- Email: [email protected]
Altered Placental Oxidative Stress Status in Gestational Diabetes Mellitus
Placenta (2004), 25, 78–84 doi:10.1016/S0143-4004(03)00183-8
Altered Placental Oxidative Stress Status in Gestational Diabetes Mellitus M. T. Coughlan a,*, P. P. Vervaart b, M. Permezel a, H. M. Georgiou a and G. E. Rice a a
Mercy Perinatal Research Centre, The University of Melbourne, Mercy Hospital for Women, Clarendon St, East Melbourne, Victoria 3002, Australia; b Department of Complex Biochemistry, Royal Children’s Hospital, Melbourne, Australia
Paper accepted 7 July 2003
Oxidative stress has been clearly linked to type 2 diabetes mellitus, however, limited data are available on the involvement of oxidative stress in gestational diabetes mellitus (GDM), a disease of similar pathophysiology. The aim of this study was to investigate the status of placental oxidative stress in healthy pregnant women and women with GDM. The hypothesis to be tested was that tissue markers of oxidative stress are significantly increased in GDM compared to normal placental tissues. Markers of oxidative stress measured were the release of 8-isoprostane (8-epi-prostaglandin F2) from human term placental explants (n=11), the activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase (n=10), and protein carbonyl content (n=12). Placental release of 8-isoprostane was 2-fold greater from women with GDM (P<0.001) compared to healthy pregnant women. Superoxide dismutase activity and protein carbonyl content were elevated in placentae obtained from women with GDM (P<0.04 and P<0.004 respectively), whilst there was no significant difference in the activity of glutathione peroxidase. These data demonstrate the presence of oxidative stress in the placenta from women with GDM, in addition to the induction of a key antioxidant, collectively indicating a state of existing oxidative stress in this condition. Placenta (2004), 25, 78–84 2003 Elsevier Science Ltd. All rights reserved.
INTRODUCTION Gestational diabetes mellitus (GDM) is defined as a glucose intolerance of varying severity with onset or first recognition during pregnancy [1]. It complicates approximately 2–4 per cent of pregnancies [2] and is a significant cause of fetal macrosomia, perinatal mortality and maternal long-term risk of developing type 2 diabetes mellitus [3]. Gestational diabetes mellitus is characterized by hyperglycaemia, insulin resistance and hyperlipidaemia: biochemical abnormalities that are common to type 2 diabetes mellitus. Gestational diabetes mellitus has been considered a ‘pre-diabetic’ state [4] and the pathophysiology of the two are clearly related. Oxidative stress refers to a disturbance in the balance between the production of reactive oxygen species (ROS) and antioxidant defences, that may lead to tissue injury [5]. The link between diabetes, particularly type 2, and oxidative stress has been well described [6,7]. Available evidence includes studies that have identified increased biomarkers of oxygen radical damage and reports that find abnormalities in the antioxidant defences of diabetic patients. An impairment of antioxidant defence has been identified, with lowered circulating enzymatic and non-enzymatic antioxidants in individuals with type 2 diabetes mellitus [7]. *
To whom correspondence should be addressed. Tel.: +61-3-92702556; Fax: +61-3-9417-5406; E-mail: [email protected] 0143-4004/04/$–see front matter
Limited data are available regarding the presence of oxidative stress in women with GDM. The placenta provides the interface of the maternal and fetal circulations, and it may play a crucial role in protecting the fetus from adverse effects of the maternal diabetic milieu, while disturbances in placental function may exacerbate this state. We have reported increased in vitro secretion of tumour necrosis factor- (TNF-) from the placenta and adipose tissue from women with GDM under conditions of high glucose [8] and propose that an altered oxidative homeostasis in women with GDM may be involved. Reactive oxygen species measurement is difficult given their high reactivity, very short half-life and low concentration. Therefore indirect markers are commonly used to evaluate secondary products of ROS damage. The induction of oxidative stress can result in cellular damage, including modification to DNA, proteins and lipids. Lipid peroxidation is the oxidative deterioration of polyunsaturated fatty acids and results in the formation of end products that may themselves pose a further oxidative risk. For example, 8-isoprostane (8-IP) (8-epi prostaglandin F2 or 15-F2t-IsoP) is an F2 isoprostane, one of a unique series of prostaglandin-like compounds formed in vivo from the free-radical-catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase enzyme [9]. It is biologically active, capable of inducing vasoconstriction in the placenta [10] and modulating the function of platelets [11,12]. 2003 Elsevier Science Ltd. All rights reserved.
Coughlan et al.: Placental Oxidant Stress in Gestational Diabetes
In addition, the teratogenicity of 8-IP has recently been demonstrated [13]. 8-isoprostane has been reported to be a stable and sensitive indicator of in vivo lipid peroxidation [14]. Circulating and urinary concentrations of 8-isoprostane are elevated in patients with type 2 diabetes [15–17] and in pregnant rats with streptozotocin-induced diabetes [18]. Oxygen radicals and other ROS cause modifications of proteins [19] and damage to enzymes, receptors, signal transduction pathways and transport proteins can occur. This damage can occur by direct attack of ROS or by secondary damage involving end attack by products of lipid peroxidation. Oxidative stress can give rise to protein carbonyl derivatives, which serve as a more universal biomarker of oxidative stress [20] and have been regarded as the most general and well-used biomarker of severe oxidative protein damage [21]. Protein carbonyl content in erythrocyte membranes is significantly greater in individuals with type 2 diabetes mellitus and correlates with impaired glycaemic control [22]. In addition, protein carbonylation in the plasma of pregnant rats with streptozotocin-induced diabetes was shown to be elevated [23]. To avoid ROS-induced damage of cellular components, several biochemical safety mechanisms are present in the placenta, including defence enzymes and antioxidants. In normal pregnancy, there is an increase in oxidative stress and lipid peroxidation compared with non-pregnant women but there is also a concomitant increase in antioxidant protection that compensates for the increased oxidative stress [24]. The first line of defence against superoxide and hydrogen peroxidemediated injury are the antioxidant enzymes superoxide dismutases (SOD), catalase and glutathione peroxidase (GSHPx). Both copper and zinc-containing SOD (CuZn-SOD) and manganese-containing SOD (Mn-SOD) have been described in the human placenta [25] and SOD increases throughout gestation [26]. Superoxide dismutases are important initial components in the cellular defense against oxygen toxicity, neutralizing superoxide radicals. Superoxide radicals react with hydrogen peroxide to generate single oxygen and hydroxy radicals, which are even more reactive and cytotoxic than superoxide radicals or hydrogen peroxide, and superoxide radicals can initiate lipid peroxidation. Glutathione peroxidase and catalase both neutralize hydrogen peroxide. The aim of this study was to investigate the oxidative stress status of the placenta from women with GDM in comparison to pregnant women with normal glucose tolerance. The hypothesis to be tested was that that tissue markers of oxidative stress are significantly increased in GDM compared to normal placental tissues. Quantification of 8-isoprostane release from the placenta and protein carbonyl content as indices of oxidative stress and the activity of the prominent placental antioxidant enzymes SOD and GSH-Px was determined. MATERIALS AND METHODS Participants Placentae were obtained from a total of 49 pregnant women (25 normal and 24 with GDM) at the time of term Caesarean
79
section. Indications for Caesarean section included repeat Caesarean section (n=35), breech presentation (n=12) or macrosomia (n=2). All of the women were non-smokers and were not administered any antenatal medications. Women with any adverse underlying medical condition were excluded, for example, asthma, arthritis, pre-eclampsia and macrovascular complications. All participants received lumbar epidural analgesia for pain relief during Caesarean section except for one woman who received general analgesia. Women with GDM were diagnosed according to the current criteria of the Australasian Diabetes in Pregnancy Society by either a fasting venous plasma glucose level of R5.5 mmol/l glucose, and/or R8.0 mmol/l glucose 2 h after a 75 g oral glucose load at approximately 28 weeks’ gestation [27]. Thirteen women with GDM were clinically managed by diet alone and 11 women were prescribed insulin in addition to dietary management. The majority of participants were Caucasian, whilst a small number were from various ethnic groups including the Middle East, Asia and the Mediterranean. Approval for this study was obtained from the Mercy Hospital for Women’s Human Research and Ethics Committee and informed consent was obtained from all participating subjects.
Reagents All reagents were purchased from BDH Chemicals Australia (Kilsyth, Vic, Australia) unless otherwise stated. The following reagents were obtained from Sigma Chemical Company (St Louis, MO, USA): -NADH (disodium salt), type II pyruvic acid (dimer free), penicillin/streptomycin and RPMI 1640. The 8-IP enzyme immunoassay kit was from Cayman Chemical Company (Ann Arbor, MI, USA). Phosphate buffered saline (PBS) was purchased from Gibco BRL (Rockville, MD, USA) and Pierce (Rockford, IL, USA) supplied the protein assay kit. The Ransod and Ransel kits were from Randox Laboratories (Crumlin, Northern Ireland, UK). The protein carbonyl kit was obtained from Zenith Technology (Dunedin, New Zealand).
Tissue collection and explant culture Placentae were collected from term elective Caesarean sections performed prior to the onset of labour. Tissue processing commenced within 15 min of delivery and involved the removal of a placental lobule from the central region of the placenta, and dissection free of the chorionic tissue and removal of the basal plate, to obtain villous tissue from the middle cross-section. Eleven placentae from healthy women and eleven from women with GDM were used for explant incubation. Placental villous tissue explants were obtained by blunt dissection and removal of visible connective tissue, vessels and calcium deposits, and were rinsed in PBS (37(C) then RPMI 1640 (37(C). Approximately 200 mg (wet weight/ well) of placental tissue was placed into a 12-well tissue culture
80
plate (Becton Dickinson Labware, NJ, USA) with 2 ml of RPMI 1640, pH 7.2. All incubations were performed in duplicate. The plate was incubated at 37(C in a humidified atmosphere of carbogen (95 per cent O2/5 per cent CO2) in a shaking water bath for 1 h to obtain tissue equilibration. Subsequently, tissue explants were incubated with 2 ml of RPMI 1640, pH 7.2, for 24 h and the incubation medium and tissue were collected and frozen at 80(C until assayed for 8-IP and total protein content. Additional samples of placenta (2–3 g) were collected at time=0 after Caesarean section, briefly rinsed in PBS, snap frozen in liquid nitrogen and stored at 80(C for subsequent analysis. A small number of these placental samples were from placentae that were also used for explant incubation, plus further samples were collected as required exclusively for the determination of antioxidant enzyme activity and/or protein carbonyl content. Thus the total number of placentae obtained in this study was 25 from normal women and 24 from women with GDM.
Total protein determination Measurement of total protein in placental explants was performed as described previously [8]. Total protein content of the tissue was determined using the bicinchoninic acid (BCA) method (Pierce, Rockford, IL, USA) following the microwell plate protocol as described by the manufacturer. The absorbance was quantified using a 96-well microtitre plate reader at 595 nm (Bio-Rad Laboratories, Hercules, CA, USA). The intra- and inter-assay coefficients of variation were 5.0 per cent and 5.5 per cent (over 8 assays) respectively.
Tissue viability To assess tissue viability during in vitro incubation, the release of the intracellular enzyme lactate dehydrogenase (LDH) into the incubation medium was determined as described previously [28]. The absorbance was quantified using a 96-well microtitre plate reader at 340 nm (Bio-Rad Laboratories, Hercules, CA, USA). LDH release was expressed as a per cent of total tissue control which was calculated as LDH activity in the medium divided by total tissue LDH activity multiplied by 100.
8-Isoprostane determination Determination of 8-IP accumulation in the explant incubation medium was performed using a competitive enzyme immunoassay kit (Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer’s specifications. Samples were diluted with RPMI-1640 as necessary within a range of neat to 1/30 depending on the tissue sample and treatment. The absorbance was quantified using a 96-well
Placenta (2004), Vol. 25
microtitre plate reader at 405 nm (Bio-Rad Laboratories, Hercules, CA, USA). The intra- and inter-assay coefficients of variation were 7.2 per cent and 18.5 per cent respectively (over 6 assays) and the limit of detection of the assay was 5 pg/ml. Protein carbonyl determination The protein carbonyl content of the placenta was determined using a commercially available enzyme immunoassay from Zenith Technology (Dunedin, New Zealand). Tissue (200 mg) that was obtained at time=0, was homogenized in ice-cold homogenizing buffer (250 m sucrose, 20 m Tris–HCl, 1 m dithiothreitol, pH 7.4) and centrifuged at 19 000 g for 20 min. Samples and standards were diluted in PBS to a protein concentration of 4 mg/ml. The procedure was followed according to the manufacturer’s instructions. All samples were measured in a single assay. The intra-assay coefficient of variation was 13 per cent and the limit of detection of the assay was 0.05 nmol/mg protein. Total protein of the placental tissue was measured as described above. Superoxide dismutase and glutathione peroxidase determination Enzymatic activity of the antioxidant enzymes SOD and GSH-Px was measured using commercially available kits from Randox Laboratories (Crumlin, Northern Ireland, UK) according to the manufacturer’s instructions. Tissue (obtained at time=0) homogenates (300 mg/ml wet weight) was prepared as follows. Placental tissue, kept on ice, was homogenized using a metal-blade tissue homogenizer (S25N-8G, IKA-Werke GMBH and Co., KG Staufen, Germany) for 1 min in ice-cold PBS, pH 7.4. The homogenate was centrifuged for 25 min at 3373 g and the resulting supernatant was diluted with PBS to 100 mg/ml wet weight. The activities of the antioxidant enzymes were measured on a Cobas Mira autoanalyzer (Roche, Graz, Austria). The SOD assay measures the Cu-ZnSOD isoform only, as Mn-SOD is inactivated by the pH of the assay. Statistical analysis Statistical computations were performed using a commercially available statistics analysis software (Statgraphics Plus for Windows, version 3.1, Statistical Graphics Corp., Rockville, MD, USA). Student’s t-test was used to assess statistical significance between normally distributed data otherwise nonparametric Mann–Whitney U (Wilcoxen) tests were used. Pearson’s correlation coefficient determinations were used in the correlation analyses. Statistical significance was assumed when P<0.05. Grouped results are reported as meanSD. RESULTS The clinical characteristics of all participants involved in the investigation are collated in Table 1. Fasting and 2-h plasma
Coughlan et al.: Placental Oxidant Stress in Gestational Diabetes
81
Table 1. Characteristics of all participants involved in the study
Age (years) Maternal BMI (kg/m2)** Gestational age at birth (weeks) Birth weight (g) Parity Fasting plasma glucose (mmol/l) Two hour plasma glucose (mmol/l)
Normal pregnancy (n=25)
GDM pregnancy (n=24)
32.43.9 24.44.1 38.51.1 3311558 1.90.8 4.20.3 5.31.0
33.06.1 29.99.1* 38.50.7 3513511 2.51.6 5.30.8* 8.81.9*
Data represent meanSD. * P<0.05 compared to normal women. **Based on first antenatal visit at approximately 12 weeks.
glucose values at oral glucose tolerance test were significantly higher in women with GDM compared to healthy pregnant women (P<0.001). The only other significant difference between the study groups was maternal body mass index (BMI), which was greater in the women with GDM (P=0.04). The release of LDH (a marker of cell membrane integrity) into the incubation medium over 24 h was determined for all explant incubations (n=11 in each group). In vitro incubation did not adversely affect LDH activity in the incubation medium and there was no significant difference between tissues from the two participant groups during the 24 h incubation period (5.581.48 per cent of total tissue control for GDM vs 5.051.38 per cent of total tissue control for normal). LDH release from the placental explants is consistent with previously documented levels [29]. Figure 1 illustrates the 8-IP release into the incubation medium over 24 h. Basal release of 8-IP from the placentae of women with GDM was significantly greater compared to normal women (1720721 pg/mg protein for GDM vs 738177 pg/mg protein for normal, P<0.001, n=11). There was a significant correlation between 8-IP release from the placenta and maternal 2-h plasma glucose concentrations after an oral glucose challenge at the time of diagnosis (r2=0.26, P<0.03, Figure 2), but no correlation was apparent between 8-IP release and maternal fasting plasma glucose. Protein carbonyl content of the placenta was significantly elevated in women with GDM compared to pregnant women with normal glucose tolerance (0.1480.153 nmol/mg protein for GDM vs 0.0620.011 nmol/mg protein for normal, P<0.004, Figure 3, n=12). The activities of the antioxidant enzymes CuZn-SOD and GSH-Px were determined in placental tissue obtained from both women with normal glucose tolerance and women with GDM. The activity of CuZn-SOD was significantly higher in the placenta obtained from women with GDM compared to normal women (1.770.23 U/mg protein for GDM vs 1.580.11 U/mg protein for normal, Figure 4, P<0.04, n=10). There was no significant difference in the activity of GSH-Px in placental tissue between healthy pregnant and GDM participants (Figure 5). In addition, there was no difference in any of the assayed placental variables in mothers
Figure 1. Release of 8-isoprostane (pg/mg protein) from human term placental explants obtained from healthy pregnant women (n=11) and women with GDM (n=11) incubated in RPMI 1640 for 24 h. Horizontal lines indicate the mean values and dots represent each data point. P<0.001.
with GDM who were managed by diet alone compared to women who were treated with diet and insulin. The ratio of CuZn-SOD activity to 8-IP release from the placenta from normal women compared to that of women with GDM was 2.1 : 1. The same ratio was found between CuZnSOD to protein carbonyl when comparing normal to GDM.
DISCUSSION The aim of this study was to investigate the placental oxidative stress status of women with GDM by testing the hypothesis that tissue markers of oxidative stress are significantly
82
Placenta (2004), Vol. 25
Figure 2. Correlation between 8-isoprostane release from term placenta and maternal plasma glucose 2 h after a glucose challenge at oral glucose tolerance test. Open squares indicate healthy pregnant women (n=8) and closed squares indicate women with GDM (n=11). r2=0.26, P=0.03.
Figure 3. Protein carbonyls (nmol/mg protein) in the placenta from healthy pregnant women (n=12) at term and women with GDM (n=12). Horizontal lines indicate the mean values and dots represent each data point. P<0.004.
*OXWDWKLRQH 3HUR[LGDVH 8PJ SURWHLQ
Figure 4. Activity of superoxide dismutase (U/mg protein) in the placenta from healthy pregnant women (n=10) at term and women with GDM (n=10). Horizontal lines indicate the mean values and dots represent each data point. P<0.04.
1RUPDO increased in GDM compared to normal placental tissues. This was determined by quantifying the release of 8-IP from placental explants and placental protein carbonyl content as indices of oxidative stress, and determining the activity of the endogenous antioxidant enzymes CuZn-SOD and GSH-Px in placental tissue from women with GDM and healthy women at term.
*'0
Figure 5. Activity of glutathione peroxidase (U/mg protein) in the placenta from healthy pregnant women (n=10) at term and women with GDM (n=10). Horizontal lines indicate the mean values and dots represent each data point.
In this study, the release of 8-IP from placental explants obtained from women with GDM was 2-fold greater than that of healthy pregnant women. 8-isoprostane has been reported to
Coughlan et al.: Placental Oxidant Stress in Gestational Diabetes
be a stable and sensitive indicator of in vivo lipid peroxidation, a central feature of oxidant stress [14]. The marked increase in 8-IP release from placental tissue obtained from women with GDM suggests that these women have increased underlying placental lipid peroxidation as a consequence of increased oxidant stress. In addition, we found a significant positive correlation between plasma glucose (2 h after a glucose challenge at the time of diagnosis) and term placental release of 8-IP. It was interesting to note that the fasting plasma glucose concentrations did not correlate with term placental release of 8-IP, suggesting that lipid peroxidation may be, at least partly, associated with the determinants of glycaemic control. This is consistent with previous findings that demonstrated in vitro glucose-induced 8-IP production in porcine vascular smooth muscle cells [30] and whole rat embryos [31]. Another study by Davi et al. [17] found a positive correlation between urinary 8-IP and plasma glucose in individuals with type 2 diabetes; treatment of hyperglycaemia decreased 8-IP. Oxidative stress can give rise to protein carbonyl derivatives via various mechanisms including direct oxidation of amino acid residues, by reaction with products of lipid peroxidation, and by interaction of reducing sugars or their oxidation products (glycation and glycoxidation products) with lysine residues of proteins [20]. Thus, increased concentrations of protein carbonyls represent a more general marker of oxidative stress than 8-IP, a marker of lipid peroxidation. In this study, we demonstrated greater protein carbonyl content in the placenta from women with GDM compared to healthy pregnant women indicating further the presence of oxidative stress in this condition. In addition, we demonstrated significantly higher CuZnSOD activity in GDM placentae compared to placentae from healthy pregnant women. CuZn-SOD, the cytosolic isoform, is mainly compartmentalized in trophoblast cells within the placenta and may thus serve an important function at the maternal-fetal interface [32]. In the women with GDM, placental CuZn-SOD activity was increased, which may reflect a compensatory protective response to existing oxidative stress in GDM pregnancy. However, the relative ratio of CuZn-SOD to oxidative stress markers (8-IP and protein carbonyl) was lower in the GDM placentae compared to that of normal. This suggests that SOD is not sufficiently compensating for the increased oxidative stress, further clarifying the existence of the oxidative stress in this condition. Superoxide dismutase and catalase activity in the placenta increases as gestation proceeds, whilst GSH-Px activity does not change [26]. It has been suggested that GSH-Px defence against ROS-induced lipid peroxidation may not be as important [33]. In this study, GSH-Px activity was comparable in the placenta of women with GDM and healthy women.
83
This is the first report, to our knowledge, of elevated 8-IP release from, and protein carbonyl concentration in the placenta in GDM. There is a paucity of data characterizing the status of oxidative stress in women with GDM. The data that are available do not afford a consistent profile. Kinalski et al. [34] reported evidence of increased lipid peroxidation but decreased SOD activity in GDM placentae compared to controls. In contrast, Pustovrh et al. [35] were unable to identify any difference in either placental lipid peroxidation or SOD between women with GDM and normal pregnant women. Kamath et al. [36] found elevated erythrocyte malondialdehyde in babies born to mothers with GDM. Consistent with the current study, Loven et al. [37] demonstrated an increase in erythrocyte CuZn-SOD in maternal age-matched pairs of women with GDM compared to healthy pregnant women. The increase in oxidative activity in GDM in this study appears not to be secondary to a deficiency of the antioxidant enzyme defence systems, but may be due to the presence of impaired glycaemic control. There is considerable evidence that oxidative stress generated as a result of hyperglycaemia is implicated in the development of diabetic complications [6]. Several pathways have been proposed for the production of ROS in the presence of high glucose concentration [7]. In the current study, the BMI of the women with GDM was significantly higher than that of the normal women. This is not unexpected, as obesity is a major risk factor for the development of GDM [38]. Although obesity has been recently associated with enhanced lipid peroxidation [39], it is not known if the increased BMI in the women with GDM in this study affected their oxidative stress status. However, there was no significant correlation between BMI and any of the indices of oxidative stress. Oxidative stress can cause vascular dysfuntion in the placenta, leading to fetal compromise [40]. The elevation in 8-IP secretion from the placenta in women with GDM may induce pathophysiological effects that contribute to adverse pregnancy outcome. To what extent oxidative stress can induce metabolic and developmental dysregulation in GDM is unknown and needs to be clarified. In this study we provide evidence for increased oxidative stress in GDM, as indicated by increased 8-IP release, increased protein carbonyl concentration and the induction of CuZn-SOD activity in placentae complicated by GDM. This state of altered oxidative homeostasis may exacerbate metabolic abnormalities in the maternal milieu, potentially leading to fetal compromise and may represent an additional therapeutic target for pharmacological intervention.
ACKNOWLEDGEMENTS This study was supported by the Medical Research Foundation for Women and Babies, Australia; Diabetes Australia Research Trust number Y00-0085 and an NHMRC Project Grant number 114106. GER was in receipt of a Principle Research Fellowship from the National Health and Medical Research Council of Australia. The authors gratefully acknowledge the assistance of clinical research nurses Valerie Bryant, Angie Denning, Joanna McKay, Lyn Tuttle and Melissa Ryan and the obstetric and midwifery staff at the Mercy Hospital for Women for their cooperation.
84 REFERENCES [1] Metzger BE, Coustan DR. Proceedings of the fourth international workshop-conference on gestational diabetes mellitus. Diabetes Care 1998;21(Suppl. 2):B1–167. [2] King H. Epidemiology of glucose intolerance and gestational diabetes in women of childbearing age. Diabetes Care 1998;21(Suppl. 2):B9–13. [3] Dornhorst A, Bailey PC, Anyaoku V, Elkeles RS, Johnston DG, Beard RW. Abnormalities of glucose tolerance following gestational diabetes. QJM 1990;284:1219–28. [4] Pendergrass M, Fazioni E, De Fronzo R. Non-insulin-dependent diabetes mellitus and gestational diabetes mellitus: same disease, another name? Diabetes Rev 1995;3:566–83. [5] Halliwell B. Antioxidants and human disease: A general introduction. Nutr Rev 1997;55:S44–52. [6] Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes 1991;40:405–12. [7] West IC. Radicals and oxidative stress in diabetes. Diabet Med 2000; 17:171–80. [8] Coughlan MT, Oliva K, Georgiou HM, Permezel JHM, Rice GE. Glucose-induced release of TNF- from human placental and adipose tissues in gestational diabetes mellitus. Diabet Med 2001;18:921–7. [9] Morrow JD. The isoprostanes: their quantification as an index of oxidant stress status in vivo. Drug Metab Rev 2000;32:377–85. [10] Leitch I, Osmond D, Falconer J, Clifton V, Walters W, Read M. Vasoactive effects of 8-epi-prostaglandin-F2alpha in the human placenta in vitro. J Soc Gynecol Investig 1999;6:195A. [11] Morrow JD, Minton TA, Roberts LJ. The F2-isoprostane, 8-epiprostaglandin F2 alpha, a potent agonist of the vascular thromboxane/ endoperoxide receptor, is a platelet thromboxane/endoperoxide receptor antagonist. Prostaglandins 1992;44:155–63. [12] Pratico D, Smyth EM, Violi F, FitzGerald GA. Local amplification of platelet function by 8-Epi prostaglandin F2alpha is not mediated by thromboxane receptor isoforms. J Biol Chem 1996;271:14916–24. [13] Wentzel P, Eriksson UJ. 8-Iso-PGF(2alpha) administration generates dysmorphogenesis and increased lipid peroxidation in rat embryos in vitro. Teratology 2002;66:164–8. [14] Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts LJ. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical catayzed mechanism. Proc Natl Acad Sci U S A 1990;87:9383–7. [15] Gopaul NK, Anggard EE, Mallet AI, Betteridge DJ, Wolff SP, Nourooz-Zadeh J. Plasma 8-epi-PGF2 levels are elevated in individuals with non-insulin dependent diabetes mellitus. FEBS Lett 1995; 368:225–9. [16] Devaraj S, Hirany SV, Burk RF, Jialal I. Divergence between LDL oxidative susceptibility and urinary F-2-isoprostanes as measures of oxidative stress in Type 2 diabetes. Clin Chem 2001;47:1974–9. [17] Davi G, Ciabattoni G, Consoli A, Mezzetti A, Falco A, Santarone S et al. In vivo formation of 8-iso-prostaglandin F-2 alpha and platelet activation in diabetes mellitus. Circulation 1999;99:224–9. [18] Gerber RT, Holemans K, O’Brien-Coker I, Mallet AI, van Bree R, Van Asschem FA et al. Increase of the isoprostane 8-isoprostaglandin F-2alpha in maternal and fetal blood of rats with streptozotocin-induced diabetes: evidence of lipid peroxidation. Am J Obstet Gynecol 2000; 183:1035–40. [19] Grune T, Reinheckel T, Davies KJ. Degradation of oxidized proteins in mammalian cells. FASEB J 1997;11:526–34. [20] Stadtman ER, Berlett BS. Reactive oxygen-mediated protein oxidation in aging and disease. Drug Metab Rev 1998;30:225–43.
Placenta (2004), Vol. 25 [21] Dalle-Donne I, Giustarini D, Colombo R, Rossi R, Milzani A. Protein carbonylation in human diseases. Trends Mol Med 2003;9:169–76. [22] Konukoglu D, Kemerli GD, Sabuncu T, Hatemi HH. Protein carbonyl content in erythrocyte membranes in type 2 diabetic patients. Horm Metab Res 2002;34:367–70. [23] Cederberg J, Basu S, Eriksson UJ. Increased rate of lipid peroxidation and protein carbonylation in experimental diabetic pregnancy. Diabetologia 2001;44:766–74. [24] Walsh SW. Maternal-placental interactions of oxidative stress and antioxidants in preeclampsia. Seminars In Reproductive Endocrinology 1998; 16:93–104. [25] Myatt L, Eis AL, Brockman DE, Kossenjans W, Greer IA, Lyall F. Differential localization of superoxide dismutase isoforms in placental villous tissue of normotensive, pre-eclamptic, and intrauterine growthrestricted pregnancies. J Histochem Cytochem 1997;45:1433–8. [26] Takehara Y, Yoshioka T, Sasaki J. Changes in the levels of lipoperoxide and antioxidant factors in human placenta during gestation. Acta Med Okayama 1990;44:103–11. [27] Hoffman L, Nolan C, Wilson JD, Oats JJN, Simmons D. Gestational diabetes mellitus—management guidelines. The Australasian Diabetes in Pregnancy Society. MJA 1998;169:93–7. [28] Farrugia W, Nicholls L, Rice GE. Effect of bacterial endotoxin on the in vitro release of Type II phospholipase-A2 and prostaglandin E2 from human placenta. J Endocrinol 1999;160:291–6. [29] Farrugia W, Ho PW, Rice GE, Moseley JM, Permezel M, Wlodek ME. Parathyroid hormone-related protein(1–34) in gestational fluids and release from human gestational tissues. J Endocrinol 2000;165:657–62. [30] Natarajan R, Lanting L, Gonzales N, Nadler J. Formation of an F2-isoprostane in vascular smooth muscle cells by elevated glucose and growth factors. Am J Physiol 1996;271:H159–65. [31] Wentzel P, Welsh N, Eriksson UJ. Developmental damage, increased lipid peroxidation, diminished cyclooxygenase-2 gene expression, and lowered prostaglandin E2 levels in rat embryos exposed to a diabetic environment. Diabetes 1999;48:813–20. [32] 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. [33] Awasthi YC, Dao DD. Glutathione-mediated detoxification mechanisms of human placenta. Placenta 1981;3:289–301. [34] Kinalski M, Sledziewski A, Telejko B, Kowalska I, Kretowski A, Zarzycki W et al. Lipid peroxidation, antioxidant defence and acid-base status in cord blood at birth: the influence of diabetes. Horm Metab Res 2001;33:227–31. [35] Pustovrh C, Jawerbaum A, Sinner D, Pesaresi M, Baier M, Micone P et al. Membrane-type matrix metalloproteinase-9 activity in placental tissue from patients with pre-existing and gestational diabetes mellitus. Reprod Fertil Dev 2000;12:269–75. [36] Kamath U, Rao G, Raghothama C, Rai L, Rao P. Erythrocyte indicators of oxidative stress in gestational diabetes. Acta Paediatr 1998;87:676–9. [37] Loven A, Romem Y, Pelly IZ, Holcberg G, Agam G. Copper metabolism—a factor in gestational diabetes? Clin Chim Acta 1992; 213:51–9. [38] Solomon CG, Willett WC, Carey VJ, Rich-Edwards J, Hunter DJ, Colditz GA et al. A prospective study of pregravid determinants of gestational diabetes mellitus. JAMA 1997;278:1078–83. [39] Davi G, Guagnano MT, Ciabattoni G, Basili S, Falco A, Marinopiccoli M et al. Platelet activation in obese women: role of inflammation and oxidant stress. JAMA 2002;288:2008–14. [40] Myatt L, Kossenjans W, Sahay R, Eis A, Brockman D. Oxidative stress causes vascular dysfunction in the placenta. J Matern Fetal Neonatal Med 2000;9:79–82.
Altered Placental Oxidative Stress Status in Gestational Diabetes Mellitus M. T. Coughlan a,*, P. P. Vervaart b, M. Permezel a, H. M. Georgiou a and G. E. Rice a a
Mercy Perinatal Research Centre, The University of Melbourne, Mercy Hospital for Women, Clarendon St, East Melbourne, Victoria 3002, Australia; b Department of Complex Biochemistry, Royal Children’s Hospital, Melbourne, Australia
Paper accepted 7 July 2003
Oxidative stress has been clearly linked to type 2 diabetes mellitus, however, limited data are available on the involvement of oxidative stress in gestational diabetes mellitus (GDM), a disease of similar pathophysiology. The aim of this study was to investigate the status of placental oxidative stress in healthy pregnant women and women with GDM. The hypothesis to be tested was that tissue markers of oxidative stress are significantly increased in GDM compared to normal placental tissues. Markers of oxidative stress measured were the release of 8-isoprostane (8-epi-prostaglandin F2) from human term placental explants (n=11), the activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase (n=10), and protein carbonyl content (n=12). Placental release of 8-isoprostane was 2-fold greater from women with GDM (P<0.001) compared to healthy pregnant women. Superoxide dismutase activity and protein carbonyl content were elevated in placentae obtained from women with GDM (P<0.04 and P<0.004 respectively), whilst there was no significant difference in the activity of glutathione peroxidase. These data demonstrate the presence of oxidative stress in the placenta from women with GDM, in addition to the induction of a key antioxidant, collectively indicating a state of existing oxidative stress in this condition. Placenta (2004), 25, 78–84 2003 Elsevier Science Ltd. All rights reserved.
INTRODUCTION Gestational diabetes mellitus (GDM) is defined as a glucose intolerance of varying severity with onset or first recognition during pregnancy [1]. It complicates approximately 2–4 per cent of pregnancies [2] and is a significant cause of fetal macrosomia, perinatal mortality and maternal long-term risk of developing type 2 diabetes mellitus [3]. Gestational diabetes mellitus is characterized by hyperglycaemia, insulin resistance and hyperlipidaemia: biochemical abnormalities that are common to type 2 diabetes mellitus. Gestational diabetes mellitus has been considered a ‘pre-diabetic’ state [4] and the pathophysiology of the two are clearly related. Oxidative stress refers to a disturbance in the balance between the production of reactive oxygen species (ROS) and antioxidant defences, that may lead to tissue injury [5]. The link between diabetes, particularly type 2, and oxidative stress has been well described [6,7]. Available evidence includes studies that have identified increased biomarkers of oxygen radical damage and reports that find abnormalities in the antioxidant defences of diabetic patients. An impairment of antioxidant defence has been identified, with lowered circulating enzymatic and non-enzymatic antioxidants in individuals with type 2 diabetes mellitus [7]. *
To whom correspondence should be addressed. Tel.: +61-3-92702556; Fax: +61-3-9417-5406; E-mail: [email protected] 0143-4004/04/$–see front matter
Limited data are available regarding the presence of oxidative stress in women with GDM. The placenta provides the interface of the maternal and fetal circulations, and it may play a crucial role in protecting the fetus from adverse effects of the maternal diabetic milieu, while disturbances in placental function may exacerbate this state. We have reported increased in vitro secretion of tumour necrosis factor- (TNF-) from the placenta and adipose tissue from women with GDM under conditions of high glucose [8] and propose that an altered oxidative homeostasis in women with GDM may be involved. Reactive oxygen species measurement is difficult given their high reactivity, very short half-life and low concentration. Therefore indirect markers are commonly used to evaluate secondary products of ROS damage. The induction of oxidative stress can result in cellular damage, including modification to DNA, proteins and lipids. Lipid peroxidation is the oxidative deterioration of polyunsaturated fatty acids and results in the formation of end products that may themselves pose a further oxidative risk. For example, 8-isoprostane (8-IP) (8-epi prostaglandin F2 or 15-F2t-IsoP) is an F2 isoprostane, one of a unique series of prostaglandin-like compounds formed in vivo from the free-radical-catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase enzyme [9]. It is biologically active, capable of inducing vasoconstriction in the placenta [10] and modulating the function of platelets [11,12]. 2003 Elsevier Science Ltd. All rights reserved.
Coughlan et al.: Placental Oxidant Stress in Gestational Diabetes
In addition, the teratogenicity of 8-IP has recently been demonstrated [13]. 8-isoprostane has been reported to be a stable and sensitive indicator of in vivo lipid peroxidation [14]. Circulating and urinary concentrations of 8-isoprostane are elevated in patients with type 2 diabetes [15–17] and in pregnant rats with streptozotocin-induced diabetes [18]. Oxygen radicals and other ROS cause modifications of proteins [19] and damage to enzymes, receptors, signal transduction pathways and transport proteins can occur. This damage can occur by direct attack of ROS or by secondary damage involving end attack by products of lipid peroxidation. Oxidative stress can give rise to protein carbonyl derivatives, which serve as a more universal biomarker of oxidative stress [20] and have been regarded as the most general and well-used biomarker of severe oxidative protein damage [21]. Protein carbonyl content in erythrocyte membranes is significantly greater in individuals with type 2 diabetes mellitus and correlates with impaired glycaemic control [22]. In addition, protein carbonylation in the plasma of pregnant rats with streptozotocin-induced diabetes was shown to be elevated [23]. To avoid ROS-induced damage of cellular components, several biochemical safety mechanisms are present in the placenta, including defence enzymes and antioxidants. In normal pregnancy, there is an increase in oxidative stress and lipid peroxidation compared with non-pregnant women but there is also a concomitant increase in antioxidant protection that compensates for the increased oxidative stress [24]. The first line of defence against superoxide and hydrogen peroxidemediated injury are the antioxidant enzymes superoxide dismutases (SOD), catalase and glutathione peroxidase (GSHPx). Both copper and zinc-containing SOD (CuZn-SOD) and manganese-containing SOD (Mn-SOD) have been described in the human placenta [25] and SOD increases throughout gestation [26]. Superoxide dismutases are important initial components in the cellular defense against oxygen toxicity, neutralizing superoxide radicals. Superoxide radicals react with hydrogen peroxide to generate single oxygen and hydroxy radicals, which are even more reactive and cytotoxic than superoxide radicals or hydrogen peroxide, and superoxide radicals can initiate lipid peroxidation. Glutathione peroxidase and catalase both neutralize hydrogen peroxide. The aim of this study was to investigate the oxidative stress status of the placenta from women with GDM in comparison to pregnant women with normal glucose tolerance. The hypothesis to be tested was that that tissue markers of oxidative stress are significantly increased in GDM compared to normal placental tissues. Quantification of 8-isoprostane release from the placenta and protein carbonyl content as indices of oxidative stress and the activity of the prominent placental antioxidant enzymes SOD and GSH-Px was determined. MATERIALS AND METHODS Participants Placentae were obtained from a total of 49 pregnant women (25 normal and 24 with GDM) at the time of term Caesarean
79
section. Indications for Caesarean section included repeat Caesarean section (n=35), breech presentation (n=12) or macrosomia (n=2). All of the women were non-smokers and were not administered any antenatal medications. Women with any adverse underlying medical condition were excluded, for example, asthma, arthritis, pre-eclampsia and macrovascular complications. All participants received lumbar epidural analgesia for pain relief during Caesarean section except for one woman who received general analgesia. Women with GDM were diagnosed according to the current criteria of the Australasian Diabetes in Pregnancy Society by either a fasting venous plasma glucose level of R5.5 mmol/l glucose, and/or R8.0 mmol/l glucose 2 h after a 75 g oral glucose load at approximately 28 weeks’ gestation [27]. Thirteen women with GDM were clinically managed by diet alone and 11 women were prescribed insulin in addition to dietary management. The majority of participants were Caucasian, whilst a small number were from various ethnic groups including the Middle East, Asia and the Mediterranean. Approval for this study was obtained from the Mercy Hospital for Women’s Human Research and Ethics Committee and informed consent was obtained from all participating subjects.
Reagents All reagents were purchased from BDH Chemicals Australia (Kilsyth, Vic, Australia) unless otherwise stated. The following reagents were obtained from Sigma Chemical Company (St Louis, MO, USA): -NADH (disodium salt), type II pyruvic acid (dimer free), penicillin/streptomycin and RPMI 1640. The 8-IP enzyme immunoassay kit was from Cayman Chemical Company (Ann Arbor, MI, USA). Phosphate buffered saline (PBS) was purchased from Gibco BRL (Rockville, MD, USA) and Pierce (Rockford, IL, USA) supplied the protein assay kit. The Ransod and Ransel kits were from Randox Laboratories (Crumlin, Northern Ireland, UK). The protein carbonyl kit was obtained from Zenith Technology (Dunedin, New Zealand).
Tissue collection and explant culture Placentae were collected from term elective Caesarean sections performed prior to the onset of labour. Tissue processing commenced within 15 min of delivery and involved the removal of a placental lobule from the central region of the placenta, and dissection free of the chorionic tissue and removal of the basal plate, to obtain villous tissue from the middle cross-section. Eleven placentae from healthy women and eleven from women with GDM were used for explant incubation. Placental villous tissue explants were obtained by blunt dissection and removal of visible connective tissue, vessels and calcium deposits, and were rinsed in PBS (37(C) then RPMI 1640 (37(C). Approximately 200 mg (wet weight/ well) of placental tissue was placed into a 12-well tissue culture
80
plate (Becton Dickinson Labware, NJ, USA) with 2 ml of RPMI 1640, pH 7.2. All incubations were performed in duplicate. The plate was incubated at 37(C in a humidified atmosphere of carbogen (95 per cent O2/5 per cent CO2) in a shaking water bath for 1 h to obtain tissue equilibration. Subsequently, tissue explants were incubated with 2 ml of RPMI 1640, pH 7.2, for 24 h and the incubation medium and tissue were collected and frozen at 80(C until assayed for 8-IP and total protein content. Additional samples of placenta (2–3 g) were collected at time=0 after Caesarean section, briefly rinsed in PBS, snap frozen in liquid nitrogen and stored at 80(C for subsequent analysis. A small number of these placental samples were from placentae that were also used for explant incubation, plus further samples were collected as required exclusively for the determination of antioxidant enzyme activity and/or protein carbonyl content. Thus the total number of placentae obtained in this study was 25 from normal women and 24 from women with GDM.
Total protein determination Measurement of total protein in placental explants was performed as described previously [8]. Total protein content of the tissue was determined using the bicinchoninic acid (BCA) method (Pierce, Rockford, IL, USA) following the microwell plate protocol as described by the manufacturer. The absorbance was quantified using a 96-well microtitre plate reader at 595 nm (Bio-Rad Laboratories, Hercules, CA, USA). The intra- and inter-assay coefficients of variation were 5.0 per cent and 5.5 per cent (over 8 assays) respectively.
Tissue viability To assess tissue viability during in vitro incubation, the release of the intracellular enzyme lactate dehydrogenase (LDH) into the incubation medium was determined as described previously [28]. The absorbance was quantified using a 96-well microtitre plate reader at 340 nm (Bio-Rad Laboratories, Hercules, CA, USA). LDH release was expressed as a per cent of total tissue control which was calculated as LDH activity in the medium divided by total tissue LDH activity multiplied by 100.
8-Isoprostane determination Determination of 8-IP accumulation in the explant incubation medium was performed using a competitive enzyme immunoassay kit (Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer’s specifications. Samples were diluted with RPMI-1640 as necessary within a range of neat to 1/30 depending on the tissue sample and treatment. The absorbance was quantified using a 96-well
Placenta (2004), Vol. 25
microtitre plate reader at 405 nm (Bio-Rad Laboratories, Hercules, CA, USA). The intra- and inter-assay coefficients of variation were 7.2 per cent and 18.5 per cent respectively (over 6 assays) and the limit of detection of the assay was 5 pg/ml. Protein carbonyl determination The protein carbonyl content of the placenta was determined using a commercially available enzyme immunoassay from Zenith Technology (Dunedin, New Zealand). Tissue (200 mg) that was obtained at time=0, was homogenized in ice-cold homogenizing buffer (250 m sucrose, 20 m Tris–HCl, 1 m dithiothreitol, pH 7.4) and centrifuged at 19 000 g for 20 min. Samples and standards were diluted in PBS to a protein concentration of 4 mg/ml. The procedure was followed according to the manufacturer’s instructions. All samples were measured in a single assay. The intra-assay coefficient of variation was 13 per cent and the limit of detection of the assay was 0.05 nmol/mg protein. Total protein of the placental tissue was measured as described above. Superoxide dismutase and glutathione peroxidase determination Enzymatic activity of the antioxidant enzymes SOD and GSH-Px was measured using commercially available kits from Randox Laboratories (Crumlin, Northern Ireland, UK) according to the manufacturer’s instructions. Tissue (obtained at time=0) homogenates (300 mg/ml wet weight) was prepared as follows. Placental tissue, kept on ice, was homogenized using a metal-blade tissue homogenizer (S25N-8G, IKA-Werke GMBH and Co., KG Staufen, Germany) for 1 min in ice-cold PBS, pH 7.4. The homogenate was centrifuged for 25 min at 3373 g and the resulting supernatant was diluted with PBS to 100 mg/ml wet weight. The activities of the antioxidant enzymes were measured on a Cobas Mira autoanalyzer (Roche, Graz, Austria). The SOD assay measures the Cu-ZnSOD isoform only, as Mn-SOD is inactivated by the pH of the assay. Statistical analysis Statistical computations were performed using a commercially available statistics analysis software (Statgraphics Plus for Windows, version 3.1, Statistical Graphics Corp., Rockville, MD, USA). Student’s t-test was used to assess statistical significance between normally distributed data otherwise nonparametric Mann–Whitney U (Wilcoxen) tests were used. Pearson’s correlation coefficient determinations were used in the correlation analyses. Statistical significance was assumed when P<0.05. Grouped results are reported as meanSD. RESULTS The clinical characteristics of all participants involved in the investigation are collated in Table 1. Fasting and 2-h plasma
Coughlan et al.: Placental Oxidant Stress in Gestational Diabetes
81
Table 1. Characteristics of all participants involved in the study
Age (years) Maternal BMI (kg/m2)** Gestational age at birth (weeks) Birth weight (g) Parity Fasting plasma glucose (mmol/l) Two hour plasma glucose (mmol/l)
Normal pregnancy (n=25)
GDM pregnancy (n=24)
32.43.9 24.44.1 38.51.1 3311558 1.90.8 4.20.3 5.31.0
33.06.1 29.99.1* 38.50.7 3513511 2.51.6 5.30.8* 8.81.9*
Data represent meanSD. * P<0.05 compared to normal women. **Based on first antenatal visit at approximately 12 weeks.
glucose values at oral glucose tolerance test were significantly higher in women with GDM compared to healthy pregnant women (P<0.001). The only other significant difference between the study groups was maternal body mass index (BMI), which was greater in the women with GDM (P=0.04). The release of LDH (a marker of cell membrane integrity) into the incubation medium over 24 h was determined for all explant incubations (n=11 in each group). In vitro incubation did not adversely affect LDH activity in the incubation medium and there was no significant difference between tissues from the two participant groups during the 24 h incubation period (5.581.48 per cent of total tissue control for GDM vs 5.051.38 per cent of total tissue control for normal). LDH release from the placental explants is consistent with previously documented levels [29]. Figure 1 illustrates the 8-IP release into the incubation medium over 24 h. Basal release of 8-IP from the placentae of women with GDM was significantly greater compared to normal women (1720721 pg/mg protein for GDM vs 738177 pg/mg protein for normal, P<0.001, n=11). There was a significant correlation between 8-IP release from the placenta and maternal 2-h plasma glucose concentrations after an oral glucose challenge at the time of diagnosis (r2=0.26, P<0.03, Figure 2), but no correlation was apparent between 8-IP release and maternal fasting plasma glucose. Protein carbonyl content of the placenta was significantly elevated in women with GDM compared to pregnant women with normal glucose tolerance (0.1480.153 nmol/mg protein for GDM vs 0.0620.011 nmol/mg protein for normal, P<0.004, Figure 3, n=12). The activities of the antioxidant enzymes CuZn-SOD and GSH-Px were determined in placental tissue obtained from both women with normal glucose tolerance and women with GDM. The activity of CuZn-SOD was significantly higher in the placenta obtained from women with GDM compared to normal women (1.770.23 U/mg protein for GDM vs 1.580.11 U/mg protein for normal, Figure 4, P<0.04, n=10). There was no significant difference in the activity of GSH-Px in placental tissue between healthy pregnant and GDM participants (Figure 5). In addition, there was no difference in any of the assayed placental variables in mothers
Figure 1. Release of 8-isoprostane (pg/mg protein) from human term placental explants obtained from healthy pregnant women (n=11) and women with GDM (n=11) incubated in RPMI 1640 for 24 h. Horizontal lines indicate the mean values and dots represent each data point. P<0.001.
with GDM who were managed by diet alone compared to women who were treated with diet and insulin. The ratio of CuZn-SOD activity to 8-IP release from the placenta from normal women compared to that of women with GDM was 2.1 : 1. The same ratio was found between CuZnSOD to protein carbonyl when comparing normal to GDM.
DISCUSSION The aim of this study was to investigate the placental oxidative stress status of women with GDM by testing the hypothesis that tissue markers of oxidative stress are significantly
82
Placenta (2004), Vol. 25
Figure 2. Correlation between 8-isoprostane release from term placenta and maternal plasma glucose 2 h after a glucose challenge at oral glucose tolerance test. Open squares indicate healthy pregnant women (n=8) and closed squares indicate women with GDM (n=11). r2=0.26, P=0.03.
Figure 3. Protein carbonyls (nmol/mg protein) in the placenta from healthy pregnant women (n=12) at term and women with GDM (n=12). Horizontal lines indicate the mean values and dots represent each data point. P<0.004.
*OXWDWKLRQH 3HUR[LGDVH 8PJ SURWHLQ
Figure 4. Activity of superoxide dismutase (U/mg protein) in the placenta from healthy pregnant women (n=10) at term and women with GDM (n=10). Horizontal lines indicate the mean values and dots represent each data point. P<0.04.
1RUPDO increased in GDM compared to normal placental tissues. This was determined by quantifying the release of 8-IP from placental explants and placental protein carbonyl content as indices of oxidative stress, and determining the activity of the endogenous antioxidant enzymes CuZn-SOD and GSH-Px in placental tissue from women with GDM and healthy women at term.
*'0
Figure 5. Activity of glutathione peroxidase (U/mg protein) in the placenta from healthy pregnant women (n=10) at term and women with GDM (n=10). Horizontal lines indicate the mean values and dots represent each data point.
In this study, the release of 8-IP from placental explants obtained from women with GDM was 2-fold greater than that of healthy pregnant women. 8-isoprostane has been reported to
Coughlan et al.: Placental Oxidant Stress in Gestational Diabetes
be a stable and sensitive indicator of in vivo lipid peroxidation, a central feature of oxidant stress [14]. The marked increase in 8-IP release from placental tissue obtained from women with GDM suggests that these women have increased underlying placental lipid peroxidation as a consequence of increased oxidant stress. In addition, we found a significant positive correlation between plasma glucose (2 h after a glucose challenge at the time of diagnosis) and term placental release of 8-IP. It was interesting to note that the fasting plasma glucose concentrations did not correlate with term placental release of 8-IP, suggesting that lipid peroxidation may be, at least partly, associated with the determinants of glycaemic control. This is consistent with previous findings that demonstrated in vitro glucose-induced 8-IP production in porcine vascular smooth muscle cells [30] and whole rat embryos [31]. Another study by Davi et al. [17] found a positive correlation between urinary 8-IP and plasma glucose in individuals with type 2 diabetes; treatment of hyperglycaemia decreased 8-IP. Oxidative stress can give rise to protein carbonyl derivatives via various mechanisms including direct oxidation of amino acid residues, by reaction with products of lipid peroxidation, and by interaction of reducing sugars or their oxidation products (glycation and glycoxidation products) with lysine residues of proteins [20]. Thus, increased concentrations of protein carbonyls represent a more general marker of oxidative stress than 8-IP, a marker of lipid peroxidation. In this study, we demonstrated greater protein carbonyl content in the placenta from women with GDM compared to healthy pregnant women indicating further the presence of oxidative stress in this condition. In addition, we demonstrated significantly higher CuZnSOD activity in GDM placentae compared to placentae from healthy pregnant women. CuZn-SOD, the cytosolic isoform, is mainly compartmentalized in trophoblast cells within the placenta and may thus serve an important function at the maternal-fetal interface [32]. In the women with GDM, placental CuZn-SOD activity was increased, which may reflect a compensatory protective response to existing oxidative stress in GDM pregnancy. However, the relative ratio of CuZn-SOD to oxidative stress markers (8-IP and protein carbonyl) was lower in the GDM placentae compared to that of normal. This suggests that SOD is not sufficiently compensating for the increased oxidative stress, further clarifying the existence of the oxidative stress in this condition. Superoxide dismutase and catalase activity in the placenta increases as gestation proceeds, whilst GSH-Px activity does not change [26]. It has been suggested that GSH-Px defence against ROS-induced lipid peroxidation may not be as important [33]. In this study, GSH-Px activity was comparable in the placenta of women with GDM and healthy women.
83
This is the first report, to our knowledge, of elevated 8-IP release from, and protein carbonyl concentration in the placenta in GDM. There is a paucity of data characterizing the status of oxidative stress in women with GDM. The data that are available do not afford a consistent profile. Kinalski et al. [34] reported evidence of increased lipid peroxidation but decreased SOD activity in GDM placentae compared to controls. In contrast, Pustovrh et al. [35] were unable to identify any difference in either placental lipid peroxidation or SOD between women with GDM and normal pregnant women. Kamath et al. [36] found elevated erythrocyte malondialdehyde in babies born to mothers with GDM. Consistent with the current study, Loven et al. [37] demonstrated an increase in erythrocyte CuZn-SOD in maternal age-matched pairs of women with GDM compared to healthy pregnant women. The increase in oxidative activity in GDM in this study appears not to be secondary to a deficiency of the antioxidant enzyme defence systems, but may be due to the presence of impaired glycaemic control. There is considerable evidence that oxidative stress generated as a result of hyperglycaemia is implicated in the development of diabetic complications [6]. Several pathways have been proposed for the production of ROS in the presence of high glucose concentration [7]. In the current study, the BMI of the women with GDM was significantly higher than that of the normal women. This is not unexpected, as obesity is a major risk factor for the development of GDM [38]. Although obesity has been recently associated with enhanced lipid peroxidation [39], it is not known if the increased BMI in the women with GDM in this study affected their oxidative stress status. However, there was no significant correlation between BMI and any of the indices of oxidative stress. Oxidative stress can cause vascular dysfuntion in the placenta, leading to fetal compromise [40]. The elevation in 8-IP secretion from the placenta in women with GDM may induce pathophysiological effects that contribute to adverse pregnancy outcome. To what extent oxidative stress can induce metabolic and developmental dysregulation in GDM is unknown and needs to be clarified. In this study we provide evidence for increased oxidative stress in GDM, as indicated by increased 8-IP release, increased protein carbonyl concentration and the induction of CuZn-SOD activity in placentae complicated by GDM. This state of altered oxidative homeostasis may exacerbate metabolic abnormalities in the maternal milieu, potentially leading to fetal compromise and may represent an additional therapeutic target for pharmacological intervention.
ACKNOWLEDGEMENTS This study was supported by the Medical Research Foundation for Women and Babies, Australia; Diabetes Australia Research Trust number Y00-0085 and an NHMRC Project Grant number 114106. GER was in receipt of a Principle Research Fellowship from the National Health and Medical Research Council of Australia. The authors gratefully acknowledge the assistance of clinical research nurses Valerie Bryant, Angie Denning, Joanna McKay, Lyn Tuttle and Melissa Ryan and the obstetric and midwifery staff at the Mercy Hospital for Women for their cooperation.
84 REFERENCES [1] Metzger BE, Coustan DR. Proceedings of the fourth international workshop-conference on gestational diabetes mellitus. Diabetes Care 1998;21(Suppl. 2):B1–167. [2] King H. Epidemiology of glucose intolerance and gestational diabetes in women of childbearing age. Diabetes Care 1998;21(Suppl. 2):B9–13. [3] Dornhorst A, Bailey PC, Anyaoku V, Elkeles RS, Johnston DG, Beard RW. Abnormalities of glucose tolerance following gestational diabetes. QJM 1990;284:1219–28. [4] Pendergrass M, Fazioni E, De Fronzo R. Non-insulin-dependent diabetes mellitus and gestational diabetes mellitus: same disease, another name? Diabetes Rev 1995;3:566–83. [5] Halliwell B. Antioxidants and human disease: A general introduction. Nutr Rev 1997;55:S44–52. [6] Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes 1991;40:405–12. [7] West IC. Radicals and oxidative stress in diabetes. Diabet Med 2000; 17:171–80. [8] Coughlan MT, Oliva K, Georgiou HM, Permezel JHM, Rice GE. Glucose-induced release of TNF- from human placental and adipose tissues in gestational diabetes mellitus. Diabet Med 2001;18:921–7. [9] Morrow JD. The isoprostanes: their quantification as an index of oxidant stress status in vivo. Drug Metab Rev 2000;32:377–85. [10] Leitch I, Osmond D, Falconer J, Clifton V, Walters W, Read M. Vasoactive effects of 8-epi-prostaglandin-F2alpha in the human placenta in vitro. J Soc Gynecol Investig 1999;6:195A. [11] Morrow JD, Minton TA, Roberts LJ. The F2-isoprostane, 8-epiprostaglandin F2 alpha, a potent agonist of the vascular thromboxane/ endoperoxide receptor, is a platelet thromboxane/endoperoxide receptor antagonist. Prostaglandins 1992;44:155–63. [12] Pratico D, Smyth EM, Violi F, FitzGerald GA. Local amplification of platelet function by 8-Epi prostaglandin F2alpha is not mediated by thromboxane receptor isoforms. J Biol Chem 1996;271:14916–24. [13] Wentzel P, Eriksson UJ. 8-Iso-PGF(2alpha) administration generates dysmorphogenesis and increased lipid peroxidation in rat embryos in vitro. Teratology 2002;66:164–8. [14] Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts LJ. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical catayzed mechanism. Proc Natl Acad Sci U S A 1990;87:9383–7. [15] Gopaul NK, Anggard EE, Mallet AI, Betteridge DJ, Wolff SP, Nourooz-Zadeh J. Plasma 8-epi-PGF2 levels are elevated in individuals with non-insulin dependent diabetes mellitus. FEBS Lett 1995; 368:225–9. [16] Devaraj S, Hirany SV, Burk RF, Jialal I. Divergence between LDL oxidative susceptibility and urinary F-2-isoprostanes as measures of oxidative stress in Type 2 diabetes. Clin Chem 2001;47:1974–9. [17] Davi G, Ciabattoni G, Consoli A, Mezzetti A, Falco A, Santarone S et al. In vivo formation of 8-iso-prostaglandin F-2 alpha and platelet activation in diabetes mellitus. Circulation 1999;99:224–9. [18] Gerber RT, Holemans K, O’Brien-Coker I, Mallet AI, van Bree R, Van Asschem FA et al. Increase of the isoprostane 8-isoprostaglandin F-2alpha in maternal and fetal blood of rats with streptozotocin-induced diabetes: evidence of lipid peroxidation. Am J Obstet Gynecol 2000; 183:1035–40. [19] Grune T, Reinheckel T, Davies KJ. Degradation of oxidized proteins in mammalian cells. FASEB J 1997;11:526–34. [20] Stadtman ER, Berlett BS. Reactive oxygen-mediated protein oxidation in aging and disease. Drug Metab Rev 1998;30:225–43.
Placenta (2004), Vol. 25 [21] Dalle-Donne I, Giustarini D, Colombo R, Rossi R, Milzani A. Protein carbonylation in human diseases. Trends Mol Med 2003;9:169–76. [22] Konukoglu D, Kemerli GD, Sabuncu T, Hatemi HH. Protein carbonyl content in erythrocyte membranes in type 2 diabetic patients. Horm Metab Res 2002;34:367–70. [23] Cederberg J, Basu S, Eriksson UJ. Increased rate of lipid peroxidation and protein carbonylation in experimental diabetic pregnancy. Diabetologia 2001;44:766–74. [24] Walsh SW. Maternal-placental interactions of oxidative stress and antioxidants in preeclampsia. Seminars In Reproductive Endocrinology 1998; 16:93–104. [25] Myatt L, Eis AL, Brockman DE, Kossenjans W, Greer IA, Lyall F. Differential localization of superoxide dismutase isoforms in placental villous tissue of normotensive, pre-eclamptic, and intrauterine growthrestricted pregnancies. J Histochem Cytochem 1997;45:1433–8. [26] Takehara Y, Yoshioka T, Sasaki J. Changes in the levels of lipoperoxide and antioxidant factors in human placenta during gestation. Acta Med Okayama 1990;44:103–11. [27] Hoffman L, Nolan C, Wilson JD, Oats JJN, Simmons D. Gestational diabetes mellitus—management guidelines. The Australasian Diabetes in Pregnancy Society. MJA 1998;169:93–7. [28] Farrugia W, Nicholls L, Rice GE. Effect of bacterial endotoxin on the in vitro release of Type II phospholipase-A2 and prostaglandin E2 from human placenta. J Endocrinol 1999;160:291–6. [29] Farrugia W, Ho PW, Rice GE, Moseley JM, Permezel M, Wlodek ME. Parathyroid hormone-related protein(1–34) in gestational fluids and release from human gestational tissues. J Endocrinol 2000;165:657–62. [30] Natarajan R, Lanting L, Gonzales N, Nadler J. Formation of an F2-isoprostane in vascular smooth muscle cells by elevated glucose and growth factors. Am J Physiol 1996;271:H159–65. [31] Wentzel P, Welsh N, Eriksson UJ. Developmental damage, increased lipid peroxidation, diminished cyclooxygenase-2 gene expression, and lowered prostaglandin E2 levels in rat embryos exposed to a diabetic environment. Diabetes 1999;48:813–20. [32] 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. [33] Awasthi YC, Dao DD. Glutathione-mediated detoxification mechanisms of human placenta. Placenta 1981;3:289–301. [34] Kinalski M, Sledziewski A, Telejko B, Kowalska I, Kretowski A, Zarzycki W et al. Lipid peroxidation, antioxidant defence and acid-base status in cord blood at birth: the influence of diabetes. Horm Metab Res 2001;33:227–31. [35] Pustovrh C, Jawerbaum A, Sinner D, Pesaresi M, Baier M, Micone P et al. Membrane-type matrix metalloproteinase-9 activity in placental tissue from patients with pre-existing and gestational diabetes mellitus. Reprod Fertil Dev 2000;12:269–75. [36] Kamath U, Rao G, Raghothama C, Rai L, Rao P. Erythrocyte indicators of oxidative stress in gestational diabetes. Acta Paediatr 1998;87:676–9. [37] Loven A, Romem Y, Pelly IZ, Holcberg G, Agam G. Copper metabolism—a factor in gestational diabetes? Clin Chim Acta 1992; 213:51–9. [38] Solomon CG, Willett WC, Carey VJ, Rich-Edwards J, Hunter DJ, Colditz GA et al. A prospective study of pregravid determinants of gestational diabetes mellitus. JAMA 1997;278:1078–83. [39] Davi G, Guagnano MT, Ciabattoni G, Basili S, Falco A, Marinopiccoli M et al. Platelet activation in obese women: role of inflammation and oxidant stress. JAMA 2002;288:2008–14. [40] Myatt L, Kossenjans W, Sahay R, Eis A, Brockman D. Oxidative stress causes vascular dysfunction in the placenta. J Matern Fetal Neonatal Med 2000;9:79–82.