, and they should be offered pre-pregnancy counseling to explain this risk and to encourage early attendance for specialist antenatal care. The present study found significantly elevated level of MDA and depleted level of vitamin C in preeclampsia patients than the healthy pregnant women, which supports the hypothesis that lipid peroxidation is a causative factor in the pathogenesis of preeclampsia. Antioxidant supplementation may be recommended to scavenge the free radical action of MDA, which may be useful as secondary therapy to prevent the oxidative damage in placental tissue of preeclampsia patients.
ACKNOWLEDGEMENTS The authors would staffs, nurses, and Gynecology, Noakhali port during the study. this study.
like to express their heartfelt thanks to all the physicians of the Department of Obstetrics and Medical College Hospital, Bangladesh, for their supThe authors are also thankful to all the participants of
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DECLARATION OF INTEREST This study was financially supported by University Grant Commission (UGC), Bangladesh.
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REFERENCES 1. Adeniji AO, Oparinde DP. Comparison of lipid peroxidation and anti-oxidant activities in pre-eclamptic and normal pregnancies in Nigerian population. Int J Clin Med 2013;4:239–43. 2. Sarwar MS, Ahmed S, Ullah MS, et al. Comparative study of serum zinc, copper, manganese, and iron in preeclamptic pregnant women. Biol Trace Elem Res 2013; 154:14–20. 3. World Health Organization (WHO). Recommendations for prevention and treatment of preeclampsia and eclampsia. Geneva: WHO Department of Maternal and Child Health; 2011. Available from: http://whqlibdoc.who.int/publications/2011/ 9789241548335_eng.pdf. [last accessed 15 Nov 2013]. 4. Osungbade OK, Ige OK. Public heath perspectives of preeclampsia in developing countries: implication for health system strengthening. J Pregnancy 2011;11: 481–95. 5. Direkvand-Moghadam A, Khosravi A, Sayehmiri K. Predictive factors for preeclampsia in pregnant women: aunvariate and multivariate logistic regression analysis. Acta Biochim Pol 2012;59:673–7. 6. Meera KS, Maitra S, Hemalatha R. Increased level of lipid peroxidation in preeclamptic pregnancy; a relationship with paraoxanase 1(PON1) activity. Biomed Res 2010;21:393–6. 7. Granger JP, Alexander BT, Bennett WA, Khalil RA. Pathophysiology of pregnancyinduced hypertension. Am J Hypertens 2001;14:178S–85S. 8. Llurba E, Grataco E, Martin-Gallan P, et al. A comprehensive study of oxidative stress and antioxidant status in preeclampsia and normal pregnancy. Free Radical Biol Med 2004;37:557–0. 9. Torun M, Yardım S, Gonenc A, Sargın H. Cesitli kanser vakalarında serum MDA duzeyleri. Turkish J Biochem 1995;20:1–7. 10. Nahar Z, Sarwar MS, Islam MS, et al. Determination of serum antioxidant vitamins, glutathione and MDA levels in panic disorder patients. ArzneimittelForsch 2013;63:424–8. 11. Satoh K. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin Chim Acta 1978;90:37–43. 12. Lowry H, Jeanne A, Lopez JA, Otto A. The determination of ascorbic acid in small amounts of blood serum. J Biol Chem 1945;162:609–15. 13. Ramsay JE, Jamieson N, Greer IA, Sattar N. Paradoxical elevation in adiponectin concentrations in women with preeclampsia. Hypertens 2003;42:891–4. 14. Howlader MZH, Parveen S, Tamanna S, et al. Oxidative stress and antioxidant status in neonates born to pre-eclamptic mother. J Tropical Ped 2009;55:363–7. 15. Fatemeh T, Marziyeh G, Nayereh G, et al. Maternal and perinatal outcome in nulliparious women complicated with pregnancy hypertension. J Pak Med Assoc 2010;60:707–10. 16. Dhananjay BS, Dayananda G, Sendilkumaran D, Murthy N. A study of factors affecting perinatal mortality in eclampsia. J Physiol Biomed Sci 2009;22:2–5. 17. Agida ET, Adeka BI, Jibril KA. Pregnancy outcome in eclamptics at the University of Abuja Teaching Hospital, Gwagwalada, Abuja: a 3 year review. Niger J Clin Pract 2010;13:394–8. 18. Dlamini NJ. Factors associated with pre-eclampsia and quality care of affected teenagers during labour within health region in Kwa-Zulu Natal. Curationis 1997; 20:16–22. 19. Azimi C, Lotfi M. Association of socioeconomic status with health and birth outcomes: maternal variables (Part 2). Am J Sci Res 2012;51:5–15.
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20. Vasiljevic N, Vasiljevic M, Plecas D. The role of nutritional factors in preeclampsia and eclampsia. Srp Arh Celok Lek 1996;124:156–9. 21. Roberts JM, Taylor RM, Goldfein A. Clinical and biochemical evidence of endothelial cells dysfunction in the pregnancy syndrome preeclampsia. Am J Hypertens 1991;4:700–8. 22. Jonas O, Roder D, Chan A. The association of maternal and socioeconomic characteristics in metropolitan Adelaide with medical, obstetric and labour complications and pregnancy outcomes. Aust Nz J Obstet Gyn 1992;32:1–5. 23. Wang Y, Walsh SW, Gu J, Zhang J. The imbalance between thromboxane and prostacyclin in preeclampsia is associated with an imbalance between lipid peroxides and vitamin E in maternal blood. Am J Obstet Gynecol 1991;165: 1695–700. 24. Roberts JM, Balk JL, Bodner LM, et al. Nutrient involvement in preeclampsia. J Nutr 2003;133:1684–92. 25. Atamer Y, Kocyigit Y, Yokus B, et al. Lipid peroxidation, antioxidant defense, status of trace metals and leptin levels in preeclampsia. Eur J Obstetric Gynecol Reprod Biol 2005;119:60–6. 26. Gohil JT, Patel PK, Gupta P. Evaluation of oxidative stress and antioxidant defence in subjects of preeclampsia. J Obstet Gynecol India 2011;61:638–40. 27. Suhail M, Suhail S, Gupta BK, Bharat V. Malondialdehyde and antioxidant enzymes in maternal and cord blood and their correlation in normotensive and preeclamptic women. J Clin Med Res 2011;1:150–5. 28. Kaur G, Mishra S, Sehgal A, Prasad R. Alterations in lipid peroxidation and antioxidant status in pregnancy with preeclampsia. Mol Cell Biochem 2008;313: 37–44. 29. Sharma JB, Sharma A, Bahadur A, Vimala N. Oxidative stress markers and antioxidant levels in normal pregnancy and pre-eclampsia. Int J Gynecol Obstet 2006;94:23–7.
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ORIGINAL ARTICLE
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Effect of socio-economic status and estimation of lipid peroxidation and antioxidant in preeclamptic pregnant women: a case–control study Md. Shahid Sarwar,1 Rakhesh Chandra Sarkar,2 Rumpa Bhowmick,2 Syed Masudur Rahman Dewan,2 Maizbha Uddin Ahmed,3 Abul Hasnat,3 Mamunur Rashid,1 and Mohammad Safiqul Islam2,4 1
Department of Pharmacy, Southeast University, Banani, Dhaka, Bangladesh, Department of Pharmacy, Noakhali Science and Technology University, Sonapur, Noakhali, Bangladesh, 3 Department of Clinical Pharmacy and Pharmacology, Faculty of Pharmacy, University of Dhaka, Dhaka, Bangladesh, and 4 Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Shimane, Japan 2
Objective: Increased extent of lipid peroxidation and decreased antioxidant defense has been postulated as a major contributor to the development of preeclampsia. The present study was conducted to determine the serum level of malondialdehyde (MDA) and vitamin C in these patients. Methods: This research was conducted as a case–control study to assess the extent of lipid peroxidation and antioxidant status in 150 preeclampsia patients and 174 healthy pregnant women. Lipid peroxidation was assessed by measuring serum MDA level, and antioxidant defense was evaluated for serum vitamin C by UV spectrophotometric method. Independent sample t test and Pearson’s correlation test were used for the statistical analysis with statistical software package SPSS, version 16.0 (SPSS, Inc., Chicago, IL). Results: Our study found significantly higher level of MDA (p50.05) and significantly lower level of vitamin C (p50.05) in preeclampsia patients in comparison to the control subjects. Pearson’s correlation analysis revealed that there was negative correlation between the serum level of MDA and vitamin C for both patient (r = 0.057, p = 0.669) and control groups (r = 0.128, p = 0.487). Analysis of socio-economic data revealed that preeclampsia was more prevalent among the rural pregnant than the urban pregnant women (OR 1.44, 95% CI 0.665–3.14, p = 0.352). It was also observed that primigravida pregnant were at higher risk for the development of preeclampsia than the multigravida pregnant women (OR 1.87, 95% CI 0.868–4.04, p = 0.108). Conclusions: Elevated serum MDA and depleted serum vitamin C may be associated with the development of preeclampsia. Keywords Malondialdehyde, Preeclampsia, Pregnant women, Socio-economic status, Vitamin C
Correspondence: Dr. Mohammad Safiqul Islam, Associate Professor, Department of Pharmacy, Noakhali Science and Technology University, Sonapur, Noakhali-3814, Bangladesh. Tel: +88-0321-71483. Fax: +88-0321-62788. E-mail: research_safiq @yahoo.com.
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INTRODUCTION Preeclampsia is a multisystem, multifactor disorder of pregnancy with elevated blood pressure (140/90 mmHg), proteinuria (300 mg/day) and pathological edema; usually occurring on or after 20 week of gestation (1). Its incidence is approximately 2–8% throughout the world which causes 16% of maternal death during pregnancy in Bangladesh (2). Preeclampsia is highly prevalent in first pregnancy, and its rate reduces extensively in subsequent pregnancies. It is the most common medical complication of pregnancy whose incidence has continued to increase worldwide and is associated with significant maternal morbidity and mortality, accounting for about 50 000 deaths per year throughout the world (3). Thus reducing maternal mortality by 75% between 1990 and 2015 has been considered as part of the millennium development goals of the World Health Organization (4). The prevalence of preeclampsia differs on the basis of geographic location but the most common contributing factors are maternal age below 20 or over 35 years, nulliparity, multiple gestation, twins, black race, low socio-economic status, hydatidiform mole, polyhydramnios, non-immune fetal hydrops obesity, diabetes, chronic hypertension and underlying renal disease (5). Although the exact pathophysiology of preeclampsia is unknown but it is suggested that increased extent of lipid peroxidation and decreased antioxidant profile serve as a contributing factor to preeclampsia (6). Several studies stated that placental perfusion triggers the activation and dysfunction of the maternal vascular endothelium, which leads to increased production of endothelin and thromboxane, and reduced production of vasodilators such as nitric oxide and prostacyclin (7,8). Generally, free radicals mediate lipid peroxidation within the cellular membrane which ultimately causes tissue damage (9). Free radicals generation leading to lipid peroxidation is a usual phenomenon in pregnancy which occurs with low intensity in normal pregnancy, because in normal pregnant women the extent of lipid peroxidation and the formation of antioxidants are well balanced. If this balance of oxidant and antioxidant system is interrupted, it may cause oxidative stress (1). Malondialdehyde (MDA) – the end product of lipid peroxidation – can be used as a marker of tissue damage resulting from oxidative stress (10). Antioxidant molecules act as body’s defense system to combat free radicalmediated oxidative damage. Several enzymatic (glutathione peroxidase, superoxide dismutase and catalase) and non-enzymatic (vitamin A, C, E and glutathione) antioxidants are available in human body which scavenge these free radicals, thus act as protection system against various diseases. Considering the aforementioned facts, the present study was designed to elucidate the possible relation of lipid peroxidation and antioxidant level with the pathogenesis of preeclampsia.
MATERIALS AND METHODS Materials The reagents used in the present study were collected from commercially available company. 2-Thiobarbituric acid (TBA), 2,4-dinitrophenylhydrazine (DNPH) and n-butyl alcohol and sulfuric acid (97%) were purchased from
Lipid peroxidation and antioxidant in preeclampsia
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Merck, Germany. Ascorbic acid was supplied by Globe Pharmaceuticals, Noakhali, Bangladesh, as a gift sample. Trichloroacetic acid (TCA) and hydrochloric acid (37%) were procured from Guangdong, China. The commercially available 1,1,3,3-tetraethoxypropane was purchased from SigmaAldrich, Germany, whereas metaphosphoric acid and thiourea were obtained from Loba Chemie, India. Methods Study Design and Blood Sample Collection This case–control study comprising 150 preeclamptic pregnant women as cases and 174 healthy pregnant women as controls was carried out in the Department of Obstetrics and Gynecology, Noakhali Medical College Hospital, Bangladesh. The study protocol was approved by the ethical review committee of the hospital. Preeclampsia was characterized by the presence of a maternal blood pressure above 140/90 mmHg after 20 weeks of gestation. A specialist obstetrician and gynecologist conducted the diagnosis of preeclampsia. Both the patients and controls were briefed about the objective of this study, and written consent was obtained from each of them. Prior to their inclusion in the study, detailed history was taken from patients and controls using a pre-designed questionnaire. Individuals with diabetes mellitus or other endocrine disorder, renal, liver or cardiovascular disease were excluded from the study. Patients who were unwilling to involve in the research and denied to share their clinical data were also excluded. Blood samples were collected from the antecubital vein of each patient and control in a metal-free sterile tube using a plastic syringe (JMI-Bangla Co. Ltd, disposable syringe, 5 mL) after overnight fasting condition. All blood samples were then kept at room temperature for about 30 min to clot and centrifuged at 2218 g for 15 min to extract the serum. The extracted serum was collected in Eppendorf tube and stored at 80 C for future analysis. Analysis of MDA and vitamin C were performed within few days after serum collection. Determination of Serum MDA Lipid peroxidation was assessed by measuring serum MDA level following the modified method of Satoh (11). According to this method, 0.5 mL of serum was mixed with 2.5 mL of 20 mg/dL trichloroacetic acid in a centrifuge tube and allowed to stand for 10 min at room temperature. After centrifugation of the mixture at 2218 g for 10 min, the supernatant was decanted, and the precipitate was washed with 0.05 M sulphuric acid. Then the precipitate was mixed with 2.5 mL of 0.05 M sulfuric acid and 3.0 ml of 0.2 mg/dL TBA (in 2 M sodium sulfate) which was heated in a boiling water bath for 30 min. This solution was cooled in ice cold water and extracted with 4.0 mL of n-butyl alcohol by vigorous shaking. The organic phase was separated by centrifugation at 2218 g for 10 min, and absorbance of the supernatant was measured spectrophotometrically (UV–VIS Spectrophotometer, Shimadzu, Japan) at 530 nm against a blank that contained all reagents except the serum sample. Serum MDA concentration was expressed as nmol/mL.
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Determination of Serum Vitamin C Serum vitamin C was determined by phenyl-hydrazine spectrophotometry method (12). The concentration of ascorbic acid in the serum was calculated as follows: mmol Absorbance of sample mg Concentration, C ¼ 56:78 Absorbance of standard dl L
Statistical Analysis All values were expressed as mean ± standard error mean (mean ± SEM). Statistical analysis was performed using the statistical software package SPSS, version 16.0 (SPSS Inc., Chicago, IL). Independent sample t test was done to determine the level of significance for various parameters between patient and control groups. Pearson’s correlation analysis was used to find the correlation among the study parameters.
RESULTS Socio-Demographic Profile Our study comprised a total of 324 samples with 150 preeclampsia patients and 174 healthy pregnant women. We observed that there was no significant difference for maternal age (p40.05), but statistically significant difference was found for gestational age between patients and control groups (p50.05) as represented in Figures 1 and 2, respectively. Both the systolic and diastolic blood pressure (SBP and DBP, respectively) were significantly higher in preeclampsia patients (p50.05) when compared with control group (Figure 3).
26
Maternal age (year)
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The 56.78 is conversion factor for ascorbic acid for standard international unit.
24
22
20 0 Patient
Control
Group Figure 1. Comparison of maternal age between patient and control groups.
Lipid peroxidation and antioxidant in preeclampsia
Pregnant women who were in their first pregnancy (primigravida) were of at particular risk for the development of preeclampsia (OR 1.87, 95% CI 0.868– 4.04, p = 0.108) than the multigravida pregnant women (Table 1). The comparative percentage of the primigravida and multigravida pregnancy was 62 and 38% versus 46.6 and 53.4% between patient and control groups, respectively. In this study, it was observed that pregnant women from the rural 40
* Gestational age (week)
30
25
20 0 Patient
Control
Group Figure 2. Comparison of gestational age between patient and control groups. Asterisk indicates statistically significant difference in comparison to control group (p50.05).
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Blood pressure (mm Hg)
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35
*
SBP
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* DBP
90
45
0 Paent
Control Group
Figure 3. Comparison of SBP and DBP between patient and control groups. Asterisk indicates statistically significant difference in comparison to control group (p50.05).
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M. S. Sarwar et al. Table 1. Socio-demographic profile of the study population. Value (%)
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Variables Gravidity Primigravida Multigravida Area of residence Rural Urban Occupation Housewife Service Income level Low Medium High Educational status Illiterate Primary Secondary Higher secondary Higher studies
Preeclamptic pregnant
Normal pregnant
p Value
93 (62) 57 (38)
81 (46.6) 93 (53.4)
0.108 (OR 1.87)
96 (64) 54 (36)
96 (55.2) 78 (44.8)
0.352 (OR 1.44)
138 (92) 12 (8)
153 (87.9) 21 (12.1)
0.486 (OR 1.58)
102 (68) 42 (28) 6 (4)
102 (58.62) 54 (31.03) 18 (10.34)
48 54 33 6 9
(32) (36) (22) (4) (6)
60 45 42 12 15
(34.5) (25.90) (24.1) (6.9) (8.6)
–
–
OR: Odd ratio.
area had higher prevalence for preeclampsia than those from the urban area (OR 1.44, 95% CI 0.665–3.14, p = 0.352). The relative percentages of the pregnant women from the rural and urban area were 64 and 36% versus 55.2 and 44.8% in patient and control groups, respectively. It was found that majority of them were of lower (68 versus 10.25%) economic class women followed by medium (28 versus 58.62) and high (4 versus 31.03%) economic class. This study explored that most of the patients were housewives in both patient and control groups (92 versus 87.9%), and the remaining small populations were of service holder (8 versus 12.1%) (OR 1.58, 95% CI 0.434– 5.74, p = 0.486). Educational status studies of the preeclamptic pregnant women explored that 32% of them were illiterate, while 36, 22, 4, and 6% continues their study up to primary, secondary, higher secondary and graduate or post-graduate level, respectively, whereas in the control group these were 34.5, 25.90, 24.1, 6.9 and 8.6%, respectively.
Lipid Peroxidation and Antioxidant Status Serum level of MDA was found 6.66 ± 0.16 and 5.01 ± 0.21 nmol/mL, whereas the level of vitamin C was 17.75 ± 1.09 and 25.26 ± 1.65mmol/L in patient and control groups, respectively (Table 2). Statistical analysis reveals that the level of MDA was significantly higher in preeclamptic pregnant women (p50.05), whereas significantly lower level of vitamin C was found in this group (p50.05).
Lipid peroxidation and antioxidant in preeclampsia Table 2. Serum level of MDA and Vitamin C in the study population. Values (Mean ± SEM) Parameters MDA (nmol/mL) Vitamin C (mmol/L)
Preeclamptic pregnant
Normal pregnant
p Value
6.66 ± 0.16 17.75 ± 1.09
5.01 ± 0.21 25.26 ± 1.65
p50.05** p50.05**
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**p50.05 (significant difference between patient and control groups at 95% confidence interval).
Table 3. Correlation of socio-demographic characteristics with serum MDA and vitamin C in the patient and control groups. Preeclamptic pregnant Correlation parameters Maternal age and SBP Maternal age and DBP Maternal age and MDA Maternal age and vitamin C Gestational age and SBP Gestational age and DBP Gestational age and MDA Gestational age and vitamin C MDA and SBP MDA and DBP MDA and vitamin C Vitamin C and SBP Vitamin C and DBP
Normal pregnant
r
p
r
p
0.399** 0.151 0.062 0.169 0.127 0.042 0.170 0.182 0.010 0.144 0.057 0.055 0.023
0.004 0.296 0.667 0.240 0.380 0.773 0.237 0.206 0.943 0.320 0.669 0.707 0.874
0.309** 0.240 0.028 0.031 0.188 0.147 0.188 0.054 0.045 0.132 0.128 0.122 0.043
0.018 0.069 0.837 0.820 0.377 0.272 0.158 0.689 0.740 0.322 0.487 0.362 0.751
r correlation co-efficient; p significance. Values with negative sign indicate an inverse correlation. **p50.05; correlation is significant at 0.05 level (two-tailed).
Correlation Analysis In the present study, the research interest focused on the potential role of lipid peroxidation in the pathophysiology of preeclampsia. For this reason, a wide range of correlation was established to the effect of maternal age, gestational age, SBP and DBP on serum level of MDA and vitamin C in both patient and control groups (Table 3). This study found no significant correlation between serum MDA or vitamin C level with gestational age, SBP and DBP (p40.05), but statistically significant correlation was found between maternal age and SBP in both patient and control groups (p50.05). There was a negative correlation between the serum level of MDA and vitamin C in both patient group (r = 0.057) and control group (r = 0.128).
DISCUSSION Preeclampsia is considered as one of the three leading complications of pregnancy that may affect both mother and fetus. Circulating level of several
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chemicals may greatly contribute to the development of preeclampsia. Although the principle objective of this study was to determine several biochemical parameters that may have an association with the pathogenesis of preeclampsia, but we also analyze the socio-demographic features of the patients and controls to find out the relationship of these parameters with the disease process. All these data could help in improving the maternal and perinatal morbidity and mortality in preeclampsia. Our study found no significant difference for maternal age between the patient and control groups (p40.05), but the gestational age was significantly lower in patient group than the control group (p50.05), which confirms previous studies (13–15). The relationship between gravidity of pregnancy and its complications continues as an interesting research to the obstetricians. Gravidity has been used as a risk marker of preeclampsia with primigravida pregnant, and women who had previous preeclampsia history are identified as higher risk factor of preeclampsia. Primigravida pregnant, i.e. women who were pregnant for the first time are considered to be at risk of preeclampsia, whereas the multigravida pregnant are considered to be at risk for hemorrhage, malpresentation, anemia, uterine rupture and complications associated with chronic medical problems such as diabetes and hypertension (2). In this study, it was found that primigravida pregnancies were at higher risk of developing preeclampsia than women in subsequent pregnancies or multigravida pregnancies which was also reported by other studies (16,17). The results of this study indicate that women from the rural community have more toxemias in pregnancy than those from urban community which is consistent with previous study (18). This may be due to the lack of awareness regarding the antenatal care, insufficient healthcare facilities or lack of qualified physicians in the rural community of Bangladesh. But another study found higher incidence of preeclampsia among the pregnant women from urban area in another country (19). It was also observed that preeclampsia is more common among the lower socioeconomic class women possibly due to their poor life style and dietary habit. Nutrients deficiency may act as a contributing factor to preeclampsia development as these substances can modulate oxidative stress by increasing or decreasing free radicals and antioxidants and/or by supplying substrate for the production of free radicals (20,21). Jonas et al. (22) estimated the associations of socio-economic status (SES) with medical problems and pregnancy outcomes, and their results indicate that low SES is related to a higher risk profile for the development of preeclampsia. Interpretation of the extent of lipid peroxidation and antioxidant status is the relationship of cause and effect in preeclampsia. It is well established that free radicals have a tendency to cause lipid peroxidation. Markers of lipid peroxidation (MDA) are increased during several pregnancy complications. Generally, there is a physiological balance between the lipid peroxidation and antioxidative processes in normal pregnancies (23). Alteration of normal physiological balance in pregnancy causes modification of polyunsaturated fatty acids (PUFA) leading to altered function and structure of capillary endothelial cells (24). Thus, the endothelial cell injury or damage may be the initiator of the pathophysiological events of the preeclampsia. In the present study, it was observed that lipid peroxidation product (MDA) was significantly
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Lipid peroxidation and antioxidant in preeclampsia
increased in preeclampsia when compared with control (p50.05) which signifies the excessive ongoing lipid peroxidation in preeclampsia. This result correlates with numerous studies undertaken by other researchers (25,26). Suhail et al. (27) reported that preeclamptic placenta contains higher MDA than those from normal pregnancies which may contribute to the development of preeclampsia. The increase in extent of lipid peroxidation might not be so important if there is a compensatory increase in antioxidant protection. Several exogenic and endogenic antioxidants act as defense system against the free radicalmediated oxidative damage. Normally, they prevent lipid peroxidation by inactivating free radicals and quenching activated singlet oxygen molecules and superoxide radicals (28). This study showed that the level of antioxidant vitamin C was significantly depleted in preeclampsia patients when compared with the controls (p50.05). The outcome of increasing or decreasing concentration of a single component in human body is not limited to that component only, but the level of other biological components may be affected. Thus, correlation analysis may provide the information whether the level of one parameter affect the level of other parameter either positively or negatively. In this study, Pearson’s correlation analysis confirmed that there was a negative correlation between the serum level of MDA and vitamin C in both patient and control groups. This may be due to the fact that large extent of vitamin C may be utilized to counteract lipid peroxidation of cellular components, hence resulting in the depletion of this antioxidant level (29). Moreover, endothelial dysfunction resulting from high-lipid peroxidation state leads to enhanced endothelial damage in preeclampsia patients, whereas antioxidant vitamins try to neutralize the negative impact of MDA on endothelial cell which subsequently results in depletion of antioxidant vitamins. The findings of the present study reveal that socioeconomic factors have considerable effect on the pathogenesis of preeclampsia possibly due to their life style and dietary habit. Primigravida pregnant had higher risk for the development of preeclampsia than the multigravida pregnant, and they should be offered pre-pregnancy counseling to explain this risk and to encourage early attendance for specialist antenatal care. The present study found significantly elevated level of MDA and depleted level of vitamin C in preeclampsia patients than the healthy pregnant women, which supports the hypothesis that lipid peroxidation is a causative factor in the pathogenesis of preeclampsia. Antioxidant supplementation may be recommended to scavenge the free radical action of MDA, which may be useful as secondary therapy to prevent the oxidative damage in placental tissue of preeclampsia patients.
ACKNOWLEDGEMENTS The authors would staffs, nurses, and Gynecology, Noakhali port during the study. this study.
like to express their heartfelt thanks to all the physicians of the Department of Obstetrics and Medical College Hospital, Bangladesh, for their supThe authors are also thankful to all the participants of
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DECLARATION OF INTEREST This study was financially supported by University Grant Commission (UGC), Bangladesh.
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