Amniotic Fluid Oxidative and Nitrosative Stress ...

Original Paper Neonatology 2013;103:181–186 DOI: 10.1159/000345194

Received: July 3, 2012 Accepted after revision: October 14, 2012 Published online: December 22, 2012

Amniotic Fluid Oxidative and Nitrosative Stress Biomarkers Correlate with Fetal Chronic Hypoxia in Diabetic Pregnancies Javier Escobar a Kari Teramo d Vedran Stefanovic d Sture Andersson e Miguel A. Asensi c Alessandro Arduini c Elena Cubells a Juan Sastre c Máximo Vento a, b a

Neonatal Research Unit, Health Research Centre La Fe, b Division of Neonatology, University and Polytechnic Hospital La Fe, and c Department of Physiology, Faculty of Pharmacy, University of Valencia, Valencia, Spain; d Department of Obstetrics and Gynaecology and e Division of Neonatology, Helsinki University Central Hospital, Helsinki, Finland

Key Words Diabetes ! Pregnancy ! Fetus ! Amniotic fluid ! Erythropoietin ! Oxidative stress

Abstract Background: In spite of improvement in obstetrical care, pregnancy in women with type 1 diabetes mellitus is associated with increased perinatal morbidity and mortality. Hyperglycemia during pregnancy causes excessive fetal growth and chronic fetal hypoxia as reflected in increased erythropoietin (EPO) levels in amniotic fluid (AF). Objectives: We hypothesized that the degree of fetal hypoxia would correlate with fetal oxidative and nitrosative stress as evidenced by the concentration of specific biomarkers in AF. Material and Methods: 19 pregnant women with type 1 or insulin-treated gestational diabetes mellitus were studied. AF samples were collected and processed for EPO, meta-tyrosine, nitro -tyrosine and 8-hydroxy-2-deoxiguanosine by chemiluminescent immunoassay and high-performance liquid chromatography coupled to tandem mass spectrometry methods, respectively. Results: The mean (SD) of the last HbA1c concentration before delivery was 7.7% (1.1). Median gestational age

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was 258 days (range 231–268). Birth weight was 3,868 8 695 g with a z-score 12 SD in 47% of the cases. A significant correlation was found between the concentrations of AF EPO and meta-tyrosine/phenylalanine ratio (p ! 0.001), nitro tyrosine (p ! 0.01) and 8-oxo-dG/2dG ratio (p ! 0.001). Conclusions: We confirmed that fetuses of type 1 diabetes or insulin-treated gestational diabetes pregnancies experience chronic hypoxia as reflected by increased EPO concentrations in AF near term. Moreover, EPO levels significantly correlated with the concentration of oxidative and nitrosative stress biomarkers in AF. This pro-oxidant status may predispose newborn infants to poor postnatal adaptation and early neonatal complications. Copyright © 2012 S. Karger AG, Basel

Introduction

Improvement of obstetric and neonatal care, generalization of antenatal steroids, and use of surfactant has decisively contributed to reduce mortality and morbidity in the neonatal and postneonatal periods [1]. However, perinatal mortality and morbidity in type 1 diabetic Máximo Vento, MD, PhD Division of Neonatology and Neonatal Research Unit University and Polytechnic Hospital La Fe and Health Research Centre La Fe Bulevar Sur s/n, ES–46026 Valencia (Spain) E-Mail maximo.vento @ uv.es

pregnancies has remained practically unchanged in the last two decades [2, 3]. Fetal plasma and amniotic fluid (AF) erythropoietin (EPO) levels are frequently elevated in diabetic pregnancies suggesting an increased incidence of chronic fetal hypoxia, especially in patients with poor glycemic control [4–6]. Although the pathogenesis of chronic fetal hypoxia in diabetic pregnancies is not fully understood, several maternal, placental and fetal factors have been implicated [7]. Both experimental and clinical studies have shown that maternal hyperglycemia (i.e. fetal hyperglycemia) and fetal hyperinsulinemia can independently cause chronic fetal hypoxia [5]. Prolonged hypoxia is known to be the major stimulus for EPO production in the fetus [8]. Since EPO does not cross the placenta, elevated concentrations of EPO in fetal plasma and AF reflect EPO synthesis and elimination. AF EPO concentrations correlate well with fetal plasma EPO concentrations both in normal and abnormal pregnancies [9, 10]. In addition, AF EPO levels correlate with several perinatal complications in diabetic pregnancies [6]. Pregnancy in type 1 diabetes and insulin-treated gestational diabetes are characterized by hyperglycemia and oxidative stress. Furthermore, oxidative stress may be even present in pregnant women with an adequate metabolic control which could be explained by downregulation of the antioxidant status in diabetic pregnancies [11, 12]. Interestingly, fetuses from gestational diabetic mothers have increased umbilical glucose concentrations despite normal maternal glucose levels and a reduction in oxygen saturation and O2 content together with increased lactate concentration, reflecting altered fetal metabolism [5]. Hyperglycemia leads to an increased production of reactive oxygen species (ROS) through different metabolic pathways. Briefly, hyperglycemia increases formation of advanced glycation end products, activates the hexosamine biosynthetic pathway, leading to the formation of glucosamine-6-phosphate which competes with glucose6-phosphate dehydrogenase and limits the synthesis of nicotinamide adenine dinucleotide necessary for reduced glutathione (GSH) rebuilding. Finally, activation of the polyol pathway, protein kinase C pathway, and oxidases activation may also be responsible for increased ROS production [13]. End products of abnormal glucose metabolism lead to an increased formation of ROS. When ROS production overcomes fetal and placental antioxidant capacity, transcription factors such as nuclear factor-!B, activator protein-1 and hypoxia-inducing factor-1 will become activated and lead to insulin resistance through different mechanisms. Hence, transcription factors may directly induce the expression of pro-inflammatory cy182

Neonatology 2013;103:181–186

tokines such as interleukin-6, tumor necrosis factor-" or monocyte chemoattractant protein-1 which will cause insulin resistance or indirectly through the activation of serine/threonine kinases which will interfere with key components of the insulin signaling pathway [14]. In addition, increased flux of glucose through the tricarboxylic acid cycle in the mitochondria will result in increased oxygen consumption and ROS production as well as enhanced uncoupling protein activation causing excessive proton leak and exhaustion of ATP reserves leading to cell death by apoptosis [15]. Fetal oxidative stress in diabetic pregnancy is detectable in AF [16–18]. Of note, increased concentrations of oxidative stress biomarkers in AF are associated with increased risk for preterm labor [19], preeclampsia [20], diabetic pregnancy [16], and morbidity in the immediate postnatal period [21]. In addition, hypoxia is associated with increased formation of ROS and reactive nitrogen species (RNS) which can be detected as oxidative (orthotyrosine (o-Tyr) or meta-L-tyrosine (m-Tyr)) and/or nitrosative (3-nitro-L-tyrosine (n-Tyr)) derivatives of phenylalanine (Phe) and oxidative bases of DNA. These very specific metabolites derived from hydroxyl and peroxynitrite aggression to amino acids or DNA can be accurately measured by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC/ MS/MS) [22–24]. However, a correlation between EPO concentration in AF and the degree of fetal oxidative stress had not been previously established. We hypothesized that in pregnant women with type 1 or insulintreated gestational diabetes increased fetal chronic hypoxia as reflected by increased EPO concentration in AF would correlate with increased generation of ROS and RNS and subsequently increased concentration of biomarkers of oxidative and nitrosative stress in AF. Consequently, it would be feasible to prenatally detect fetuses at high risk for intrauterine and postnatal complications [21]. To test this hypothesis we performed a study in which we analyzed AF of type 1 diabetic pregnant women or insulin-treated women with gestational diabetes before delivery for specific oxidative and nitrosative stress biomarkers and correlated the results with AF EPO levels. Material and Methods Patients This is an observational pilot study performed at the Department of Obstetrics and Gynecology of Helsinki University Central Hospital. Amniocentesis is used clinically in this hospital in order to evaluate possible chronic fetal hypoxia by measuring AF

Escobar /Teramo /Stefanovic /Andersson / Asensi /Arduini /Cubells /Sastre /Vento

Table 1. Transitions m/z, retention time and limits of detection

and quantitation for different analytes performed in AF samples obtained in pregnant women with type 1 diabetes

Analyte

Cone Collision MRM transition Retention Limit of voltage m/z time, min detection, nm

8-OH-dG 2dG Phe Tyr m-Tyr o-Tyr n-Tyr

20 20 15 20 20 20 20

20 15 15 15 15 15 15

284.4>167.9 268.4>151.9 166.3>119.6 182.4>136.2 182.4>136.2 182.4>136.2 227.4>167.9

13.34 6.16 6.20 3.25 4.22 5.51 10.66

1 1 2.5 2.5 1 1 1

EPO concentration in diabetic pregnancies. Fetal lung maturity is also assessed from the same sample. Amniocentesis was performed by a specialist in perinatology under ultrasound guidance. Approximately 10 ml of AF was obtained and two 2-ml aliquots of AF were stored at –80 ° C. AF samples from 15 type 1 and 4 insulin-treated gestational diabetic women were selected with known EPO levels ranging between 5 and 1,368 mU/ml (median 63.8). The AF samples were shipped with dry ice to the Department of Physiology (University of Valencia, Spain) for HPLC/MS/ MS (see below). The study was approved by the institutional review board of Helsinki University Central Hospital. All participants signed the informed consent form.

Positive ion electrospray tandem mass spectra were recorded using the following conditions: capillary voltage 3.5 kV, source temperature 120 ° C, cone and nebulization gases were set at 300 and 30 liter/h, respectively. The cone and collision voltages optimized for each analyte are summarized in table 1. Linear calibration curves in the 0.0001–10 # M (o-Tyr, m-Tyr and n-Tyr) and 0.0025–250 # M (Phe, Tyr) concentration range were obtained using peak area values. The multiple reaction monitoring (MRM) transition mass to charge (m/z) ratio, retention time, limits of detection and quantitation for each considered analyte are summarized in table 1.

Statistics Normal distribution of the data was determined using Kolmogorov-Smirnov’s test. Depending on the distribution, mean 8 SD (normal distribution) or median with 5 and 95% confidence intervals were used to illustrate the skewness of the data in the non-normally distributed data. Logarithmically transformed EPO values were correlated with dependent variables of oxidative and nitrosative stress markers using simple regression analysis. The Statgraphics Plus 5.0 statistical package (Statpoint Technologies, Inc., Warrenton, Va., USA) was used for the statistical analyses.

Methods EPO Determination AF EPO levels were measured on the same day of sampling using an automated chemiluminescent immunoassay method (Immulite EPO Assay; Diagnostic Products, Inc., Los Angeles, Calif., USA) as described before [6]. The assay was standardized according to the World Health Organization’s Second International Reference Preparation for EPO. Tyrosine Oxidation Determination by HPLC/MS/MS Levels of 2"-deoxiguanosine (2dG), 8-hydroxy-deoxyguanosine (8-OH-dG), m-Tyr, n-Tyr and Phe in AF samples were determined by HPLC/MS/MS. Prior to HPLC/MS/MS, AF samples were filtered using 0.2-#m nylon syringe filters (Thermo Fisher Scientific, Inc., Waltham, Mass., USA). HPLC/MS/MS was performed using a Quattro Micro triplequadruple mass spectrometer (Micromass Ltd., Manchester, UK) equipped with a Shimadzu LC-10ADvp pump, a SLC-10Avp controller system and a SIL-10ADvp autosampler. Analytical separation was carried out at room temperature using a MediterraneaTM Sea18 column (5 ! 0.21 cm, 3 #m) (Teknochroma, Sant Cugat del Vallés, Barcelona, Spain) using an injection volume of 20 #l. A gradient elution was performed at a flow rate of 0.2 ml!min–1 as follows: (min/%A/%B) (A, 0.1% formic acid; B, methanol): 0/95/5, 10/95/5, 15/5/95, then the isocratic condition was held for 5 min. Finally, a 0.1-min linear gradient was used to return to the initial conditions which were held for 10 min to re-equilibrate the system.

Fetal Oxidative Stress in Diabetic Pregnancy

Results

Clinical Results The median (range) age of the 19 diabetic pregnant women was 30 (23–42) years and the mean (SD) body mass index 25.8 (4.5). Nine women were nulliparous and 15 were delivered by cesarean section. Three of the women had mild or moderate preeclampsia and 1 had gestational hypertension. Perinatal characteristics of the offspring are shown in table 2. Six were females and 9 were born preterm. The median gestational age was 258 days (366/7 weeks), the median (range) birth weight was 3,950 g (2,815–4,775) and the median (range) birth weight z-score was +1.1 (–1.1 to 4.3) SD units. The median (range) HbA1c was 7.55% (5.6–9.8). In 11 out of 18 women (61%) the last HbA1c was 17.0%. Analytical Results Table 3 shows the analytical results of AF concentrations of EPO and biomarkers of oxidative stress m-Tyr, n-Tyr, and 8-OH-dG as well as the reference values for Phe and 2dG. o-Tyr concentration was barely detectable in spite of a repeated concentration of AF, and therefore o-Tyr results have been omitted. A significant correlation (r = 0.90; p ! 0.001) was established between log-transformed AF EPO concentration and m-Tyr/Phe ratio which reflects Phe oxidation by oxygen free radicals (fig. 1). In addition, a significant correlation (r = 0.59; p ! 0.01) was also established between Neonatology 2013;103:181–186

183

30

90

70 60

20 n-Tyr (nmol/l

m-Tyr/Phe (×1,000 ratio)

80

50 40 30

10

20 10 0

0 1

10

100

1

1,000

10

100 EPO (mU/ml)

EPO (mU/ml)

1,000

10,000

Fig. 1. Exponential correlation (p ! 0.0001) between m-Tyr/Phe ratio (representative of oxidation of circulating proteins by free radicals) and log-transformed EPO concentration in AF of pregnant women with type 1 or insulin-treated diabetes mellitus.

Fig. 2. Exponential correlation (p ! 0.0001) between n-Tyr/Phe

Table 2. Offspring characteristics of type 1 diabetic pregnant women or insulin-treated women with gestational diabetes

Table 3. AF concentrations of EPO and oxidative stress biomarkers m-Tyr, n-Tyr, 8-OH-dG and 2dG in pregnancies of type 1 or insulin-treated gestational diabetic women

Case

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Gender

Gesta- Birth tional weight age g days

F M M F M M M M F M M F M F M M M F M

248 258 268 263 263 267 244 247 256 231 263 264 267 257 261 264 253 257 256

Birth weight z-score (SD)

3,940 4,775 4,125 3,530 4,020 4,485 4,130 3,720 4,060 1,950 4,240 3,680 3,950 3,615 3,785 2,905 4,245 2,815 4,390

3.5 4.1 1.5 0.8 1.7 2.4 4.3 2.7 2.8 –0.9 2.2 1.1 1.2 1.5 1.3 –1.1 3.3 –0.6 3.3

One minute Apgar score

UA pH

UA pO2 (kPa)

NICU days

CPAP treatment

9 8 9 9 9 9 9 10 9 8 9 8 8 7 4 9 9 7 9

7.2 7.22 7.27 7.26 7.28 7.21 7.24 7.29 7.24 7.19 7.15 7.16 7.23 7.03 6.97 7.28 7.28 – 7.2

2 2.4 3.1 1.7 2.2 2 1.5 2.2 2.2 1.5 1.5 1.7 1.7 2.6 1.5 1.8 2 – 1.8

7 0 0 0 0 0 2 0 1 2 2 0 0 0 0 0 0 0 0

no no no yes yes yes yes no yes yes yes yes no no no yes no no yes

UA = Umbilical artery; NICU = neonatal intensive care unit; CPAP = continuous positive airway pressure.

184

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ratio (representative of nitrosative modification of circulating proteins by free radicals) and log-transformed EPO concentration in AF of pregnant women with type 1 or insulin-treated gestational diabetes mellitus.

Case

GA

EPO mU/ml

Phe #mol/l

m-Tyr nmol/l

n-Tyr nmol/l

8-OH-dG 2dG nmol/l nmol/l

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

0 1 1 0 1 8 0 0 0 0 0 5 5 2 2 2 7 0 6

128 90 63.8 50.5 186 13 1,368 190 645 86.2 75.8 7 10 6 5 13 19 109 8

1.19 1.13 1.00 0.87 1.82 1.62 0.88 0.70 0.51 0.91 1.07 1.38 2.07 0.91 0.88 1.23 1.53 2.07 2.44

43.19 35.03 33 32.19 100.1 30.78 – 53.20 – 38.22 31.03 13.8 57.96 20.02 8.8 8.61 10.71 70.38 12.2

10.20 12.58 0.33 0.13 – 0.33 2.15 7.90 0.79 0.35 0.24 2.65 4.75 0.26 0.46 – 12.72 8.96 0.25

0.90 1.43 0.92 1.99 2.42 1.81 0.22 1.95 0.58 1.83 0.804 11.01 2.12 2.58 1.53 2.38 2.16 1.00 1.48

38.04 36.11 16.06 21.32 14.53 10.80 17.29 9.29 14.77 9.00 1.89 19.27 13.38 9.19 3.10 2.52 0.55 9.09 10.68

GA = Gestational age when AF was collected (days before delivery).


100

8-oxo-dG/2dG (×1,000 ratio)

90 80 70 60 50 40 30 20 10 0 1

10

100

1,000

EPO (mU/ml)

Fig. 3. Exponential correlation (p ! 0.0001) between 8-oxo-

dG/2dG ratio (representative of oxidation of guanidine bases of DNA by free radicals) and log-transformed EPO concentration in AF of pregnant women with type 1 or insulin-treated gestational diabetes mellitus.

log-transformed AF EPO concentration and n-Tyr which reflects Phe oxidation by nitrogen free radicals as shown in figure 2. Finally, figure 3 shows how oxidation of guanidine bases of nuclear DNA reflected by the 8-OHdG/2dG ratio also correlated significantly correlated (r = 0.93; p ! 0.001) with log-transformed AF EPO concentration. Discussion

Type 1 diabetic pregnancies are still at high risk for fetal death and neonatal mortality and morbidity [2, 3, 6]. Fetal hyperinsulinemia is associated with fetal overgrowth, hypertrophic cardiomyopathy, birth asphyxia and neonatal hypoglycemia, which result in increased admissions to the neonatal intensive care unit [6]. Additionally, hypoxic stress in diabetic pregnancy may impair embryonic gene expression and defective development by inducing oxidative stress. Li et al. [25] have shown that neural tube defects in a mouse model of diabetic embryopathy are associated with oxidative stress which leads to deficient expression of Pax3, a gene required for neural tube closure. Maternal hyperglycemia leads to fetal hyperglycemia, ROS and RNS formation which may cause oxidative damage to the endothelium, nitration of proFetal Oxidative Stress in Diabetic Pregnancy

teins predisposing towards vasoconstriction, and modification of DNA [26, 27]. Plasma EPO levels in adults start to increase approximately 90 min after acute hypoxia and the rate and magnitude of such increase correlates directly with the intensity and duration of hypoxia [28]. High fetal plasma or serum EPO levels reflect fetal chronic hypoxia [4, 6]. Since AF EPO concentrations correlate well with cord plasma EPO values [10], increased AF EPO values also reflect fetal hypoxia. The positive correlation between AF oxidative/nitrosative biomarkers and AF EPO observed in the present study strongly suggests that fetal chronic hypoxia is associated with oxidative stress. It also emphasizes the role of fetal EPO as an important marker of intrauterine hypoxia. Under normal circumstances, fetal to neonatal transition causes physiologic oxidative stress in the newborn infant relevant to induced specific metabolic pathways [1]. However, resuscitation with oxygen may generate a burst of ROS and RNS, and cause increased morbidity and mortality [29, 30]. Moreover, in preterm infants, oxidative stress in the immediate postnatal period has been linked to long-term conditions such as bronchopulmonary dysplasia [22]. It has been suggested that newborn infants from diabetic mothers have enhanced oxidative stress and that oxidative stress would correlate with the degree of poor glycemic control [31]. Several studies have reported increased oxidative stress markers in umbilical cord blood in diabetic pregnancies [31–33]. However, other studies report no significant changes of oxidative stress markers in infants of diabetic mothers when compared to normal controls [34, 35]. In conclusion, previous studies have shown that poor glycemic control can lead to fetal chronic hypoxia, which, as shown in the present study, is associated with oxidative and nitrosative stress. Fetal oxidative stress can cause both short- and long-term deleterious effects in the newborn infant. The relevance of our findings in the pathogenesis of fetal and neonatal complications in diabetic pregnancies deserves urgently further studies.

Acknowledgements The authors would like to thank all the patients enrolled in the study for their participation would also the nurses and laboratory technicians for their inestimable help. This study has been funded with the following grants: RD008/0072/0022 to M. Vento, Sara Borrell Grant CD11/00154 to J. Escobar, and Consolider Grant CD2007-0020 to J. Sastre (Instituto Carlos III, Spanish Ministry of Economy and Competitiveness).

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