http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, 2015; 28(5): 544–547 ! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2014.924102
ORIGINAL ARTICLE
Oxidative stress and antioxidant status of pregnant rural women in north-west Nigeria: prospective cohort study Emmanuel Ugwa1, Mohammed Gwarzo2, and Adewale Ashimi1 1
Department of Obstetrics and Gynecology, Federal Medical Centre, Birnin Kudu, Jigawa State, Nigeria and 2Department of Chemical Pathology, Aminu Kano Teaching Hospital, Kano State, Nigeria Abstract
Keywords
Background/Objective: This research was undertaken to determine the oxidative stress and antioxidant status of pregnant northern Nigerian women. Methods: Prospective cohort study. The study was done at General Hospital, Dawakin Kudu LGA, Kano. Kano has a population of 9.2 million and predominantly Muslims. Two hundred consecutive women presenting for antenatal care and 100 apparently normal controls. Ethical approval was obtained. Socio-demographic and clinical information were obtained via interviewer-administered questionnaires from 200 consecutive pregnant women who presented for antenatal care. Serum levels of vitamins A, C, E and malondialdehyde were determined. Data was analyzed with SPSS version 16.0 statistical software. Descriptive statistics was used. Means were compared using analysis of variance (ANOVA). Associations between trimesters and oxidative stress/antioxidant status were tested using chi-square test and p50.05 was considered statistically significant. Result: The mean serum vitamin A, E and C levels were 13.39 ± 9.44 mg/dl, 0.35 ± 0.51 mg/dl and 5.99 ± 3.95 m/dl respectively (below normal limits). The prevalence of vitamins A, C and E deficiency were 65.5%, 79.5% and 51% respectively. The mean serum levels of malondialdehyde was 4.04 ± 0.91 nmol/l (pregnant) and 1.84 ± 0.40 nmol/l (non-pregnant) (p ¼ 0.001). There was strong negative correlation between serum levels of malondialdehyde and vitamins A, C and E. Conclusion: Vitamins A, C, E deficiency and oxidative stress is a problem among Northern Nigeria pregnant women, therefore the value of antioxidant vitamin supplementation should be explored.
Antioxidant, Nigeria, oxidative stress, pregnant, prospective cohort
Introduction Pregnancy is associated with progressive and periodic changes in metabolic and physiological status of the woman. Therefore remarkable and dramatic events occur during this period for sustaining mother and fostering the growth and maintenance of fetus [1]. Pregnancy though not a disease is associated with a high-energy demand of many bodily functions and an increased oxygen requirement [2]. During pregnancy, minute ventilation increases by 36% at the 8th week of pregnancy [3]. Pregnancy also has an effect on maternal antioxidant enzyme activities. Glutathione peroxidase and superoxide dismutase activities have been found to be reduced during the second trimester of pregnancy in humans [4]. Such conditions may be responsible for raised oxidative stress in pregnancy. Malondialdehyde (MDA) is a decomposition product of peroxidized polyunsaturated fatty acids [5,6],
Address for correspondence: Emmanuel A. Ugwa, Department of Obstetrics and Gynecology, Federal Medical Centre, Birnin Kudu, Jigawa State, Nigeria. Tel: +234 8035851872. E-mail: [email protected]
History Received 31 December 2013 Accepted 11 May 2014 Published online 5 June 2014
and is widely used in evaluating the degree of oxidative stress in various pathological conditions[7]. The production of malondialdehye and other aldehydes is used as a biomarker to measure the level of oxidative stress in an organism [8,9]. Antioxidants are substances generated endogenously or taken as exogenous compounds with the ability to block the initiation of free radical formation or scavenge the free radical formed [10–12]. Examples of such compounds with antioxidant properties are vitamins A, C and E, sulhydryl-containing compounds such as cysteine and glutathione and serum proteins, such as albumin, ceruloplasmin and transferrin and enzyme system such as superoxide dismutase. Vitamin A is a hydrophilic molecule that can scavenge several radicals, among them the hydroxyl radical is of importance [13,14]. Vitamin C also affects the overall antioxidant status of the body. Food rich in vitamin C such as strawberries and spinach are as effective in enhancing the overall antioxidant status in serum as a large dose of vitamin C [15,16]. The main function of vitamin E is as a chain-breaking, free-radical trapping antioxidant present in cell membranes and plasma lipoproteins [17]. The other relevant antioxidant systems include superoxide dismutase, albumin, glutathione and selenium.
DOI: 10.3109/14767058.2014.924102
Studies have shown that malondialdehyde levels were higher in pregnant mothers as compared to age matched nonpregnant women and at the same time antioxidant levels were found to be significantly low due to higher antioxidant requirement and intake [18], metabolic demand [19], hemodilution [20] or a combination of these. These observations call for need to determine the degree of oxidative stress and antioxidant cascade in pregnancy in our community. This present study was conducted to assess the status of oxidative stress in rural northern Nigerian pregnant women by blood levels of vitamins with antioxidant activity and lipid peroxidation product (malondialdehyde).
Materials and methods Ethical approval for the research was obtained from General Hospital, Dawakin Kudu, Kano, where the research was conducted. This was a prospective study that was done between 31 November 2009 and 30 March 2011. It involved 216 consecutive pregnant women who presented for antenatal care at the maternity section of the hospital. Those who cannot be followed-up, those who had hypertensive diseases, diabetes mellitus and other illnesses with known effect on antioxidant status were excluded. Maternal and obstetrics information was retrieved with interviewer administered questionnaire. The ages, parities and gestational ages of the women were collated. Biochemical analysis was done on serum specimens collected from these women to determine their oxidative stress and antioxidant status. One hundred apparently healthy nonpregnant subjects were used as control. Sample size was determined using under-nutrition prevalence among women of 10–40% from a previous similar study [21]. n ¼ z2 p q=d2 (n ¼ Sample Size, z ¼ Standard Normal Deviation ¼ 1.96 at 95% Confidence Limit, p ¼ Prevalence Rate ¼ 15%, q ¼ 1p ¼ 115% ¼ 0.85, d Error Margin ¼ 5%) 2
1:96 0:15 0:85 n¼ ¼ 196 0:052 Ten percent of the minimum sample size was added to the minimum sample size in anticipation of drop-out. Therefore the sample size was 216. Ten milliliters (10 ml) of whole blood sample was collected and drawn directly into a plain blood sample container. Blood was obtained from venipuncture after applying a tourniquet. The tourniquet was removed as soon as blood began to flow into the tube so as to minimize the risk of haemolysis. The blood samples were transported carefully in ice pack. Centrifugation at 2500 to 3000 rpm for 5 minutes was done to obtain plasma. They were labeled and covered with aluminum foil. Storage was in a deep freezer. Biochemical analysis was done using the methods of Bessey et al. [22] for serum vitamin A; Roe & Kuether [23] for vitamin C; Tsen [24] for vitamin E; Guiltride & Wilkins [25] and Ohakawa et al. [26] for malondialdehyde. The data obtained was analyzed using SPSS version 16.0 statistical software (Chicago, IL). Absolute numbers and simple percentages were used to describe categorical
Value of vitamin A, C, E supplementation in pregnancy
545
variables. Similarly, quantitative variables were described using measures of central tendency (mean, median) and measures of dispersion (range, standard deviation) as appropriate. Statistical significance of differences between means was determined using ANOVA. Significant association between trimesters of pregnancy and oxidative stress/antioxidant status were tested using chi-square test and p50.05 was considered significant.
Results This was a prospective study that was done between 31 November 2009 and 30 March 2011. Two-hundred and fortysix (216) pregnant women were recruited for the study. At the end of the study period 200 (92.6%) of the women were found to be evaluable. The others did not present for follow up. Table 1 shows that the mean age of the subjects were 23.67 ± 6.11 years for the pregnant and 23.22 ± 7.99 years for the non-pregnant. The mean parities were 3 ± 3 and 3 ± 2, respectively. The age and parity difference between the two groups were not statistically significant (p40.05). As shown in Table 2, 65.5% had serum vitamin A levels below normal of 15 mg/dl, while 34.5% had normal levels of 15–6 mg/dl. Similarly, for vitamin C, 79.5% had levels below normal of 0.5 mg/dl and 20.5% had normal values of 0.5– 2.0 mg/dl. For vitamin E also, 51% of the subjects had below normal level, while 49% had normal level. The mean serum levels of MDA, vitamins A, C and E were 4.04 ± 0.91 nmol/ dl, 13.39 ± 9.44 mg/dl, 0.35 ± 0.52 mg/dl and 5.99 ± 3.95 mg/dl respectively. There was a strong negative correlation between the serum levels of MDA and vitamin A (Pearson’s Coefficient of Correlation, r ¼ 0.75) and vitamin E (Pearson’s Coefficient of Correlation, r ¼ 0.71). As MDA Table 1. Socio-demographic and obstetrics information. Variables Parity 0 1–4 5 Age 520 20–39 40
Pregnant
Non-pregnant
p value
3±3
3±2
0.076
23.67 ± 6.11
23.22 ± 7.99
0.078
Table 2. Distribution ofserum antioxidant vitamins levels among pregnant women. Variable Vitamin A 515 mg/dl 15–60 mg/dl 460 mg/dl Vitamin C 50.5 mg/dl 0.5–2.0 mg/dl 42.0 mg/dl Vitamin E 55 mg/dl 5–20 mg/dl 420 mg/dl
Frequency
Percentage
Mean ± SD
131 69 0
65.5 34.5 0
13.39 ± 9.44
159 41 0
79.5 20.5 0
0.35 ± 0.51
102 98 0
51 49 0
5.99 ± 3.95
546
E. Ugwa et al.
J Matern Fetal Neonatal Med, 2015; 28(5): 544–547
Table 3. Distribution of mean serum levels of malondialdehyde and antioxidant vitamins at different trimesters.
Trimester
Malondialdehyde (nmol/L)
First Second Third
Vitamin A (mg/dl)
2.63 ± 0.42 4.04 ± 0.93 5.25 ± 1.25
Table 4. Distribution non-pregnant women. Malondialdehyde (nmol/l)
of
Vitamin E (mg/dl)
19.54 ± 9.97 0.20 ± 0.18 10.27 ± 5.05 11.94 ± 9.80 0.50 ± 0.99 5.23 ± 3.45 8.71 ± 8.55 0.35 ± 0.36 2.49 ± 3.35
MDA
Frequency
Pregnant (n ¼ 200) 1–2.99 25 3–4.99 50 5–6.99 125 Non-pregnant (n ¼ 100) 1–2.99 90 3–4.99 10 5–6.99 0
Vitamin C (mg/dl)
levels
between
pregnant
and
Percentage
Mean ± SD
p value
12.5 25 62.5
4.04 ± 0.91
0.001
90 10 0
1.84 ± 0.40
levels rise, levels of vitamins A and E fall. The association is however non-linear between MDA and vitamin C levels. As shown in Table 3, the values for vitamin A were 19.54 ± 9.97 mg/dl during the first trimester, 11.94 ± 9.80 in the second trimester and 8.71 ± 8.55 mg/dl in the third trimester. The values for vitamin C were 0.20 ± 0.18 mg/dl during the first trimester, 0.50 ± 0.99 in the second trimester and 0.35 ± 0.36 mg/dl in the third trimester. For vitamin E, the serum levels were 10.27 ± 5.05 mg/dl in the first trimester, 5.23 ± 3.45 mg/dl in the second trimester and 2.49 ± 3.35 mg/dl in the third trimester. Table 4 shows that the serum MDA level among pregnant women (4.04 ± 0.91 nmol/dl) was higher than that of nonpregnant (1.84 ± 0.40 nmol/dl). This difference was statistically significant (p ¼ 0.001).
Discussion This present study showed low serum levels of vitamins A, C and E among pregnant women. A poor dietary intake of antioxidant nutrients or a low body level is becoming important because recent evidences suggest that maternal oxidative stress during pregnancy plays an important role in the pathophysiology of low birth weight [27,28]. The birth weight of the babies has been shown by this study to be below average for developing regions of the world, a probable consequence of maternal macro- and micronutrient deficiency in keeping with previous research findings [27,28]. In particular, maternal nutrition during pregnancy has been regarded as an important determinant for fetal growth [29]. In this study there was a significant reduction in serum vitamins A, C and E concentration throughout the period of pregnancy. This agrees with previous studies [30–32]. For vitamins A and E, this reduction worsened as the pregnancy advanced reaching lowest levels in the third trimester. However with vitamin C, the lowest levels were in the first trimester with a significant difference when compared with
levels in the second trimester being highest. This agrees with finding in previous research [31]. In the recent past, it has been shown that there is a significantly higher oxidative stress in pregnant women compared to non-pregnant control [33]. In this study also we found a corresponding increase in the serum levels of malondialdehyde with gestation and this agrees also with previous studies [32,34]. The increased utilization of these antioxidant vitamins to neutralize the toxicity of the resulting reactive oxygen species consequent to increased oxidative stress can account for the low serum concentration of these vitamins. Vitamin C is also involved in maintaining the tocopherol cycle, necessary for the peroxyl radical scavenging action of vitamin E in the biological lipid phase [35]. The plasma concentration of MDA was high among majority of the women in the study population. Higher MDA levels than non-pregnant control has been reported in pregnancy complicated by pregnancy induced hypertension [22]. This has been reported to be due to increased generation of oxygen radicals following increased oxygen demand during pregnancy [36], reduction in the activities of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase [28], and reduction in plasma vitamin C and vitamin E concentration during pregnancy [37,38]. This study also showed a strong negative correlation between the levels of MDA and vitamins A, C and E. This is also in support of an earlier study where a correlation between serum levels of malondialdehyde and vitamin E was established [33], but it went further to determine a similar correlation between serum levels of malondialdehyde and vitamins A and C. Ingestion of vitamin C during pregnancy was observed to reduce the generation of MDA [31]. Vitamin C is a primary water-soluble antioxidant in plasma and within cells, but it can also interact with the plasma membrane by donating electrons to the a-tocopheroxyl radical and a transplasma membrane oxidoreductase activity. Vitamin C supplementation is particularly important in pregnant women as its deficiency has been shown to affect placental structure and facilitates placental infection both of which results in increased risk of premature rupture of placental membranes and premature births [39]. In addition the supplementation could help prevent the development of such complications of pregnancy like gestational hypertension, intrauterine growth retardation and gestational diabetes, all known to be associated with high levels of oxidative stress, in addition to the numerous other benefits of this vitamin in human metabolism and health [40]. Generally, antioxidant vitamin supplementation in pregnancy has been recommended by some studies [30]. Vitamins A, C and E deficiency are prevalent among rural Northern Nigerian women. In view of the reduction in the concentration of these antioxidant vitamins with corresponding increase in the level of malondialdehyde, there is need to incorporate nutritional advocacy of food that contain not only antioxidant vitamins, but also those that can induce the upregulation of the endogenous antioxidants in the body in order to eliminate or reduce the adverse consequences of pregnancy and its outcome associated with undernutrition. The value of Vitamin A, C and E supplementation should be explored in future research.
DOI: 10.3109/14767058.2014.924102
Declaration of interest The authors have no conflict of interests. This research was funded by the authors.
Author contribution (1) Ugwa EA, research design, data collection, analysis and main body of research. (2) Gwarzo MY, data collection and proof-reading. (3) Ashimi A, research design and proof-reading.
Ethical approval Ethical approval was obtained from Ethical Committee of General Hospital, Dawakin Kudu Local Government Area, Kano State, Nigeria.
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16. Van Den Broek NR, Letsky AE. Aetiology of anaemia in pregnancy in South Malawi. Am J Clin Nutr 2000;72:247S–56S. 17. Herrera E, Barbas C. Vitamin E: action, metabolism and perspective. J Physiol Biochem 2000;57:43–56. 18. Alberti-Fidanza A, Di Renzo GC, Burini G, et al. Diet during pregnancy and total antioxidant capacity in maternal and umbilical cord blood. J Matern Fetal Neonatal Med 2002;12:59–63. 19. Soni ND, Sharma P, Kiran C, et al. Leucocyte and serum ascrobic acid levels in mothers and their newborn in semi arid Western India. Indian J Physiol Pharmac 1989;33:239–42. 20. Pritchard JA. Changes in the blood volume during pregnancy and delivery. Anesthesiology 1965;26:393–9. 21. Ene-Obong HN, Enugu GI, Uwaegbute AC. Determinants of health and nutritional status of rural Nigerian women. J Health Popul Nutr 2001;19:320–30. 22. Bessey OA, Lowry OH, Brock MJ, Lopez JA. The determination of vitamin a and carotene in small quantities of blood serum. J Biol Chem 1946;166:77–8. 23. Roe JH, Kuether CA. The determination of ascorbic acid in whole blood and urine through the 2,4-dinitrophenylhydrazine derivative of dehydroascorbic acid. J Biol Chem 1943;147:399–400. 24. Tsen CC. An improved spectrophotometric method for determination of tocopherols using 4,7-diphenyl-1,10-phenanthroline. Anal Chem 1961;33:849–51. 25. Guilteridge JMC, Wilkins S. Copper-dependent hydroxyl radical damage to ascorbic acid: formation of a thiobarbituric acid-reactive product. FEBS Lett 1982;137:327–30. 26. Ohakawa H, Oshishi N, Yagi K. Assay for lipid peroxidation in animal tissue by thiobarbituric acid reaction. Anal Biochem 1979; 75:351–8. 27. Scholl TO, Stein TP. Oxidant damage to dna and pregnancy outcome. Matern Fetal Med 2001;10:182–5. 28. Wang Y, Walsh SW. Increased superoxide generation is associated with decreased superoxide dismutase activity and mRNA expression in placental trophoblast cells in pre-eclampsia. Placenta 2001; 22:206–12. 29. Godfrey K, Barker DJ, Robinson S, Osmond C. Maternal birthweight and diet in pregnancy in relation to the infant’s thinness at birth. Br J Obstet Gynaecol 1997;104:663–7. 30. Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in female reproduction. Reprod Biol Endocrinol 2005;3:28–48. 31. Garba G, Ubom D, Gatsing R, et al. L-ascorbic acid status of pregnant women and its potential role in pregnancy-induced stress. Internet J Nutr Wellness 2005;1:2. 32. Mohanty PK, Mandal PC, Panda A. Evaluation of oxidative stress in pregnancy-induced hypertension. Indian J Clin Biochem 2006; 21:101–5. 33. Updadhyaya C, Mishra S, Singh PP, Sharma P. Antioxidant status and peroxidative stress in mother and newborn – a pilot study. Indian J Clin Biochem 2005;20:30–4. 34. Sies H, Stahl W. Vitamins E and C, beta-carotene, and other carotinoids as antioxidants. Am J Clin Nutr 1995;62:1315–21. 35. Patil SB, Kodliwadmath MV, Kodliwadmath SM. Lipid peroxidation and nonenzymatic antioxidants in normal pregnancy. J Obstet Gynecol India 2006;56:399–401. 36. Halliwell B, Gutteridge JMC. The antioxidants of human extracellular fluids. Arch Biochem Biophys 1990;280:1–8. 37. Kumer CA, Des WW. Oxidant stress in pre-eclampsia and essential hypertension. J Assoc Physicians India 2002;50:1372–5. 38. Kharb S. Vitamin E and C in preeclampsia. Eur J Obstet Gynecol Reprod Biol 2000;93:37–9. 39. Casanueva E, Viteri, FE. Iron and oxidative stress in pregnancy. J Nutr 2003;133:1700S–8S. 40. Rumbold AC, Crowther CA. Vitamin C supplementation in pregnancy. The Cochrane Database Syst Rev 2005;1:CD004072.
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ORIGINAL ARTICLE
Oxidative stress and antioxidant status of pregnant rural women in north-west Nigeria: prospective cohort study Emmanuel Ugwa1, Mohammed Gwarzo2, and Adewale Ashimi1 1
Department of Obstetrics and Gynecology, Federal Medical Centre, Birnin Kudu, Jigawa State, Nigeria and 2Department of Chemical Pathology, Aminu Kano Teaching Hospital, Kano State, Nigeria Abstract
Keywords
Background/Objective: This research was undertaken to determine the oxidative stress and antioxidant status of pregnant northern Nigerian women. Methods: Prospective cohort study. The study was done at General Hospital, Dawakin Kudu LGA, Kano. Kano has a population of 9.2 million and predominantly Muslims. Two hundred consecutive women presenting for antenatal care and 100 apparently normal controls. Ethical approval was obtained. Socio-demographic and clinical information were obtained via interviewer-administered questionnaires from 200 consecutive pregnant women who presented for antenatal care. Serum levels of vitamins A, C, E and malondialdehyde were determined. Data was analyzed with SPSS version 16.0 statistical software. Descriptive statistics was used. Means were compared using analysis of variance (ANOVA). Associations between trimesters and oxidative stress/antioxidant status were tested using chi-square test and p50.05 was considered statistically significant. Result: The mean serum vitamin A, E and C levels were 13.39 ± 9.44 mg/dl, 0.35 ± 0.51 mg/dl and 5.99 ± 3.95 m/dl respectively (below normal limits). The prevalence of vitamins A, C and E deficiency were 65.5%, 79.5% and 51% respectively. The mean serum levels of malondialdehyde was 4.04 ± 0.91 nmol/l (pregnant) and 1.84 ± 0.40 nmol/l (non-pregnant) (p ¼ 0.001). There was strong negative correlation between serum levels of malondialdehyde and vitamins A, C and E. Conclusion: Vitamins A, C, E deficiency and oxidative stress is a problem among Northern Nigeria pregnant women, therefore the value of antioxidant vitamin supplementation should be explored.
Antioxidant, Nigeria, oxidative stress, pregnant, prospective cohort
Introduction Pregnancy is associated with progressive and periodic changes in metabolic and physiological status of the woman. Therefore remarkable and dramatic events occur during this period for sustaining mother and fostering the growth and maintenance of fetus [1]. Pregnancy though not a disease is associated with a high-energy demand of many bodily functions and an increased oxygen requirement [2]. During pregnancy, minute ventilation increases by 36% at the 8th week of pregnancy [3]. Pregnancy also has an effect on maternal antioxidant enzyme activities. Glutathione peroxidase and superoxide dismutase activities have been found to be reduced during the second trimester of pregnancy in humans [4]. Such conditions may be responsible for raised oxidative stress in pregnancy. Malondialdehyde (MDA) is a decomposition product of peroxidized polyunsaturated fatty acids [5,6],
Address for correspondence: Emmanuel A. Ugwa, Department of Obstetrics and Gynecology, Federal Medical Centre, Birnin Kudu, Jigawa State, Nigeria. Tel: +234 8035851872. E-mail: [email protected]
History Received 31 December 2013 Accepted 11 May 2014 Published online 5 June 2014
and is widely used in evaluating the degree of oxidative stress in various pathological conditions[7]. The production of malondialdehye and other aldehydes is used as a biomarker to measure the level of oxidative stress in an organism [8,9]. Antioxidants are substances generated endogenously or taken as exogenous compounds with the ability to block the initiation of free radical formation or scavenge the free radical formed [10–12]. Examples of such compounds with antioxidant properties are vitamins A, C and E, sulhydryl-containing compounds such as cysteine and glutathione and serum proteins, such as albumin, ceruloplasmin and transferrin and enzyme system such as superoxide dismutase. Vitamin A is a hydrophilic molecule that can scavenge several radicals, among them the hydroxyl radical is of importance [13,14]. Vitamin C also affects the overall antioxidant status of the body. Food rich in vitamin C such as strawberries and spinach are as effective in enhancing the overall antioxidant status in serum as a large dose of vitamin C [15,16]. The main function of vitamin E is as a chain-breaking, free-radical trapping antioxidant present in cell membranes and plasma lipoproteins [17]. The other relevant antioxidant systems include superoxide dismutase, albumin, glutathione and selenium.
DOI: 10.3109/14767058.2014.924102
Studies have shown that malondialdehyde levels were higher in pregnant mothers as compared to age matched nonpregnant women and at the same time antioxidant levels were found to be significantly low due to higher antioxidant requirement and intake [18], metabolic demand [19], hemodilution [20] or a combination of these. These observations call for need to determine the degree of oxidative stress and antioxidant cascade in pregnancy in our community. This present study was conducted to assess the status of oxidative stress in rural northern Nigerian pregnant women by blood levels of vitamins with antioxidant activity and lipid peroxidation product (malondialdehyde).
Materials and methods Ethical approval for the research was obtained from General Hospital, Dawakin Kudu, Kano, where the research was conducted. This was a prospective study that was done between 31 November 2009 and 30 March 2011. It involved 216 consecutive pregnant women who presented for antenatal care at the maternity section of the hospital. Those who cannot be followed-up, those who had hypertensive diseases, diabetes mellitus and other illnesses with known effect on antioxidant status were excluded. Maternal and obstetrics information was retrieved with interviewer administered questionnaire. The ages, parities and gestational ages of the women were collated. Biochemical analysis was done on serum specimens collected from these women to determine their oxidative stress and antioxidant status. One hundred apparently healthy nonpregnant subjects were used as control. Sample size was determined using under-nutrition prevalence among women of 10–40% from a previous similar study [21]. n ¼ z2 p q=d2 (n ¼ Sample Size, z ¼ Standard Normal Deviation ¼ 1.96 at 95% Confidence Limit, p ¼ Prevalence Rate ¼ 15%, q ¼ 1p ¼ 115% ¼ 0.85, d Error Margin ¼ 5%) 2
1:96 0:15 0:85 n¼ ¼ 196 0:052 Ten percent of the minimum sample size was added to the minimum sample size in anticipation of drop-out. Therefore the sample size was 216. Ten milliliters (10 ml) of whole blood sample was collected and drawn directly into a plain blood sample container. Blood was obtained from venipuncture after applying a tourniquet. The tourniquet was removed as soon as blood began to flow into the tube so as to minimize the risk of haemolysis. The blood samples were transported carefully in ice pack. Centrifugation at 2500 to 3000 rpm for 5 minutes was done to obtain plasma. They were labeled and covered with aluminum foil. Storage was in a deep freezer. Biochemical analysis was done using the methods of Bessey et al. [22] for serum vitamin A; Roe & Kuether [23] for vitamin C; Tsen [24] for vitamin E; Guiltride & Wilkins [25] and Ohakawa et al. [26] for malondialdehyde. The data obtained was analyzed using SPSS version 16.0 statistical software (Chicago, IL). Absolute numbers and simple percentages were used to describe categorical
Value of vitamin A, C, E supplementation in pregnancy
545
variables. Similarly, quantitative variables were described using measures of central tendency (mean, median) and measures of dispersion (range, standard deviation) as appropriate. Statistical significance of differences between means was determined using ANOVA. Significant association between trimesters of pregnancy and oxidative stress/antioxidant status were tested using chi-square test and p50.05 was considered significant.
Results This was a prospective study that was done between 31 November 2009 and 30 March 2011. Two-hundred and fortysix (216) pregnant women were recruited for the study. At the end of the study period 200 (92.6%) of the women were found to be evaluable. The others did not present for follow up. Table 1 shows that the mean age of the subjects were 23.67 ± 6.11 years for the pregnant and 23.22 ± 7.99 years for the non-pregnant. The mean parities were 3 ± 3 and 3 ± 2, respectively. The age and parity difference between the two groups were not statistically significant (p40.05). As shown in Table 2, 65.5% had serum vitamin A levels below normal of 15 mg/dl, while 34.5% had normal levels of 15–6 mg/dl. Similarly, for vitamin C, 79.5% had levels below normal of 0.5 mg/dl and 20.5% had normal values of 0.5– 2.0 mg/dl. For vitamin E also, 51% of the subjects had below normal level, while 49% had normal level. The mean serum levels of MDA, vitamins A, C and E were 4.04 ± 0.91 nmol/ dl, 13.39 ± 9.44 mg/dl, 0.35 ± 0.52 mg/dl and 5.99 ± 3.95 mg/dl respectively. There was a strong negative correlation between the serum levels of MDA and vitamin A (Pearson’s Coefficient of Correlation, r ¼ 0.75) and vitamin E (Pearson’s Coefficient of Correlation, r ¼ 0.71). As MDA Table 1. Socio-demographic and obstetrics information. Variables Parity 0 1–4 5 Age 520 20–39 40
Pregnant
Non-pregnant
p value
3±3
3±2
0.076
23.67 ± 6.11
23.22 ± 7.99
0.078
Table 2. Distribution ofserum antioxidant vitamins levels among pregnant women. Variable Vitamin A 515 mg/dl 15–60 mg/dl 460 mg/dl Vitamin C 50.5 mg/dl 0.5–2.0 mg/dl 42.0 mg/dl Vitamin E 55 mg/dl 5–20 mg/dl 420 mg/dl
Frequency
Percentage
Mean ± SD
131 69 0
65.5 34.5 0
13.39 ± 9.44
159 41 0
79.5 20.5 0
0.35 ± 0.51
102 98 0
51 49 0
5.99 ± 3.95
546
E. Ugwa et al.
J Matern Fetal Neonatal Med, 2015; 28(5): 544–547
Table 3. Distribution of mean serum levels of malondialdehyde and antioxidant vitamins at different trimesters.
Trimester
Malondialdehyde (nmol/L)
First Second Third
Vitamin A (mg/dl)
2.63 ± 0.42 4.04 ± 0.93 5.25 ± 1.25
Table 4. Distribution non-pregnant women. Malondialdehyde (nmol/l)
of
Vitamin E (mg/dl)
19.54 ± 9.97 0.20 ± 0.18 10.27 ± 5.05 11.94 ± 9.80 0.50 ± 0.99 5.23 ± 3.45 8.71 ± 8.55 0.35 ± 0.36 2.49 ± 3.35
MDA
Frequency
Pregnant (n ¼ 200) 1–2.99 25 3–4.99 50 5–6.99 125 Non-pregnant (n ¼ 100) 1–2.99 90 3–4.99 10 5–6.99 0
Vitamin C (mg/dl)
levels
between
pregnant
and
Percentage
Mean ± SD
p value
12.5 25 62.5
4.04 ± 0.91
0.001
90 10 0
1.84 ± 0.40
levels rise, levels of vitamins A and E fall. The association is however non-linear between MDA and vitamin C levels. As shown in Table 3, the values for vitamin A were 19.54 ± 9.97 mg/dl during the first trimester, 11.94 ± 9.80 in the second trimester and 8.71 ± 8.55 mg/dl in the third trimester. The values for vitamin C were 0.20 ± 0.18 mg/dl during the first trimester, 0.50 ± 0.99 in the second trimester and 0.35 ± 0.36 mg/dl in the third trimester. For vitamin E, the serum levels were 10.27 ± 5.05 mg/dl in the first trimester, 5.23 ± 3.45 mg/dl in the second trimester and 2.49 ± 3.35 mg/dl in the third trimester. Table 4 shows that the serum MDA level among pregnant women (4.04 ± 0.91 nmol/dl) was higher than that of nonpregnant (1.84 ± 0.40 nmol/dl). This difference was statistically significant (p ¼ 0.001).
Discussion This present study showed low serum levels of vitamins A, C and E among pregnant women. A poor dietary intake of antioxidant nutrients or a low body level is becoming important because recent evidences suggest that maternal oxidative stress during pregnancy plays an important role in the pathophysiology of low birth weight [27,28]. The birth weight of the babies has been shown by this study to be below average for developing regions of the world, a probable consequence of maternal macro- and micronutrient deficiency in keeping with previous research findings [27,28]. In particular, maternal nutrition during pregnancy has been regarded as an important determinant for fetal growth [29]. In this study there was a significant reduction in serum vitamins A, C and E concentration throughout the period of pregnancy. This agrees with previous studies [30–32]. For vitamins A and E, this reduction worsened as the pregnancy advanced reaching lowest levels in the third trimester. However with vitamin C, the lowest levels were in the first trimester with a significant difference when compared with
levels in the second trimester being highest. This agrees with finding in previous research [31]. In the recent past, it has been shown that there is a significantly higher oxidative stress in pregnant women compared to non-pregnant control [33]. In this study also we found a corresponding increase in the serum levels of malondialdehyde with gestation and this agrees also with previous studies [32,34]. The increased utilization of these antioxidant vitamins to neutralize the toxicity of the resulting reactive oxygen species consequent to increased oxidative stress can account for the low serum concentration of these vitamins. Vitamin C is also involved in maintaining the tocopherol cycle, necessary for the peroxyl radical scavenging action of vitamin E in the biological lipid phase [35]. The plasma concentration of MDA was high among majority of the women in the study population. Higher MDA levels than non-pregnant control has been reported in pregnancy complicated by pregnancy induced hypertension [22]. This has been reported to be due to increased generation of oxygen radicals following increased oxygen demand during pregnancy [36], reduction in the activities of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase [28], and reduction in plasma vitamin C and vitamin E concentration during pregnancy [37,38]. This study also showed a strong negative correlation between the levels of MDA and vitamins A, C and E. This is also in support of an earlier study where a correlation between serum levels of malondialdehyde and vitamin E was established [33], but it went further to determine a similar correlation between serum levels of malondialdehyde and vitamins A and C. Ingestion of vitamin C during pregnancy was observed to reduce the generation of MDA [31]. Vitamin C is a primary water-soluble antioxidant in plasma and within cells, but it can also interact with the plasma membrane by donating electrons to the a-tocopheroxyl radical and a transplasma membrane oxidoreductase activity. Vitamin C supplementation is particularly important in pregnant women as its deficiency has been shown to affect placental structure and facilitates placental infection both of which results in increased risk of premature rupture of placental membranes and premature births [39]. In addition the supplementation could help prevent the development of such complications of pregnancy like gestational hypertension, intrauterine growth retardation and gestational diabetes, all known to be associated with high levels of oxidative stress, in addition to the numerous other benefits of this vitamin in human metabolism and health [40]. Generally, antioxidant vitamin supplementation in pregnancy has been recommended by some studies [30]. Vitamins A, C and E deficiency are prevalent among rural Northern Nigerian women. In view of the reduction in the concentration of these antioxidant vitamins with corresponding increase in the level of malondialdehyde, there is need to incorporate nutritional advocacy of food that contain not only antioxidant vitamins, but also those that can induce the upregulation of the endogenous antioxidants in the body in order to eliminate or reduce the adverse consequences of pregnancy and its outcome associated with undernutrition. The value of Vitamin A, C and E supplementation should be explored in future research.
DOI: 10.3109/14767058.2014.924102
Declaration of interest The authors have no conflict of interests. This research was funded by the authors.
Author contribution (1) Ugwa EA, research design, data collection, analysis and main body of research. (2) Gwarzo MY, data collection and proof-reading. (3) Ashimi A, research design and proof-reading.
Ethical approval Ethical approval was obtained from Ethical Committee of General Hospital, Dawakin Kudu Local Government Area, Kano State, Nigeria.
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