Influence of Smoking on Maternal and Neonatal Serum Malondialdehyde, Superoxide Dismutase, and Glutathione Peroxidase Levels

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1 Annals of Clinical & Laboratory Science, vol. 34, no. 4, Influence of Smoking on Maternal and Neonatal Serum Malondialdehyde, Superoxide Dismutase, and Glutathione Peroxidase Levels Bahri Ermis, 1 Rahmi Ors, 2 Abdulkadir Yildirim, 3 Ayhan Tastekin, 2 Fatih Kardas, 2 and Fatih Akcay 3 1 Department of Pediatrics, School of Medicine, Karaelmas University, Zonguldak,Turkey; 2 Department of Pediatrics and 3 Department of Biochemistry, School of Medicine, Ataturk University, Erzurum, Turkey Abstract. This cohort study investigated postnatal serum malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) levels in 14 active-smoking, 14 passive-smoking, and 15 nonsmoking mothers and their newborns on day 7 post-partum. No significant differences were noted among the study groups with respect to MDA (p = 0.63) or SOD levels (p = 0.98) in either the mothers or their infants. However, there were significant differences among the study groups with respect to serum GPx activities in both the mothers (p = 0.028) and the infants (p = 0.039). When GPx activities were analyzed separately in both mothers and infants, a significant difference was noted only between the infants of smoking mothers and the infants of non-smoking mothers (p = 0.015). In conclusion, there was a significant increase in GPx activities of smoking mothers and their infants, suggesting that they may have been exposed to more oxidant stress. Key words: smoking, pregnancy, newborns, malondialdehyde, glutathione peroxidase, superoxide dysmutase Introduction Oxidative stress has been reported to play a role in some physiological and pathological conditions, including pregnancy [1] and its complications [2]. In normal pregnancy, oxidative stress and lipid peroxidation are increased; on the other hand, antioxidant protection is also increased [3]. Cigarette smoking has been suggested as a source of oxidant stress in pregnant women and their infants exposed in utero. It has been demonstrated that antioxidant vitamin concentrations are lower in smokers than in non-smokers, which could be due either to an inadequate diet or an inadequate intake of antioxidants in smokers [4]. The deficiency of antioxidant vitamins may also promote oxidative stress. Address correspondence to Bahri Ermis, M.D., Department of Pediatrics, School of Medicine, Zonguldak Karaelmas University, Kozlu/Zonguldak, Turkey; tel ; fax ; bahriermis@yahoo.com. In the English literature, there is only one study of plasma antioxidant vitamin status of mothers and their newborns at delivery and in the immediate neonatal period in relation to maternal smoking [5]. Serum superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, which are the important components of the antioxidant systems in the body, have not been studied until now. The aim of this study was to evaluate the levels of serum malondialdehyde (MDA), SOD, and GPx in mothers and their infants in relation to maternal smoking during pregnancy. Materials and Methods Subjects. Forty-three mothers (age yr) and their infants (7 days old) were enrolled in the study. This study was approved by the local Ethics Committee. Mothers were informed and their consent was obtained before enrollment. We attempted to recruit as many smoking mothers as /04/ $ by the Association of Clinical Scientists, Inc. 405_

2 406 Annals of Clinical & Laboratory Science, vol. 34, no. 4, 2004 possible when the infants were brought for routine physical examination at 7 days old. Passive-smoking and non-smoking mothers and their infants were recruited randomly during the same period. Mothers were interviewed to obtain their smoking histories, including the number of cigarettes smoked per day and the number of years of smoking. Detailed histories of pregnancy and delivery were obtained from birth records. Urinary nicotine levels were not tested to confirm the smoking status of the mothers. Based on interviews and birth records, 43 mothers and their infants met the eligibility criteria and were classified into 1 of 3 groups: (a) the activesmoking group (n = 14) in which both the mother (5-10 cigarettes/day in pregnancy) and father (20 cigarettes/day) had been smoking up to date; (b) the passive-smoking group (n = 14) in which only the father (20 cigarettes/day) had been smoking in the house; and (c) the non-smoking group (n = 15) in which the parents never smoked either before or during pregnancy. Inclusion criteria. The following criteria were used to accept the mothers and their infants into the study: no maternal chronic illness, no alcohol consumption, no gestational problems (eg, hypertension, preeclampsia, diabetes mellitus, or infection), no congenital anomalies, no neonatal complications (asphyxia, meconium aspiration, etc), no intrauterine growth retardation, no caesarean section, no jaundice requiring phototherapy, no formula feeding, birth weight >2500 g, and gestational age >37 weeks. Biochemical assays. Venous blood samples were collected in Vacutainer tubes without any additive and were centrifuged at 3500 x g (5 min, 4 C). Serum aliquots were immediately stored at -80 C until the biochemical analyses were performed. All chemicals and reagents were obtained from Sigma Chemical Co (St Louis, MO, USA). Spectrophotometric measurements were performed using a model DU 530 spectrophotometer (Beckman Instruments, Brea, CA, USA). Malondialdehyde (MDA) assay. MDA, a product of lipid peroxidation, was determined spectrophotometrically by a method similar to that described by Jain et al [6]. Briefly, 0.2 ml of serum was mixed thoroughly with 0.8 ml of phosphate buffered saline (ph 7.4) and ml of butylated hydroxytoluene solution (8.8 g/l). After addition of 0.5 ml of 30% trichloroacetic acid, the samples were placed on ice for 2 hr and then centrifuged at 2000 x g for 15 min. One ml of supernatant was mixed with ml of 0.1 M EDTA and 0.25 ml of 1% thiobarbituric acid in 0.05 N NaOH. The samples were placed in boiling water for 15 min, cooled to room temperature, and the absorbance was determined at 532 nm. Total thiobarbutiric acid reactive substances were expressed as MDA, using molar extinction coefficient for MDA of 1.56 x 10 5 cm -1 M -1. The results were expressed as µmol/l. Superoxide dismutase (SOD) assay. CuZnsuperoxide dismutase (SOD) activity was detected by the method of Sun et al [7]. Exactly 2.45 ml of assay reagent (containing 0.3 mm xanthine, 0.6 mm Na 2 EDTA, 0.15 mm nitroblue tetrazolium (NBT), 0.4 M Na 2 CO 3, 1 g/l bovine serum albumin) was combined with 100 µl of serum sample. Xanthine oxidase (50 µl, 167 U/L) was added to initiate the reaction and the reduction of NBT by superoxide anion radicals, which are produced by the xanthinexanthine oxidase system, was determined by measuring the absorbance at 560 nm. CuZn-SOD activity was expressed in units of SOD per liter, where 1 U is defined as that amount of enzyme causing half-maximal inhibition of NBT reduction. Glutathione peroxidase (GPx) assay. GPx activity was measured by the method of Paglia and Valentine [8]. To a 0.5 ml serum sample, an equal volume of double strength Drabkin s reagent was added and mixed. This mixture (50 µl) was combined with 100 µl of 8 mm NADPH, 100 ml of 150 mm glutathione (reduced form), 20 ml of glutathione reductase (30 U/ml), 20 µl of 0.12 M sodium azide solution, and 2.65 ml of 50 mm potassium phosphate buffer (ph 7.0, 5 mm EDTA) and the tubes incubated for 30 min at 37 C. The reaction was initiated with the addition of 100 µl of 2 mm H 2 O 2 solution, mixed rapidly by inversion, and the conversion of NADPH to NADP was measured 405_

3 Influence of smoking on oxidant/antioxidant status 407 spectrophotometrically for 5 min at 340 nm. The enzyme activity was expressed as U/L. Statistical analysis. All data were entered into an SPSS database (SPSS Inc, Chicago, IL, USA). Nonparametric tests were used due to the small number of the groups. The significance of the differences among the 3 groups was evaluated by Kruskal-Wallis one-way analysis. When the p value was <0.05, it was considered a statistically significant difference among the 3 groups. For comparisons between 2 groups, the Mann-Whitney U test was used, and p values smaller than (<0.05/3) were considered to show a significant difference. The chisquare (χ 2 ) test was also used where appropriate. Results The characteristics of the mothers and their infants are shown in Table 1. No significant difference was noted among the 3 groups in regard to maternal age, maternal weight, primigravida, education, birth weight, or gestational age. Table 2 shows serum levels of MDA, SOD, and GPx in the mothers. There was no significant difference in MDA values among the 3 groups (p = 0.63). Serum SOD activities did not show any significant difference among the study groups (p = 0.98). There was significant difference among the study groups in GPx activities (p = 0.028). When GPx values were analyzed separately by the Mann- Table 1. Maternal and neonatal characteristics of the study groups (mean±sd or %). Smoking Passive-smoking Non-smoking group p value group (n = 14) group (n = 14) group (n = 15) among groups Maternal age (yr) 26.2± ± ± Maternal weight (kg) 66.5± ±3.8 66± Maternal education (>11 yr, %) 14% 14% 20% 0.95 Primigravida (%) 50% 50% 53% 0.94 Birth weight (g) 3165± ± ± Gestational age (wk) 39.5± ± ± Table 2: Serum levels of MDA, SOD, and GPx in mothers (mean±sd). Smoking Passive-smoking Non-smoking group p value group (n = 14) group (n = 14) group (n = 15) among groups MDA (µmol/l) 4.09± ± ± SOD (U/ml) 3.31± ± ± GPx (u/l) a 128.3± ± ± a Smoking vs passive-smoking (p = 0.023); smoking vs non-smoking (p = 0.019); passive-smoking vs non-smoking (p = 0.85). Table 3: Serum levels of MDA, SOD, and GPx in infants (mean±sd). Infants of smoking Infants of passive- Infants of non-smoking p value group (n = 14) smoking group (n = 14) group (n = 15) among groups MDA (µmol/l) 2.66± ±0.72± 2.61± SOD (U/ml) 5.1± ± ± GPx (U/L) a 446.4±60.4 a 390.2± ± a Infants of smoking vs non-smoking (p = 0.015), smoking vs passive-smoking (p = 0.075), passive-smoking vs non-smoking groups (p = 0.48). 405_

4 408 Annals of Clinical & Laboratory Science, vol. 34, no. 4, 2004 Whitney U test, significant differences were detected between study groups [smoking vs passive-smoking groups (p = 0.023), smoking vs non-smoking groups (p = 0.019), but not between the passive smoking vs non-smoking groups (p = 0.85)]. Table 3 shows serum values of MDA, SOD, and GPx in infants. There was no significant difference among the study groups in regard to serum MDA levels (p = 0.96). No significant difference was detected in SOD levels among the study groups (p = 0.73). However, significant differences were found in serum GPx activities among the study groups (p = 0.039). When the GPx values were analyzed separately by Mann-Whitney U test, no significant differences were noted between the infants of smoking vs passive-smoking groups (p = 0.075), or the passive-smoking vs non-smoking groups (p = 0.48), but the smoking vs non-smoking groups showed a significant difference (p = 0.015). Discussion The antioxidative defense system has been reported to be impaired in adult smokers compared to nonsmokers. Some studies revealed that serum SOD and GPx activities are lower in smokers than in nonsmokers [9,10]. In contrast to these findings, some investigators have not found any change in the tissue oxidant defense system [11,12], whereas others have reported increased activities of these antioxidant systems [13,14]. Lipid peroxidation, as measured by production of thiobarbituric acid reactive substances (TBARS) or MDA in blood, was also found to be increased in adult smokers [10,15]. Pregnancy is a physiological state accompanied by high metabolic demand and elevated requirements for tissue oxygen, resulting in over-production of reactive oxygen species and lipid peroxidation [3]. The antioxidative defense system is modified during pregnancy to limit oxidative stress [3]. In our study, we investigated serum levels of MDA, SOD, and GPx in active-smoking, passivesmoking and non-smoking mothers and their newborns. Because the process of labor itself may be associated with production of oxygen free radicals, we evaluated antioxidant status at 7 days after delivery. We did not find any significant differences among the study groups with respect to MDA and SOD levels in either mothers or infants. However, serum mean SOD activities seem to be lower in infants in the smoking group than in the others, suggesting an oxidative stress. On the other hand, there were significant differences among the study groups with respect to serum GPx activities in the mothers (p = 0.028) and the infants (p = 0.039). When GPx activities were analyzed separately in both mothers and infants, a significant difference was noted only between the infants of smoking vs non-smoking groups (p = 0.015). There are a few studies suggesting that cigarette smoking is a source of oxidant stress in pregnant women and their newborns. Smoking even a single cigarette may cause transient uteroplacental vasoconstriction [3]. Placental glutathione uptake is decreased in pregnant smokers, resulting in reduction of fetal glutathione levels [3]. Schwarz et al [16] showed that the breath ethane test, which is an indicator of oxidant stress, is increased in both pregnant smokers and in infants of smoking mothers compared to non-smoking controls. Vitamin E levels are lower in tissues of smokers than in non-smokers. Infants of smoking mothers have low vitamin E levels, consistent with neonatal oxidative stress [5]. In mothers and newborns, the most detailed study of plasma MDA and antioxidant vitamin status was carried out by Bolisetty et al [5]. In that study, although the mean plasma MDA levels were higher at delivery than in the immediate postnatal period in both mothers and infants, no significant differences were observed between smoking and non-smoking groups with respect to MDA, as confirmed by our study. The increased levels of MDA seen at delivery were attributed to increased oxidative stress during labor and delivery. They found that plasma vitamin E levels were lower in smokers than in non-smokers in both mothers and newborns. These results suggest an increased oxidative stress in the smoking group, resulting in increased consumption of vitamin E. In contrast to the Bolisetty study [5], we assayed serum activities of SOD and GPx, which are major components of the body s antioxidant defense system. Sajjad et al [17] compared the cord blood activities of SOD and GPx between spontaneous 405_

5 Influence of smoking on oxidant/antioxidant status 409 vaginal delivery and cesarean section cases, and they found increased GPx and decreased SOD activities in normal vaginal deliveries compared to cesarean sections. They claimed that the fetus responds to the oxidative stress by increasing its GPx activity. Also, increased utilization of SOD may explain the low values of SOD in normal vaginal deliveries compared to cesarean sections. Initially, these results may appear inconsistent. However, GPx is responsible for removing lipoperoxidation products, which are formed over a longer period, whereas SOD is responsible for removing oxygen free radicals which occur over a short period of time [17]. GPx limits the accumulation of lipid peroxides and utilizes glutathione as its cofactor to convert lipid peroxides into less harmful substances. In conclusion, there was significant increase in serum GPx activities of smoking mothers and their infants, suggesting that they may have been exposed to more oxidant stress than non-smoking mothers and their infants. References 1. Rogers MS, Mongelli JM, Tsang KH, Wang CC, Law KP. Lipid peroxidation in cord blood at birth: the effect of labour. Br J Obstet Gynaecol 1998;105: Poranen AK, Ekbald U, Uotila P, Ahoptupa M. Lipid peroxidation and antioxidants in normal and preeclamptic pregnancies. Placenta 1996;17: Gitto E, Reiter RJ, Karbownik M. Causes of oxidative stress in the pre-and perinatal period. Biol Neonate 2002; 81: Ortega RM, Lopez-Sobaler AM, Martinez RM, Andres P, Quintas ME. Influence of smoking on vitamin E status during the third trimester and on breast-milk tocopherol concentrations in Spanish women. Am J Clin Nutr 1998; 68: Bolisetty S, Naidoo D, Lui K, et al. Postnatal changes in maternal and neonatal plasma antioxidant vitamins and the influence of smoking. Arch Dis Child Fetal Neonatal Ed 2002;86:F Jain SK, McVie R, Duett J, Herbst JJ. Erythrocyte membrane lipid peroxidation and glycosylated hemoglobin in diabetes. Diabetes 1989;38: Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988;34: Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70: Hulea SA, Olinescu R, Nita S, Crocnan D, Kummerow FA. Cigarette smoking causes biochemical changes in blood that are suggestive of oxidative stress: a case-control study. J Environ Pathol Toxicol Oncol 1995;14: Kim SH, Kim JS, Shin HS, Keen CL. Influence of smoking on markers of oxidative stress and serum mineral concentrations in teenage girls in Korea. Nutrition 2003; 19: Wurzel H, Yeh CC, Gairola C, Chow CK. Oxidative damage and antioxidant status in the lungs and bronchoalveolar lavage fluid of rats exposed chronically to cigarette smoke. J Biochem Toxicol 1995;10: Gupta MP, Khanduja KL, Sharma RR. Effect of cigarette smoke inhalation on antioxidant enzymes and lipid peroxidation in the rat. Toxicol Lett 1998; 41: Abou-Seif MA. Blood antioxidant status and urine sulfate and thiocyanate levels in smokers. J Biochem Toxicol 1996;11: Hilbert J, Mohsenin V. Adaptation of lung antioxidants to cigarette smoking in humans. Chest 1996;110: Durak I, Elgun S, Bingol NK. Effects of cigarette smoking with different tar content on erythrocyte oxidant/ antioxidant status. Addict Biol 2002;7: Schwarz KB, Cox JM, Sharma S, et al. Prooxidant effects of maternal smoking and formula in newborn infants. J Pediatr Gastroenterol Nutr 1997;24: Sajjad Y, Leonard M, Doyle M. Antioxidant levels in the cord blood of term fetus. J Obstet Gynaecol 2000;20: _