Placental antioxidant enzyme status and lipid peroxidation in pregnant women with type 1 diabetes: The effect of vitamin C and E supplementation

    Placental antioxidant enzyme status and lipid peroxidation in pregnant women with type 1 diabetes: the effect of vitamin C and E supplementation Philip C. Johnston, David R. McCance, Valerie A. Holmes, Ian S. Young, Ann McGinty PII: DOI: Reference:

S1056-8727(15)00391-8 doi: 10.1016/j.jdiacomp.2015.10.001 JDC 6560

To appear in:

Journal of Diabetes and Its Complications

Received date: Revised date: Accepted date:

13 May 2015 7 September 2015 3 October 2015

Please cite this article as: Johnston, P.C., McCance, D.R., Holmes, V.A., Young, I.S. & McGinty, A., Placental antioxidant enzyme status and lipid peroxidation in pregnant women with type 1 diabetes: the effect of vitamin C and E supplementation, Journal of Diabetes and Its Complications (2015), doi: 10.1016/j.jdiacomp.2015.10.001

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ACCEPTED MANUSCRIPT Placental antioxidant enzyme status and lipid peroxidation in pregnant women with type 1 diabetes: the effect of vitamin C and E supplementation

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Philip C Johnston1, David R McCance1, Valerie A Holmes2, Ian S Young2, Ann McGinty2 1

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Dr Ann McGinty Nutrition and Metabolism Group Pathology Building Grosvenor Road Centre for Public Health Queen’s University Belfast Belfast BT12 6BA UK

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Address for Correspondence:

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Regional Centre for Endocrinology and Diabetes, Royal Victoria Hospital, Belfast, 2Nutrition and Metabolism Group, Centre for Public Health, Queen’s University Belfast, UK

Tel: +(044) 2890632730

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E Mail: [email protected]

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Fax: +(044) 2890235900

Acknowledgements: We would like to thank all the women who participated in the study. We would also like to thank Dr Caroline Mercer, Cyril McMaster and Kathy Pogue for technical help with placental analysis and Dr Chris Cardwell for advice on statistical analysis. Disclosure of interests: The authors have no conflict of interest to declare Ethics approval: West Midlands Multi-Centre Research Committee (MREC/02/7/16) Funding: Northern Ireland Research and Development office (EAT/3474/06) Key words: placenta, antioxidant status, pre-eclampsia, Vitamin C and E supplementation, lipid peroxidation, type 1 diabetes mellitus

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ACCEPTED MANUSCRIPT Abbreviations Diabetes and Pre-eclampsia Interventional Trial Enzyme Linked Immunosorbent Assay Glutathione Peroxidase Glutathione Reductase International Society for the Study of Hypertension in Pregnancy International Units Sodium Chloride Sodium Orthovanadate Normotension Optical Density Phosphate Buffered Saline Pre-eclampsia Potential for Hydrogen Ion Concentration PhenylMethaneSulfonylFluoride Systolic Blood Pressure Superoxide Dismutase Standard Deviation Statistical Package for the Social Sciences Micromoles per Litre

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DAPIT ELISA Gpx Gred ISSHP IU NaCl NA3VO4 NT OD PBS PE pH PMSF SBP SOD SD SPSS uMOL/L

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ACCEPTED MANUSCRIPT ABSTRACT

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Aim: In view of the increased rates of pre-eclampsia observed in diabetic pregnancy and the lack of ex vivo data on placental biomarkers of oxidative stress in T1 diabetic pregnancy, the aim of the current investigation was to examine placental antioxidant enzyme status and lipid peroxidation in pregnant women with type 1 diabetes. A further objective of the study was to investigate, the putative impact of vitamin C and E supplementation on antioxidant enzyme activity and lipid peroxidation in type 1 diabetic placentae.

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Methods: The current study measured levels of antioxidant enzyme [glutathione peroxidase (Gpx), glutathione reductase (Gred), superoxide dismutase (SOD) and catalase] activity and degree of lipid peroxidation (aqueous phase hydroperoxides and 8-iso-Prostaglandin F2α) in matched central and peripheral samples from placentae of DAPIT (n=57) participants. Levels of vitamin C and E were assessed in placentae and cord blood.

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Results: Peripheral placentae demonstrated significant increases in Gpx and Gred activities in pre-eclamptic in comparison to non pre-eclamptic women. Vitamin C and E supplementation had no significant effect on cord blood or placental levels of these vitamins, nor on placental antioxidant enzyme activity or degree of lipid peroxidation in comparison to placebo-supplementation.

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Conclusion: The finding that maternal supplementation with vitamin C/E does not augment cord or placental levels of these vitamins is likely to explain the lack of effect of such supplementation on placental indices including antioxidant enzymes or markers of lipid peroxidation.

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ACCEPTED MANUSCRIPT 1. Introduction

Pre-eclampsia is defined by the new onset of hypertension during pregnancy

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accompanied by the development of proteinuria, it is associated with significant

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maternal and perinatal morbidity and mortality. Although our understanding of the pathogenesis of pre-eclampsia has improved significantly, the exact aetiology is still

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not fully understood, however the pathophysiology involves abnormal placental development during the first trimester with resulting placental insufficiency and the

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subsequent release of placental factors into maternal circulation resulting in the clinical syndrome of pre-eclampsia [1,2]. Oxidative stress plays an important role in the normal inflammatory response of pregnancy, however, evidence has been provided that pregnancies complicated by pre-eclampsia are associated with

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increased concentrations of oxidative stress markers including lipid peroxidation

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products, and a reduction in antioxidant concentrations [3,4]. Most studies have examined systemic markers of oxidative stress and antioxidant status, limited

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analyses have suggested that placental oxidative stress is also present in pre-

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eclampsia [5]. The antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidise (Gpx), glutathione reductase (Gred) and catalase are essential in scavenging hydrogen peroxide which potentiates injury at the cellular level [6]. Numerous studies have examined the relationship between exogenous antioxidant supplementation and their impact on reducing pre-eclampsia risk, however these studies were derived mainly from non-diabetic populations [7-11]. Rates of pre-eclampsia are 2-4 times higher in diabetic pregnancy in comparison to their non-diabetic counterparts, furthermore, pregnancy outcomes in the UK remain worse for women with pre-existing diabetes [12]. The Diabetes and Pre-eclampsia Intervention Trial (DAPIT) addressed the impact of vitamin C/E supplementation on 4

ACCEPTED MANUSCRIPT the risk of pre-eclampsia in a large population of type 1 diabetic women [13]. In DAPIT the authors also demonstrated that in women who developed pre-eclampsia,

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glycaemic control was significantly higher before and during pregnancy compared

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with women who did not develop pre-eclampsia [14]. The DAPIT study also showed that poor glycaemic control was associated with the development of pre-eclampsia

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but not pregnancy induced hypertension. These data are important in terms of their support for putative relevance of poor glycaemic control to increased production of

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free radical species (lipid peroxidation) with inadequate antioxidant defence mechanisms in the pathogenesis of pre-eclampsia.

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A systematic review and meta-analysis of nine RCTs of supplementation with vitamins C/E for the prevention of pre-eclampsia has concluded that such There is a lack of ex vivo

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supplementation does not prevent pre-eclampsia [15].

data on placental biomarkers of oxidative stress in T1 diabetic pregnancy and limited

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evidence regarding the impact of antioxidant vitamin supplementation on placental

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indices in pre-eclamptic women. Therefore, the aim of the current study using placentae from a DAPIT sub-cohort was to investigate placental antioxidant enzyme (Gpx, Gred, SOD and catalase) activity and the degree of lipid peroxidation, as measured by aqueous phase hydroperoxides and 8-iso-Prostaglandin F2α, in nonpre-eclamptic pregnancies and those complicated by pre-eclampsia. A further aim of the study was to investigate the putative impact of vitamin C and E supplementation on antioxidant enzyme activity and lipid peroxidation in type 1 diabetic placentae.

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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1 Subjects

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Pre-eclampsia was defined as gestational hypertension with proteinuria using the

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International Society for the Study of Hypertension in Pregnancy guidelines [16]. Each case of hypertensive pregnancy was reviewed by the staff of the Trial Co-

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ordinating Centre, and the diagnosis was confirmed by three senior clinicians,

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working independently and unaware of treatment allocation. Women received placebo or vitamin supplementation (vitamin C [1000 mg] and vitamin E [400 IU], administered on a daily basis from between 8 and 22 weeks gestation until delivery. The DAPIT inclusion and exclusion criteria as well as maternal serum HbA1c, PAI-1

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and PAI-II as well as cord Vitamin C and E analyses and placental nitrotyrosine and

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vitamin C and E has been described previously [13,17].

2.2 Placental collection and preparation

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Placentae (n=57) were collected from a DAPIT sub-cohort which comprised of

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consecutive deliveries in women with Type 1 diabetes at the Royal Maternity Hospital Belfast, Northern Ireland by trained study personnel from January 2004 to December 2008. Approval from the West Midlands Multi-Centre Research Committee (MREC/02/7/16) was obtained to allow for placental collection at this single site. Placental collection has been described in detail previously; one placental block (2 cm3 section) was obtained from both the centre and periphery immediately after delivery (to reduce any inherent structural disparity in placental composition) snap frozen in liquid nitrogen and stored at -200C for analyses of antioxidant enzyme activity and lipid peroxidation markers [17].

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ACCEPTED MANUSCRIPT 2.3 Placental antioxidant enzyme activities

A portion of stored placenta was removed and washed in 0.1M PBS (pH 7.2, 0.15M

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NaCl containing 0.5mM EDTA) then blotted dry. Placental lysates (central and

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peripheral components were then prepared for antioxidant enzyme analyses by homogenising placental tissue for 3 min in a 0.1M PBS, 1mM EDTA, 0.05 % Triton X

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solution. Sonication was performed for 10 min on a Lucas Dawe Sonicator (Lucas Dawe Ultrasound, London, UK). Homogenates were vortexed for 10 sec and left on

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ice for 5 min for a total of 2 cycles. Following this, placental lysates were centrifuged at 4472 x G for 20 min at 40C. Aliquots of the resulting supernatant were used for antioxidant enzyme assay.

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Glutathione peroxidise, glutathione reductase and superoxide dismutase activities

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were measured using commercially available kits on an ILAB 600 clinical analyser supplied by Instrumentation Laboratory, (Lexington, Massachusetts). Glutathione

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peroxidase activity in placental lysates was assessed by kinetic analysis using a

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commercially available Ransel kit (catalogue no:RS505), glutathione reductase was also measured by kinetic analysis using a commercially available kit (catalogue no:GR2368) [both supplied by Randox Laboratories Antrim, N. Ireland]. Gpx and Gred sample absorbance were read at 340nm. Superoxide dismutase activity in placental lysates was measured by kinetic analysis using a commercially available Ransod kit (catalogue no:SD125), Randox Laboratories and sample absorbance was read at 505nm. Gpx, Gred and SOD activities were expressed as U/l/mg. Catalase activity in placental lysates was measured by means of a commercially available EIA Kit [(catalogue no:707002),Cayman Chemicals]. Sample absorbance was read at 540nm on a BioRad Benchmark Microplate reader with data expressed as 7

ACCEPTED MANUSCRIPT nmol/min/mg. The intra-assay CV was 1.45 % for GPX, 3.53 % for SOD and 1.58 % for GRED. The intra-assay CV was 2.4 %, and the inter-assay CV was 11.4 % for

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Catalase.

2.4 Placental lipid peroxidation

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Aqueous phase hydroperoxide

Stored placentae were removed and washed in 0.1M PBS (pH 7.2, 0.15M NaCl

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containing 0.5mM EDTA) blotted dry. After this, placental lysates were then prepared for aqueous phase hydroperoxide analysis by homogenising placental tissue for 3 min in a 1:100 butylated hydroxytoluene [200µM;BHT/PBS] solution. Sonication was performed for 10 minutes on a Lucas Dawe Sonicator (Lucas Dawe Ultrasound,

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London, UK). Placental supernatant was then vortexed for 10 seconds and

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centrifuged at 4472 x G for 5 minutes at 23 0C. Placental aqueous phase hydroperoxide levels were analysed using the Ferrous Oxidation-Xylenol Orange

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(FOX-1) assay. Samples were then read at 560nm on a BioRad Benchmark

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Microplate Reader with data expressed as µmol/mg. The calculated manual intraassay CV was 5.1 % and the inter-assay CV was 10 % for aqueous phase hydroperoxides.

8-iso-Prostaglandin F2α

Placental lysates were prepared for isoprostane analysis by homogenising placental tissue at 40C for 3 min. Following this, 4M NaOH (sodium hydroxide) was added by volume (1:1;v/v]

and heated at 450C for 2 hours to ensure hydrolysis.

After

hydrolysis, samples were cooled at room temperature and treated with an equal volume of 2M HCL (hydrochloric acid) resulting in a final 1:4 dilution. The neutralised 8

ACCEPTED MANUSCRIPT sample were centrifuged at 4472 x G at room temperature for 5 minutes, the pH of the sample was in the range 6-8. Placental 8-iso-Prostaglandin F2α was measured

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by means of a commercially available EIA kit (catalogue no:900-091), supplied by

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Assay Designs Ann Arbor, (Michigan, U.S.A), according to the manufacturer’s instructions. The direct 8-iso-PGF2α EIA antibody (catalogue no:80-1172) is highly

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specific for 8-iso-Prostaglandin F2α (8-iso-PGF2α). All data were handled by an immunoassay software package utilizing a 4 parameter logistic curve fitting program

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(Triturus Cayman Chemicals, U.S.A.) and expressed as pg/mg. The intra-assay CV was 2 %, the inter-assay CV was 9.1% for 8-iso-Prostaglandin F2α.

2.5 Statistics

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Clinical outcomes were compared between patients receiving placebo and vitamin-

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supplementation and between subgroups (non-pre-eclampsia and pre-eclampsia) for all placental and maternal serum analyses using independent T test. To determine if

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an association existed between each separate placental analysis, as well as

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between maternal characteristics both at baseline and throughout pregnancy Pearson’s Correlation was used. Prior to analysis; data for aqueous phase hydroperoxides and 8-iso-Prostaglandin F2α were Log10 transformed to normalize the data. All results were expressed as mean ± standard deviation. All statistical tests were performed using the SPSS for Windows statistical software package version 20.0.

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ACCEPTED MANUSCRIPT 3. Results 3.1 Patient characteristics

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The DAPIT sub-cohort (n=57) was derived from a relatively homogenous population

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of Caucasian women in Northern Ireland. Maternal baseline co-variates were similar in both vitamin and placebo-supplemented groups including age, BMI, and

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gestational age of delivery (Table 1). A longer duration of Type 1 diabetes (p=0.03)

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and an increased rate of previous pre-eclampsia (p=0.001) were present in the placebo group in comparison to vitamin-supplemented women. Rates of current smoking were similar between groups and one patient in the vitamin supplemented group received aspirin.

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In the DAPIT sub-cohort; glycaemic control in both placebo and vitaminsupplemented groups was similar with a reduction in HbA1c observed as pregnancy

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progressed, this was comparable to glycaemic control in the DAPIT study as a whole

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[12,13], and reflects the intensive effort to improve glycemic control in T1DM

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pregnancy. Caesarean-section was performed in 49 women, and vaginal births in 8. Vitamin-supplementation was not associated with any adverse maternal or neonatal outcomes, including gestational age at delivery, pre-term delivery and gestational foetal weight. Vitamin supplementation resulted in a demonstrable rise in maternal plasma vitamin C and serum vitamin E throughout pregnancy in comparison to placebo (Table 1). The rate of pre-eclampsia was 16% (n=9), which is comparable to the full DAPIT cohort, and other published studies among women with type 1 diabetes [18,19]. Gestational age at delivery was significantly lower in the pre-eclamptic group than in

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ACCEPTED MANUSCRIPT the non-pre-eclamptic group (mean age 35.5 ± 2.01 vs 37.15 ± 2.18 weeks,

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p=0.049).

3.2 Placental antioxidant status and lipid peroxidation in pre-eclampsia

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Placental tissue sections (n=114: central 57, peripheral 57) were examined for antioxidant status by measuring antioxidant enzymes Gpx, Gred, SOD and catalase

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activities. Placental lipid peroxidation was measured by assessing levels of aqueous phase hydroperoxides and 8-iso-Prostaglandin F2α. Neither central nor peripheral placentae demonstrated a significant difference in 8-iso-Prostaglandin F2α between

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pre-eclamptic and non pre-eclamptic women, however, levels of aqueous phase hydroperoxides were higher in peripheral samples from pre-eclamptic placentae and

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the difference approached significance (p=0.07), (Table 2). Antioxidant enzyme activity did not differ in the central placentae between the two groups (non pre-

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eclamptic v pre-eclamptic), however, in the peripheral placentae Gpx (U/l/mg: 45.46 [n=9]; p=0.02) and Gred (U/l/mg: 23.90 ± 7.20

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± 13.81 [n=48] v 57.74 ± 15.20

[n=48] v 29.31 ± 7.69 [n=9] v; p=0.04) activities were significantly higher in preeclamptic in comparison to non pre-eclamptic women, respectively. SOD and catalase activities were similar in both central and peripheral placenta between the two groups.

Maternal circulating levels of plasminogen activator inhibitor type 1 (PAI-I) and type II (PAI-II) were measured in DAPIT participants [13]. PAI-II is produced predominantly in the placenta, by trophoblasts, and is considered to to be a marker of placental function throughout pregnancy [20].

A significant correlation was found with 11

ACCEPTED MANUSCRIPT maternal serum levels of PAI-II and central aqueous phase hydroperoxide levels

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(p=0.04, r= -0.82).

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3.3 Comparison of placental antioxidant enzyme activity and lipid peroxidation

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markers in placebo and vitamin-supplemented women

Placental antioxidant enzyme activity or degree of lipid peroxidation were not significantly different in women receiving Vitamin C and E supplementation in comparison to those receiving placebo (Table 3).

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3.4 Impact of mode of delivery and ante-natal steroids on placental antioxidant

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enzyme activity and lipid peroxidation markers

The potential confounding effect of mode of delivery on antioxidant enzyme activity

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and markers of lipid peroxidation (C-section v vaginal birth) was examined. Central

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Gred activity was significantly increased (U/l/mg: 25.78 ± 7.97 [C-section, n=49] v 18.66 ± 9.16 [vaginal birth, n=8], p=0.03), while increases in central Gpx activity with delivery by caesarean section approached significance (U/l/mg: 48.79 ± 14.07 [Csection, n=49] v 38.25 ± 14.39 [vaginal birth, n=8], p=0.05). The administration of ante-natal steroids also had a significant effect on placental antioxidant enzyme activity, specifically, both central and peripheral Gred activity was increased by the use of ante-natal steroids (U/l/mg: central; 22.33 ± 7.60 [no steroids, n=34] v 28.41 ± 8.47 [steroids, n=23]; p=0.01. Peripheral; 22.45 ± 6.66 [no steroids, n=34] v 28.09 ± 7.47 [steroids, n=23]; p=0.01). Central and peripheral activity of Gpx were also increased by the use of ante-natal steroids (U/l/mg: central; 43.73 ± 12.86 [no 12

ACCEPTED MANUSCRIPT steroids] v 52.61± 15.34 [steroids]; p=0.02. Peripheral; 42.05 ± 12.32 [no steroids] v 55.14± 14.44 [steroids]; p=0.01).

No significant difference between levels of

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markers of lipid peroxidation were found with administration of ante-natal steroids or

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with mode of delivery.

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3.5 Relationship between antioxidant status, lipid peroxidation and glycaemic control

For each respective antioxidant enzyme and lipid peroxide marker the placental

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central and peripheral activities correlated significantly. Central Gpx correlated with central Gred and SOD but not catalase, the same was true for the peripheral placentae. Central Gred correlated with central Gpx and SOD, again significant

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correlations were seen for peripheral placentae (data not included). HbA1c at baseline was positively associated with Gred (p=0.01, r= +0.391) and Gpx

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(p=0.01, r=+0.355) activities in central placentae. HbA1c at 26 weeks was also positively associated with central Gred activity (p=0.04, r=+0.289). Glycaemic control

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during the last trimester did not correlate with any antioxidant enzymes or markers of

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lipid peroxidation. Placental aqueous phase hydroperoxide levels did not correlate with any placental antioxidant enzymes. Central placental 8-iso-Prostaglandin F2α was negatively associated with central (p=0.01, r=-0.377) and peripheral (p=0.04, r=0.272) Gred. Peripheral 8-iso-Prostaglandin F2α was negatively associated with central Gpx (p=0.04, r=-0.278) and Gred (p=0.02, r=-0.299) but positively associated with peripheral SOD (p=0.02, r=+0.308).

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ACCEPTED MANUSCRIPT 4. DISCUSSION

The current study represents the first significant analysis of placentae from women

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from a key RCT of antioxidant vitamin supplementation for the prevention of pre-

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eclampsia, involving as it does measurement of four key antioxidant enzyme activities and two markers of lipid peroxidation in both central and peripheral sections The results of the

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from fifty-seven placentae from women with Type 1 diabetes.

current study provide mechanistic ex vivo evidence regarding placental oxidative

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status in high risk pregnancy. The current study found some evidence of augmented oxidative stress (increased antioxidant enzyme activity and a trend towards increased levels of peripheral aqueous phase hydroperoxides) in the placenta from pregnancies

complicated

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pre-eclampsia.

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antioxidant

vitamin

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supplementation was not found to augment placental or cord vitamin levels, nor to

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modulate placental antioxidant enzyme activity or levels of lipid peroxidation markers, thereby providing novel evidence that increases in circulating levels of

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these vitamins has limited impact on placental indices.

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A finding of the current study is that some evidence of increased oxidative stress was found in the peripheral, but not central placental samples from pregnancies complicated by pre-eclampsia.

Specifically, Gpx and Gred activities were

significantly higher and levels of aqueous phase hydroperoxides were higher, albeit that this difference did not achieve significance. The majority of the DAPIT sub cohort were delivered by Caesarean section, with 14% delivered vaginally. It has been suggested that the mode of delivery may result in differing levels of placental oxidative stress with dynamic changes in the levels of antioxidants and free radicals [21], increases were seen in central Gred activity, but not levels of lipid peroxidation. 14

ACCEPTED MANUSCRIPT Similarly, administration of ante-natal steroids was found to significantly augment both central and peripheral Gpx and Gred activities. It is important to note that 7/9

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(77%) of the pre-eclamptic group received steroids, in comparison to 16/48 (33%) of

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the non-pre-eclamptic group. Given that multivariate analysis with logistic regression could not be performed in the current study due to the fact that there were less than

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10 cases of pre-eclampsia, it is not possible to determine the relative contributions of pre-eclampsia vs steroid administration to the observed increases in peripheral Gpx

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and Gred. Notwithstanding this limitation, the finding that peripheral, but not central, Gpx and Gred activities, were significantly increased in pre-eclamptic placentae, provides evidence that the placenta may not be acting as a homogenous entity with

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regards to an oxidative stress environment and that placental architecture differs between the central and peripheral components in pre-eclamptic placentae. In favour

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of this a trend towards increased lipid peroxidation and significantly increased antioxidant enzyme activity indicative of an oxidative stress environment was

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observed in peripheral and not central placentae from pre-eclamptic women. In the

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current study placental histomorphology was not performed. In comparison to non-diabetic pregnancy, diabetic pregnancy has been shown to have increased levels of lipid peroxidation, one possible explanation for this is that increased glycated proteins present in diabetic women could act as sources of free radicals which may play a role in lipid peroxidation [22,23].

The hyperglycaemic

milieu in diabetic pregnancy resulting in inherent oxidative stress has been observed in uncomplicated normotensive T1DM pregnancy as demonstrated by placental nitrotyrosine immunopositivity reflecting the pro-oxidant peroxynitrite production [17]. The lack of differences found in placental SOD and catalase activities between nonpre-eclamptic and pre-eclamptic pregnancies, may be due to the relative degree of 15

ACCEPTED MANUSCRIPT hyperglycaemia in T1DM, and the overall increased oxidative stress environment, resulting in increased difficulty in distinguishing or detecting differences in placental It has been

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markers of oxidative stress, including these anti-oxidant enzymes.

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reported that elevated levels of lipid peroxides in diabetes mellitus inhibit superoxide dismutase in erythrocyte membranes leading to accumulation of superoxide radicals

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which are responsible for maximum lipid peroxidation and endothelial dysfunction

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[24,25].

Numerous studies regarding the role of antioxidant enzymes and markers of lipid peroxidation in the pathogenesis of pre-eclampsia have been performed, with the majority directly comparing normotension and pre-eclampsia in uncomplicated low

population.

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risk pregnancies [24,26-31], the majority of which were from a non-diabetic Evidence of oxidative stress in the placentae from women with pre-

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eclampsia has been well documented [32-37],

however, conflicting results of

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individual studies relating to antioxidant enzymes and markers of lipid peroxidation, most likely reflect the varying populations and different sources of material studied

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including maternal serum, cord and placentae. The current study provides a unique insight into placental oxidative stress from a high risk cohort of women all of whom had Type 1 diabetes. The original hypothesis underlying the DAPIT trial was that the addition of antioxidants vitamin C and E during high risk diabetic pregnancy might enhance overall antioxidant status and reduce free radical production or reactive oxygen species, thereby ameliorating the deleterious impact of oxidative stress and the subsequent risk of developing pre-eclampsia. It has previously been shown in this DAPIT sub-cohort that maternal vitamin C and E supplementation compared with placebo, significantly increased maternal serum levels of these respective

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ACCEPTED MANUSCRIPT vitamins during pregnancy, however, this effect was not seen in cord blood or placentae [Table 1].

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This finding could be attributed to the observation that placental tissue is only slightly

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permeable to vitamin E and that low levels of lipoproteins are observed in the foetal circulation, as vitamin E circulates in blood attached to lipoproteins [38]. In addition

Clearly, if supplementation with vitamin C/E

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the presence of hyperglycaemia [39].

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the placental transport of vitamin C is Na+K+ATPase dependant and is inhibited in

augments maternal, but not cord or placental levels of these vitamins, it is to be expected that there would be limited effect of such supplementation on placental indices, including antioxidant enzymes or markers of lipid peroxidation. A systematic

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review and meta-anlysis of nine RCTs of supplementation with vitamins C/E for the prevention of pre-eclampsia has concluded that such supplementation does not Several possible reasons for the lack of effect have

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prevent pre-eclampsia [15].

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been suggested, including the fact that oxidative stress may be pathophysiologically relevant, but not causal in the development of pre-eclampsia [39,40 ]. Alternatively,

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oxidative stress may only be of relevance to a subgroup of women and not the general population. Arguing against the latter, however, stratified analysis by risk category at trial entry by Conde-Agudelo et al., indicated that vitamin C/E supplementation did not decrease the risk of pre-eclampsia in a range of risk categories [15].

There is some evidence that gestational supplementation with

vitamin C/E may reduce the risk of pre-eclampsia in women with a low baseline antioxidant status, in this regard the DAPIT study as a whole found that women with a low antioxidant status at baseline (plasma ascorbate <10 µmol/L or serum αtocopherol ≤5 µmol/mmol cholesterol) who received vitamins C and E, had a reduced risk of pre-eclampsia in comparison to placebo, although the numbers were 17

ACCEPTED MANUSCRIPT small and the differences did not achieve statistical significance [13].

In broad

agreement with these findings, a randomised, placebo-controlled trial of 110

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pregnant women (between 8-12 weeks gestation) who had low baseline antioxidant

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status were treated with either antioxidants or control diets daily until 2 weeks’ postpartum. The study reported significant differences between the supplementation

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and control group for the incidence of pre-eclampsia (2.0% versus 14.5%) ,this study also found that mRNA levels of plasma SOD were significantly higher in the vitamin

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supplemented vs placebo supplemented women, providing evidence for a systemic antioxidant effect of these vitamins [41].

A strength of the current study is that it is the first analysis of placentae from a trial of

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gestational vitamin C/E supplementation in women at risk of pre-eclampsia (namely, pre-gestational diabetes). In view of the fact that the initiation of vitamin

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supplementation varied between 8 and 22 weeks of gestation, vitamin supplemented subjects were categorised according to whether supplementation began in week 8-

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12 or week 13-22. No significant differences in the various outcome measures were

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found between these sub-groups, indicating that the gestational point at which vitamin supplementation began did not impact on antioxidant enzyme status or lipid peroxidation markers (data not shown). A further benefit of the study was the measurement of both central and peripheral placental components which enabled the impact of placental homogeneity to be assessed. Additionally, women were derived from a relatively homogenous population and all had T1DM with similar glycaemic control before and during pregnancy. A limitation of the study was that multivariate analysis with logistic regression could not be performed due to the fact that there were less than 10 cases of preeclampsia, in addition rates of previous pre-eclampsia and longer duration of 18

ACCEPTED MANUSCRIPT diabetes were more common in the placebo treated group, which might explain why rates of pre-eclampsia were more common in those given placebo, however,

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glycaemic control was similar at baseline and throughout gestation in the two

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treatment groups [42]. This limited the ability to make definitive conclusions albeit, as discussed, the rate of pre-eclampsia in the DAPIT sub-cohort was comparable to

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that of the complete DAPIT cohort and that found in other studies. Post hoc analysis of both placental lipid peroxides and antioxidant enzyme analysis between the three

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groups pre-eclampsia normotension and pregnancy induced hypertension were perfomed (data not included), in general there was no difference between these groups for these respective placental markers.

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5. Conclusion

In summary the current study found some evidence of augmented oxidative stress in

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the placentae from pregnancies complicated by pre-eclampsia. Antioxidant vitamin supplementation was not found to modulate placental antioxidant enzyme activity or

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levels of lipid peroxidation markers.

Duality of interest

The authors declare that they have no competing interest.

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ACCEPTED MANUSCRIPT Contribution statement

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AmcG devised and managed the study, supervised PCJ, and oversaw manuscript preparation and submission. DMcC co-supervised PCJ and co-wrote the manuscript. VH and IY co-wrote the manuscript. PCJ undertook the laboratory analyses for this study and co-wrote the manuscript. All authors read and approved the final manuscript.

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ACCEPTED MANUSCRIPT Table 1

Vitamins C and E

Placebo

n=27

n=30

31. ± 5.0 12 ± 8.5 28 ± 5.0 6 4

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Age (years) Duration of diabetes (years) 2 BMI (kg/m ) Nulliparity Antihypertensive treatment before pregnancy Previous pre-eclampsia Gestation (weeks) Vaginal births C-Section HbA1c (%) Baseline 26* weeks 34* weeks Renal status at randomization Normal Microalbuminuria Current smoker Ante-natal steroids Aspirin at randomisation Non-pre-eclampsia Pre-eclampsia

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________________________________________________________________________ 31 ± 4.9 + 17 ± 8.9 28 ± 4.9 8 5 #

5 36.6 ± 2.4 3 27

7.1 ± 1.2 6.6 ± 0.9 6.6 ± 0.8

7.2 ± 1.0 6.6 ± 0.7 6.5 ± 0.5

24 1 6 8 1 24 3

26 1 5 ± 15 0 24 6

51.1 ± 24.5 # 73.5 ± 32.7 # 58.6 ± 34.1

44.7 ± 22.0 41.2 ± 17.2 36.3 ± 17.1

6.1 ± 1.2 # 8.6 ± 3.0 # 7.3 ± 2.3

6.2 ± 1.4 5.7 ± 1.1 5.8 ± 1.3

Cord Vitamin C (Umol/l) Vitamin E γ-tocopherol (Umol/l) Vitamin E α-tocopherol (Umol/l)

84.2 ± 21.6 0.68 ± 0.16 9.8 ± 2.6

86.4 ± 29.0 0.63 ± 0.1 8.78 ± 2.1

Placenta Central Vitamin C (Umol/mg) Vitamin E (Umol/mg)

43.8 ± 40.8 1.7 ± 1.2

46.4 ± 39.1 2.1 ± 1.0

Placenta Peripheral Vitamin C (Umol/mg) Vitamin E (Umol/mg)

44.4 ± 36.7 1.8 ± 1.1

47.5 ± 35.2 2.2 ± 1.5

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1 37.1 ± 1.9 5 22

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Maternal plasma ascorbate (µmol/l) Baseline 26* weeks 34* weeks

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Maternal serum αtocopherol†(µmol/mmol chol) Baseline 26* weeks 34* weeks

Data are presented as mean ± SD, * within 2 weeks, †α-tocopherol corrected to serum cholesterol (μmol/mmol), available cord vitamin C and E data (placebo;n=28, vitamin;n=23), +;P=0.03, #;P=0.01, ±;P=0.02 21

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Non- Pre-eclampsia N=48

Pre-eclampsia N=9

51.90 ± 14.04 26.58 ± 8.81 0.20 ± 0.05 162.20 ± 36.67 4.35 ± 1.14 4.22 ± 1.29

0.30 0.49 0.76 0.89 0.70 0.92

45.46 ± 13.81 23.90 ± 7.20 0.18 ± 0.05 167.14 ± 85.26 4.45 ± 0.92 4.36 ± 1.12

57.74 ± 15.20 29.31 ± 7.69 0.21 ± 0.02 154.91 ± 77.14 5.10 ± 0.80 4.26 ± 1.64

0.02 0.04 0.23 0.69 0.06 0.82

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46.45 ± 14.53 24.45 ± 8.42 0.19 ± 0.06 159.05 ± 68.43 4.49 ± 0.95 4.25 ± 0.99

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Central Gpx (U/l/mg) Gred (U/l/mg) SOD (U/l/mg) Catalase (nmol/min/mg) AHP⃰ (µmol/mg) 8-Iso-Pros-F2α⃰ (pg/mg)

Peripheral

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Gpx (U/l/mg) Gred (U/l/mg) SOD (U/l/mg) Catalase (nmol/min/mg) AHP⃰ (µmol/mg) 8-Iso-Pros-F2α⃰ (pg/mg)

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Table 2

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Values are mean ± SD, Gpx:glutathionine peroxidase Gred:glutathione peroxidase SOD:superoxide dismutase AHP:aqueous phase hydroperoxide, ⃰Log10

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ACCEPTED MANUSCRIPT Table 3

Vitamins C and E N=27

Placebo N=30

45.60 ± 14.04 22.93 ± 8.70 0.20 ± 0.08 166.29 ± 74.27

48.86 ± 14.91 26.45 ± 7.97 0.19 ± 0.04 153.72 ± 54.50

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Variable

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GPpx (U/l/mg) Gred (U/l//mg) SOD (U/l/mg) Catalase (nmol/min/mg) APH⃰ (µmol/mg) 8-Iso-Pros-F2α⃰ (pg/mg)

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Central

4.43 ± 1.11 4.39 ± 1.12

4.50 ± 0.84 4.12 ± 0.94

0.78 0.33

45.08 ± 13.56 23.08 ± 7.34 0.19 ± 0.05 156.42 ± 78.92

49.62 ± 15.46 26.34 ± 7.39 0.18 ± 0.04 172.95 ± 87.91

0.24 0.10 0.52 0.46

4.46 ± 0.88 4.42 ± 1.17

4.61 ± 0.97 4.28 ± 1.25

0.55 0.67

Peripheral

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Gpx (U/l/mg) Gred (U/l/mg) SOD (U/l//mg) Catalase (nmol/min/mg) APH⃰ (µmol/mg) 8-Iso-Pros-F2α⃰ (pg/mg)

0.40 0.11 0.79 0.47

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Values are presented as mean ± SD, GPX:glutathionine peroxidase GRED:glutathione peroxidase SOD:superoxide dismutase, APH:aqueous phase hydroperoxide, ⃰ Log10

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REFERENCES

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ACCEPTED MANUSCRIPT 17. Johnston, P.C., Powell, L.A., McCance, D.R., Pogue, K., et al. (2013). Placental protein tyrosine nitration and MAPK in type 1 diabetic pre-eclampsia: impact of antioxidant vitamin supplementation. Journal. Of. Diabetes. and. its. Complications,27,322-327.

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24.Wang, Y., Walsh, S.W. (1996). Antioxidant activities and mRNA expression of superoxide dismutase, catalase, and glutathione peroxidase in normal and preeclamptic placentas. Journal. Of. The. Society. For. Gynecologic. Investigation,3,179-184. 25.Suryawanshi, N.P., Bhutey, A.K., Nagdeote, A.N., Jadhav, A.A., et al. (2006). Study of lipid peroxide and lipid profile in diabetes mellitus. Ind. J. Clin. Biochem,27,126-130.

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26 Regan, C.L., Levine, R.J., Baird, D.D., Ewell, M.G., et al. (2001). No evidence for lipid peroxidation in severe preeclampsia. American. Journal. Of. Obstetrics. And. Gynecology,185,572-578.

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27.Ilhan, N., Ilhan, N., Simsek, M. (2002). The changes of trace elements, malondialdehyde levels and superoxide dismutase activities in pregnancy with or without preeclampsia. Clin. Biochem, 35,393-397.

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28. Atamer, Y., Kocyigit, Y., Yokus, B., Atamer, A., et al. (2005). Lipid peroxidation, antioxidant defense, status of trace metals and leptin levels in preeclampsia. European. Journal. Of. Obstetrics. Gynecology. And. Reproductive. Biology,119,60-66. 29.Pasaoglu, H., Bukan, N., Bulduk, G., Celen, S. (2003). Lipid peroxidation, nitrate and nitrite levels in eclamptic and intrauterine growth retarded pregnancies. Turk. J. Med. Sci ,33,89-93. 30. Sharma, J.B., Sharma, A., Bahadur, A., Vimala, N., et al. (2006). Oxidative stress markers and antioxidant levels in normal pregnancy and pre-eclampsia. Journal. Of. Obstetrics. And. Gynecology, 94,23-27. 31. Hubel, C.A., Mc Laughlin, M.K., Evans, R.W., Hauth, B.A., et al. (1996). Fasting serum triglycerides, free fatty acids and malondialdehyde are increased in pre-eclampsia, are positively correlated and decrease within 48 hours post partum. Am. J. Obstet. Gynecol ,174,975-982. 32. Myatt, L., Eis, A.L.W., Brockman, D.E., Kossenjans, W., et al. (1997). Differential localisation of superoxide dismutase isoforms in placental villous tissue of normotensive, pre-eclamptic and intrauterine growth restricted pregnancies. The. Journal. Of. Histochemistry. And. Cytochemistry ,45,1433-1438. 33. Walsh, S.W., Wang, Y. (1995). Trophoblast and placental villous core production of lipid peroxides,thromboxane, and prostacyclin in preeclampsia. J. Clin. Endocrinol. Metab, 80,1888–1893. 25

ACCEPTED MANUSCRIPT 34. Staff, A.C., Halvorsen, B., Ranheim, T., Henriksen, T. (1999). Elevated levels of free 8-isoprostanglandin F2 alpha in the decidua basalis of women with pre-eclampsia. Am. J. Obstet. Gynecol, 181,1211-1215. 35. Walsh, S.W., Vaughan, J.E., Wang, Y., Roberts, L.J. (2000). Placental isoprostane is significantly increased in preeclampsia. FASEB. J, 14,1289-1296.

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36. Vanderlie, J., Venardos, K., Clifton, V.L., Gude, N.M., et al. (2005). Increased biological oxidation and reduced anti-oxidant enzyme activity in pre-eclamptic placentae. Placenta, 26,53-58. 37. Many, A., Hubel, C.A., Fisher, S.J., Roberts, J.M., et al. (2000). Invasive cytotrophoblasts manifest evidence of oxidative stress in preeclampsia. Am. J. Pathol, 156,321-331.

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39. Prasad, P., Leibach, F., Ganapathy, V. (1997). Transplacental transport of water soluble vitamins: A review. Placenta, 19,243-257. 40. Poston, L., Igosheva, N., Mistry, H.D., Seed, P.T., et al. (2011). Role of oxidative stress and antioxidant supplementation in pregnancy disorders. Am. J. Clin. Nutr, 94,1980S-1985S. 41.Wibowo, N., Purwosunu, Y., Sekizawa, A., Farina, A., et al. (2012). Antioxidant supplementation in pregnany women with low antioxidant status. J. Obstet. Gynaecol. Res, 38,1152-1161.

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LEGENDS

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Table 1 Maternal baseline characteristics and outcomes in placebo and vitamin-supplemented women Table 2 Comparison of placental antioxidant enzymes and lipid peroxidation from non-pre-eclamptic and pre-eclamptic pregnancies

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Table 3 Comparison of placental antioxidant enzymes and lipid peroxidation from vitamin and placebo supplemented women

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