The effect of the mode of delivery on the maternal–neonatal erythrocyte membrane acetylcholinesterase activity

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Clinical Biochemistry 41 (2008) 818 – 823

The effect of the mode of delivery on the maternal–neonatal erythrocyte membrane acetylcholinesterase activity Dimitrios G. Vlachos a , Kleopatra H. Schulpis b , Theodore Parthimos a , Spyros Mesogitis c , George D. Vlachos c , George A. Partsinevelos c , Aris Antsaklis c , Stylianos Tsakiris a,⁎ a b

Department of Physiology, Medical School, Athens University, P.O. Box 65257, Athens 15401, Greece Institute of Child Health, Research Centre, “Aghia Sophia” Children's Hospital, Athens 11527, Greece c 1st Department of Obstetrics and Gynecology, Medical School, Athens University, Greece Received 10 January 2008; received in revised form 24 March 2008; accepted 6 April 2008 Available online 18 April 2008

Abstract Free radical production and high catecholamine levels are implicated with the modulation of acetylcholinesterase (AChE) activity. Objective: To investigate the effect of the mode of delivery on maternal–neonatal erythrocyte membrane AChE activity. Subjects and methods: Some women with normal pregnancy (N = 30) were divided into two groups: group A (N = 16) with normal labour and vaginal delivery and group B (N = 14) with scheduled Cesarean section, twenty non-pregnant women were the controls. Blood was obtained from controls and from mothers pre- vs post-delivery as well as from the umbilical cord (CB). Total antioxidant status (TAS), membrane AChE activities and catecholamine blood levels were measured with a commercial kit, spectrophotometrically and HPLC methods, respectively. Results: TAS and catecholamine levels as well as membrane AChE activities were similar in the two groups of mothers pre-delivery and in controls. TAS levels were reduced whereas AChE activities and catecholamine levels were increased post-delivery in mothers of group A and unaltered in group B at the same times of study. AChE activity was similarly lower in the CB of neonates than those of their mothers pre-delivery. Conclusions: During a normal delivery process, the low TAS, the increased levels of catecholamines and the increased AChE activity, postdelivery, may be due to the increased stress due to the participation of uterus and skeletal muscles as during endurance exercise. The low AChE activity in newborns may be related to perinatal immaturity. © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Catecholamines; Delivery; Newborn; Acetylcholinesterase; Erythrocyte membranes

Introduction Prolonged submaximal exercise has been shown to induce oxidative stress [1]. During prolonged aerobic exercise the increase in free radical production is primarily due to a dramatic increase in oxygen uptake. The latter has been associated with a major increase in plasma catecholamines levels [1,2]. Labour and delivery are functions of nature during which both skeletal and uterus muscles participate. However, it has been documented that physical exercise augments the production of reactive oxygen species (ROS) in exercising muscles [2], by shunting blood away from the non-exercising areas to the working ⁎ Corresponding author. Fax: +30 2107462871. E-mail address: [email protected] (S. Tsakiris).

muscles, inducing the formation of ROS. If the production of free radicals is great enough to overcome antioxidant defense, oxidative stress will ensure increased levels of the end products of oxidative damage, which are observed in blood and tissues after exercise [3]. The release of catecholamines is mediated predominantly by central mechanisms from brain motor centres. During prolonged exercise the importance of peripheral stimulation of the neuroendocrine system is enhanced [4–6]. The placenta, which is regarded as a source of such molecules [7], produces, thioredoxin in normal pregnancies [8]. Recently, it has been reported that oxidative stress causes vascular dysfunction in the placenta, as found in pre-eclampsia [9,10]. In addition, the relationship between lipid peroxidation (LPO) and antioxidant activity in normal pregnancy is unclear. Plasma lipids normally rise in pregnancy with an accompanying increase in

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low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) content [11,12]. Lipoproteins are highly susceptible to oxidation, and lipoprotein oxidation has been proposed to play a role in the pathogenesis of pregnancy complications, such as preeclampsia [13–15], even though an in vitro study [16] showed that β-estradiol inhibits LDL oxidation. Additionally, there is limited knowledge in the literature investigating lipoprotein oxidation in normal pregnancy, and no literature defines any change during labour and its effect on the newborn. Moreover, brain tissue is especially prone to the deleterious effects of free radicals for various reasons, including modest antioxidant defenses [17]. Activation of neuronal nitric oxide synthase, and, thus, NO formation because of the high Ca2+ traffic across neuronal membranes [18] and high oxygen demand, can lead to increased formation of free radicals [19,20]. Acetylcholinesterase (AChE) (E.C. 3.1.1.7) is a biologically significant component of the membrane, contributing to its integrity and to permeability changes occurring during synaptic transmission and conduction. It is a membrane-bound enzyme with its active side exposed in the external leaflet of the bilayer (ectoenzyme). Because of the widespread distribution of cholinergic functions, if acetylcholine (ACh) action is enhanced, toxic effects involve the sympathetic, parasympathetic, motor and central nervous systems [21,22]. Our previous studies [23,24] showed that normal and/or prolonged labour and deliveries are implicated with low total antioxidant status (TAS) in mothers, post-delivery. In addition, erythrocyte membrane AChE activity was found modulated in athletes after endurance exercise in whom their TAS levels were measured low in their blood and their plasma catecholamines remarkably increased [25–27]. Since TAS levels are closely related to labour and delivery and AChE activity, catecholamines to autoxidation and endurance training, we aimed to investigate the effect of the mode of delivery on the enzyme activity pre- vs post-delivery in mothers and their newborns. Subjects and methods

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“Alexandra” Maternity Hospital. Gestation age was determined based on the menstrual history and ultrasound obstetrical findings. The participants were divided into two groups according to the mode of labour and delivery: group A (N = 16) women with normal labour and vaginal delivery, group B (N = 14) with scheduled Cesarean section (CS). CSs were performed with spinal anaesthesia without O2. Twenty (N = 20) non-pregnant medical students of comparable age were the controls. On admission, just before entering the delivery room, blood samples (pre-delivery) were obtained for routine laboratory examinations of complete blood count, BUN, uric acid etc., and the measurement of serum total cholesterol (t-Chol), triglycerides (TG), HDL-C, LDL-C and VLDL-C, creatine kinase (CK), and total antioxidant status (TAS). In addition blood was obtained for the evaluation of erythrocyte membrane AChE activity. Controls underwent the above laboratory investigations once. Immediately after delivery, blood from the umbilical cord and from the mothers (post-delivery) was also collected. Sera, plasma or erythrocytes were separated and kept frozen (− 70°C) until analysis for the same biochemical parameters within a maximum of 72h. Analytical procedure Blood chemistry including determinations of serum pyruvate transaminase (ALT), oxalate transaminase (AST), alkaline phosphatase (ALP), CK, glucose, and γ-glutamyl transaminase (γGT), was performed using the Bayer ADVIA 1650 Clinical Chemistry Analyzer (Bayer Corp., Tarrytown, NY, USA). Internal quality control of the lipids was carried out according to the laboratory manual of the Lipid Research Clinics Programme. Cholesterol bound to VLDL and LDL was estimated by the Friedewald equation [28]. Inter- and intra-assay variations were 3.8% and 4.3%, respectively. TAS measurements were performed spectrophotometrically at 450nm in microtiter plates, as described previously, using commercial kits available from Immundiagnostik AG (Bensheim, Germany). Inter- and intra-assay variations were 2.4 and 2.4%, respectively [29].

Subjects Plasma biogenic amines measurement The study was performed in accordance with the Helsinki Declaration of 1964 (as amended in 1983 and 1989), and approved by the Institutional Ethics Committee of Athens. Written consents were taken from all the participants of this study. Throughout a period of 3months, 30 serum samples were collected from mothers at the beginning of labour (pre-delivery) 3–4 min after membrane rupture, at the end of delivery (post-delivery), and from the cord blood (CB) of healthy full-term newborn infants, according to the following criteria: [1] singleton live birth; [2] gestational age between the beginning of the 37th week and the end of the 41st week; [3] body weight of the newborn 2500 and 4000g and [4] Apgar scores of ≥ 9 at the first to fifth minute. A history of the pregnancies and deliveries was obtained from the notes in the records made by obstetricians and pediatricians, according to the strict routine “follow-up” practice of the First Dept of Obstetrics and Gynecology of Athens University in the

Plasma catecholamines, adrenaline (A), noradrenaline (NA) and dopamine (DA) concentrations were evaluated by HPLC Table 1 Clinical characteristics in mothers and their newborns

Age (yrs) Gestation (weeks) Placenta (g) Birth wt (g) Labour + delivery (h) Range

Group A (N = 16)

Group B (N = 14)

26 ± 3 36.0 ± 1.0 476 ± 36 2980 ± 120 10 ± 3 (6–13)

22 ± 2 34.0 ± 1.0 468 ± 40 3010 ± 130 0.45 ± 0.12 (0.25–0.50)

Values are expressed as mean ± SD. Group A: normal pregnancy and delivery. Group B: scheduled Cesarean section.

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Table 2 Biochemical characteristics in the mothers pre- and post-delivery, in the cord blood (CB) of their newborns and in controls Group A (N = 16)

AST (U/L) ALT (U/L) Al P (U/L) γGt (U/L) CK (U/L) t.protein (g/L) t-Chol (mmol/L) Trigl (mmol/L) HDL (mmol/L) LDL (mmol/L) VLDL (mmol/L) TAS (μmol/L)

Group B (N = 14)

Controls (N = 20)

Pre-

Post-

CB

Pre-

Post-

CB

27 ± 7 21 ± 8 74 ± 24 12 ± 3 80 ± 12a 7.2 ± 0.4 6.7 ± 0.5 2.3 ± 0.2 1.7 ± 0.4 3.2 ± 0.8 0.5 ± 0.1 425 ± 36a

39.0 ± 9 36 ± 28 86 ± 7 15 ± 7 240 ± 15b 6.9 ± 0.5 6.7 ± 0.4 2.5 ± 0.2 1.6 ± 0.4 3.2 ± 0.9 0.5 ± 0.1 235 ± 33b

40 ± 9 42 ± 8 92 ± 8 20 ± 9 84 ± 15c 6.6 ± 0.9 1.8 ± 0.3 0.3 ± 0.1 0.8 ± 0.2 0.8 ± 0.2 0.1 ± 0.0 326 ± 34c

29 ± 7 25 ± 8 70 ± 21 14 ± 3 86 ± 12d 7.0 ± 0.9 6.6 ± 0.4 2.4 ± 0.3 1.6 ± 0.5 3.1 ± 0.8 0.5 ± 0.1 420 ± 35d

31 ± 9 40 ± 25 80 ± 7 15 ± 8 97 ± 15e 6.8 ± 0.8 6.5 ± 0.5 2.4 ± 0.2 1.6 ± 0.5 3.0 ± 0.6 0.5 ± 0.1 416 ± 34e

34 ± 7 44 ± 8 86 ± 10 20 ± 10 76 ± 12f 6.4 ± 0.5 1.7 ± 0.3 0.3 ± 0.1 0.9 ± 0.2 0.8 ± 0.2 0.1 ± 0.0 362 ± 36f

30 ± 9 25 ± 8 70 ± 22 14 ± 3 86 ± 12a 7.0 ± 0.5 6.7 ± 0.5 2.2 ± 0.2 1.6 ± 0.4 3.1 ± 0.8 0.5 ± 0.1 434 ± 38a

Values are expressed as mean ± SD. Statistics: a/b, a/c, a/f, b/c, b/d, b/e, b/f, c/d, c/e, e/f, p b 0.001; a/a, a/d, a/e, c/f, d/e, d/f, p N 0.05 (NS).

(Hewlett-Packard, USA) with electrochemical detection as previously described [30], CV for A, NA, and DA were 3.4%, 2.9%, and 3.2%, respectively. Erythrocyte membrane preparation Blood samples (7.0 mL) were collected into heparinized blood collection tubes. Within 2 h of collection, the erythrocytes were sedimented by centrifugation at 2000×g for 30 min at 4 °C, and were washed three times after three similar centrifugations with buffer solution (250 mM Tris–HCl, pH 7.4, 140 mM NaCl, 1 mM MgCl2, and 10 mM glucose). The erythrocytes were then resuspended in 1.0 mL of the same buffer and stored at 4 °C for up to 24 h before erythrocyte membrane preparation. Washed erythrocytes were lysed after freeze-thawing (− 80/+ 50 °C) five times, as described by Galbraith and Watts [31] and Kamber et al. [32]. The hemolyzate was centrifuged four times at 35,000×g for 30 min with 40–60 volumes of cold 0.1M Tris–HCl, pH 7.4 until a white–pink color appeared. Membranes were suspended in 0.1M Tris–HCl, pH 7.4 to a final concentration of 2mg protein/mL. The protein content was determined using the Lowry method [33]. Membranes stored at − 40°C retained enzyme activities for at least 2weeks. The minor amount of hemoglobin that remained attached to the membrane surface was measured with a 527-A kit (Sigma, St. Louis, MO, USA), and the value was subtracted from the total protein concentration.

Measurement of erythrocyte membrane AChE activity AChE activity was determined according to the method of Ellman et al. [34], as modified by Tsakiris et al. [35] and Schulpis et al. [27]. The reaction mixture (1mL) contained 50 mM Tris– HCl, pH 8.0, 240 mM sucrose and 120 mM NaCl in the presence of 80–100μg of protein from erythrocyte membranes. Quinidine sulfate (2 × 10− 2mM) was added to the mixture to inhibit pseudocholinesterase activity. Finally, 0.030mL of 5,5′-dithionitro-benzoic acid (DTNB) and 0.050mL of actylthiocholine iodide (substrate) were added and the reaction was started. The final concentrations of DTNB and substrate were 0.125 and 0.5 mM, respectively. The reaction was measured spectroP photometrically by the increase in absorbance (DOD) at 412nm. Statistics Data are presented as means ± SD. Two-way ANOVA was utilized for the analyses of the results with SPSS 13.0 statistical package on an IBM computer. p values b 0.05 were considered statistically significant. Results As shown in Table 1, the age of the mothers, their gestation, the weight of placenta and the birth weights of their newborns

Table 3 Catecholamines, adrenaline (A), nor-adrenaline (NA) and dopamine (DA) blood levels in mothers pre- vs post-delivery, in the cord blood (CB) of their neonates and in controls Group A (N = 16)

Group B (N = 14)

Controls (N = 20)

Catecholamines

Pre-

Post-

CB

Pre-

Post-

CB

A (pmol/L) NA (nmol/L) DA (pmol/L)

230 ± 30a 1.60 ± 0.4a 52 ± 15a

988 ± 130b 6.2 ± 0.8b 174 ± 32b

900 ± 160c 6.0 ± 0.8c 184 ± 30c

220 ± 35d 1.45 ± 0.5d 50 ± 16d

236 ± 45d 1.58 ± 0.6d 54 ± 16d

240 ± 36d 1.6 ± 0.6d 56 ± 16d

Values are expressed as mean ± SD. Statistics: A: a/b, a/c, a/b, b/d, b/c, c/d, p b 0.001; a/d, d/d, a/a, p N 0.05. NA: a/b, a/c, b/c, b/d, c/d, p b 0.001; a/a, a/d, d/d, p N 0.05. DA: a/b, a/c, b/c, b/d, c/d, p b 0.001; a/a, a/d, d/d, p N 0.05.

238 ± 30a 1.64 ± 0.4a 58 ± 15a

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did not differ among the groups in the study. In contrast, the durations of labour were remarkably higher in group A than those in group B. The above-mentioned, common laboratory tests, including complete blood count, glucose, urea, uric acid etc., were normal for all groups (data not shown). Liver enzymes and lipid profile did not differ pre- vs postdelivery in the groups of mothers (Table 2) and in controls. In addition, the lipid levels measured in the CB were statistically significantly lower than those pre- and post-delivery, but did not differ among the groups. The muscle enzyme, CK, and serum levels were significantly increased in all the studied groups post-delivery, especially in group A and in the CB of the newborns, except in those of group B and controls. TAS levels measured were statistically significantly lower in group A post-delivery as compared to those of group B at the same time of study and in controls. Additionally, TAS levels were remarkably higher in the CB of group B than those in the CB of group A. The studied biogenic amine levels were similar in the studied groups of mothers pre-delivery and in controls (Table 3). In addition, catecholamine (A, NA, DA) blood levels were remarkably increased in mothers of group A pre- vs postdelivery and in their infants, as compared to those of group B at the same times of study. These catecholamine levels remained unchanged in group B at all times of study. Additionally, these biogenic amine levels were remarkably higher in the CB of group A than those in the CB of group B. As shown in Table 4, the studied enzyme activities did not differ pre-delivery in the groups of mothers and controls. In contrast, AChE activity was remarkably increased (~ 35%) in mothers of group A post-delivery, whereas the enzyme activity remained unaltered in group B at the same time of study. AChE activity was similar in the two groups of neonates. In addition, the enzyme activity was significantly lower in neonates as compared to that of their mothers predelivery.

Table 4 Erythrocyte membrane AChE activities in mothers of group A and group B prevs post-delivery, in their neonates and in controls ¯¯ /min × mg protein) AChE (ΔOD Group A (N = 16) PrePostCB

2.53 ± 0.10a 3.42 ± 0.17b (+ 35%) 1.92 ± 0.06c (− 23%)

Group B (N = 14) PrePostCB

2.47 ± 0.012d 2.33 ± 0.14e (− 6%) 1.92 ± 0.04f (− 22%)

Controls (N = 20)

2.49 ± 0.12g

Values are expressed as mean ± SD. Statistic: a/b, a/c, b/c, b/d, b/e, b/f, c/d, c/e, c/g, d/f, f/g, p b 0.001; a/d, a/e, d/e, d/g, a/g, p N 0.05 (NS). NS: not statistically significant.

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Discussion In this study, the dramatically decreased TAS levels in women of group A at the end of their labour (post-delivery), may be due to the depletion of the antioxidants during their lasting process. This finding is frequently observed in athletes after prolonged training [36–38], and in women with preeclampsia, showing that labour and delivery might temporarily present the same abnormal biochemical parameters as found in pre-eclampsia conditions [39,40]. In support of this suggestion, we can present the unaltered TAS levels (pre- vs post-delivery) measured in the women of group B, who underwent a scheduled CS. Additionally, in an in vitro study [40], direct evidence was shown for exercise intensity — dependent increase in free radical outflow across an active muscle bed, that was associated with an increase in sarcolema membrane permeability. Furthermore, to increase mitochondrial electron flux subsequent to an increase in O2 extraction and flow, exercise-induced free radical generation may also be regulated in hydrogen ion generation, high levels of norepinephrine autoxidation, peroxidation of lipids and damage tissue and xanthine oxidase activation [2,3]. Additionally Yao et al. [41] reported that ACh release induced ROS generation. From this view, ACh production during uterus and skeletal muscle contractions may contribute to increase oxidative stress in a normal delivery process. Additionally, in a very recent study, it was reported that umbilical arterial lipids are more susceptible to peroxidation than umbilical venous lipids, indicating high oxidative stress in the fetal circulation irrespective of the mode of delivery [36]. In this study, the absence of a serious TAS reduction in the blood of the neonates, especially in those of group B, which also indicates the peroxidation of lipids, may be due to the presence of low levels of lipids, and, especially, to the very low LDL levels in the CB of the infants, and/or the placenta protective effect against oxidative stress [23,24]. Furthermore, during the last years, gene knockouts of various types of adrenoreceptors or enzymes involved in metabolism of norepinephrine have provided new possibilities for addressing the role of noradrenergenic pain modulatory mechanisms at the system level. Development of various types of pathophysiological pain models in experimental animals allowed investigating noradrenergic pain modulation not only in physiological but also in pathophysiological conditions [42]. Additionally, plasma norepinephrine (NA) level was remarkably higher in patients with painful than non-painful diabetic polyneuropathy [43], suggesting that supraspinal adrenoreceptors have a role in pain control [44]. Obviously, labour and delivery are well-known painful processes and elevated catecholamine blood levels were expected to increase in the blood of mothers of group A, postdelivery, without excluding the increased stress accompanying normal delivery not seen in the scheduled Cesarean section. Additionally, the participation of uterus and skeletal muscles, as in endurance exercise, [45–47] may also enhance the production of catecholamines showing an activation of their neuroendocrine system in this physiological process. Moreover, a noradrenergic pain modulatory system was shown to interact at the spinal cord level to produce more

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powerful antinociception [43,44]. A synergic interaction at the spinal level was shown to take place between alpha-2adrenoreceptor agonists and serotonin 5-HT2 and 5HT4B receptor actions [46]. Similarly, the high catecholamine levels, in the CB of group A may mirror those of their mothers post-delivery and/or may be due to the same causes, as mentioned above. The above explanations are further reinforced by the unaltered biogenic amine levels determined in mothers and their neonates of group B who underwent scheduled Cesarean section. Previous in vitro study [48] showed that oestrogen addition may increase the epithelial rat AChE activity. In contrast, no alteration of muscarinic function in the guinea-pig uterine artery was observed during pregnancy [49]. In agreement with the latter study, we found that erythrocyte AChE activity predelivery was similar to controls. This finding may be due to a different isoform of the enzyme (epithelial vs erythrocyte membrane and/or rat vs human). In addition, the observed remarkable increase of the membrane AChE activity in mothers of group A, post-delivery, may be related to the increase of their plasma catecholamine levels as we found in athletes postendurance exercise [25–27] in whom catecholamine levels were also increased in their blood stream. Similarly, in our previous study [50], we observed decreased the erythrocyte membrane AChE activity in phenylketonuric patients “off diet” with low catecholamine levels in their blood. Additionally, increased enzyme activity was also reported post-exercise several years ago [51]. Obviously, we cannot exclude that increased AChE activity may be measured post-endurance exercise due to high ACh production during increased neuromuscular activity [51,52]. These suggestions are further supported by the practically unaltered plasma biogenic amine levels as well as the unchanged membrane AChE activity in mothers of group B post-delivery who underwent a scheduled Cesarean section. Furthermore, the low AChE activity observed in the CB of neonates, in both groups of study, may be due to perinatal immaturity. Low AChE activity was measured in the rat brain during lactation and weaning period [53]. In addition, erythrocyte AChE activity was low in neonates of group A, even though their catecholamine levels were increased. This finding may be due the placental production and/or the membrane resistance of their immature erythrocytes to external modifications (e.g. the nature of Hb (HβF) which covers these cells). Conclusions (a) Low TAS determined in the blood of mothers and their newborns of group A may be due not only to the strenuous participation of their uterus and skeletal muscles during the delivery process but also to the autoxidation of remarkably high catecholamine blood levels. (b) The high catecholamine blood levels measured in mothers of group A at the same time of study may be due to the painful noradrenergic process of their delivery. Catecholamine concentrations determined in the cord blood of the neonates may mirror those of their mothers post-delivery.

(c) Erythrocyte membrane AChE activity was similar in pregnant vs non-pregnant women. The observed remarkably increased membrane enzyme activity, post-normal delivery process, may be due to the increased catecholamine levels and/or the increased ACh production in relation to the high neuromuscular activity of mothers due to uterine and skeletal muscle contractions. (d) The similarly low membrane AChE activity measured in neonates of both groups of this study may be due to perinatal immaturity. Acknowledgments The authors thank Mrs Anna Stamatis for her careful typing of this manuscript. Moreover, many thanks are expressed to the medical students Anastasios Pantazopoulos, Vasilios Memtsas and Marios Arvanitis for their assistance. References [1] Balakrishnan SD, Anuradha CV. Exercise, depletion of antioxidants and antioxidant manipulation. Cell Biochem Funct 1998;16:269–75. [2] Rokitzki L, Logemann E, Sagredos AN, Murphy M, Wetzel-Roth W, Keul J. Lipid peroxidation and antioxidative vitamins under extreme endurance stress. Acta Physiol Scand 1994;151:149–58. [3] Groussard C, Rannou-Bekono F, Machefer G, et al. Changes in blood lipid peroxidation markers and antioxidants after a single sprint anaerobic exercise. Eur J Appl Physiol 2002;89:14–20. [4] Galbo H. Autonomic neuroendocrine responses to exercise. Scand J Sport Sci 1986;8:3–11. [5] Fallo F. Renin–angiotensin–aldosterone system and physical exercise. J Sports Med Phys Fitness 1993;33:306–12. [6] Little RE, Gladen BC. Levels of lipid peroxides in uncomplicated pregnancy: a review of the literature. Reprod Toxicol 1999;13:347–52. [7] Nakamura H, Nakamura K, Yodoi J. Redox regulation of cellular activation. Annu Rev Immunol 1997;15:351–69. [8] Ejima K, Nanri H, Toki N, Kashimura M, Ikeda M. Localization of thioredoxin reductase and thioredoxin in normal human placenta and their protective effect against oxidative stress. Placenta 1999;20:95–101. [9] Many A, Hubel CA, Fisher SJ, Roberts JM, Zhou Y. Invasive cytotrophoblasts manifest evidence of oxidative stress in preeclampsia. Am J Pathol 2000;156:321–31. [10] Hubel CA, Shakir Y, Gallaher MJ, McLaughlin MK, Roberts JM. Low-density lipoprotein particle size decreases during normal pregnancy in association with triglyceride increases. J Soc Gynecol Investig 1998;5: 244–50. [11] Uotila J, Tuimala R, Aarnio T, Pyykko K, Ahotupa M. Lipid peroxidation products, selenium-dependent glutathione peroxidase and vitamin E in normal pregnancy. Eur J Obstet Gynecol Reprod Biol 1991;42:95–100. [12] Brizzi P, Tonolo G, Esposito F, et al. Lipoprotein metabolism during normal pregnancy. Am J Obstest Gynecol 1999;181:430–4. [13] Llurba E, Gratacos E, Martin-Gallan R, Cabero L, Dominguez C. A comprehensive study of oxidative stress and antioxidant status in preeclampsia and normal pregnancy. Free Radic Biol Med 2004;37: 557–70. [14] Wakatsuki A, Ikenoue N, Okatani Y, Shinohara K, Fukaya T. Lipoprotein particles in preeclampsia: susceptibility to oxidative modification. Obstet Gynecol 2000;96:55–9. [15] Sattar N, Greer IA, Louden J, et al. Lipoprotein subfraction changes in normal pregnancy: threshold effect of plasma triglyceride on appearance of small, dense low density lipoprotein. J Clin Endocrinol Metab 1997;82: 2483–91. [16] Huber L, Scheffler E, Poll T, Ziegler R, Dresel H. 17 beta-estradiol inhibits LDL oxidation and cholesteryl ester formation in cultured macrophages. Free Radic Res Commun 1990;8:167–73.

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