Arch Gynecol Obstet (2013) 287:19–24
DOI 10.1007/s00404-012-2502-4
MATERNAL-FETAL MEDICINE
Trace elements and oxidative stress in hypertensive disorders
of pregnancy
Vanja Fenzl • Zlata Flegar-Meštrić •
Sonja Perkov • Luka Andrišić • Franz Tatzber •
Neven Žarković • Željko Duić
Received: 25 March 2012 / Accepted: 24 July 2012 / Published online: 10 August 2012
Ó Springer-Verlag 2012
Abstract
Purpose Due to increased metabolic requests, pregnancy
can be considered as metabolic stress, especially if associ-
ated with oxidative stress triggered by disbalance of pro/
antioxidants. The aim of the study was to determine serum
concentrations of the trace elements iron (Fe), zinc (Zn) and
copper (Cu) important in growth regulation and pro/anti-
oxidant homeostasis, in relation to the total serum oxidant
V. Fenzl
Ž. Duić
Department of Gynecology and Obstetrics, University Hospital
‘‘Merkur’’, Zajčeva 19, 10000 Zagreb, Croatia
V. Fenzl
University of Applied Health Studies, Mlinarska Cesta 38,
10000 Zagreb, Croatia
V. Fenzl (&)
Bijenicka 52, 10000 Zagreb, Croatia
e-mail: vanja.radic@inet.hr
Z. Flegar-Meštrić
S. Perkov
Institute of Clinical Chemistry, University Hospital ‘‘Merkur’’,
Zajčeva 19, 10000 Zagreb, Croatia
Z. Flegar-Meštrić
Faculty of Pharmacy and Biochemistry, Zagreb University,
Ante Kovačića 1, 10000 Zagreb, Croatia
L. Andrišić
N. Žarković
Division of Molecular Medicine, Rud̄er Bošković Institute,
Bijenička 54, 10000 Zagreb, Croatia
F. Tatzber
Department of Biochemical Engineering, University of Applied
Sciences, Technikum Wien, Hoechstaedtplatz 5, 1200 Vienna,
Austria
Ž. Duić
Faculty of Medicine, Rijeka University, Braće Branchetta 20,
51000 Rijeka, Croatia
capacity (TOC) and total serum antioxidant capacity (TAC)
in pregnant women with preeclampsia (n = 30) or with
gestational hypertension (n = 30) and in healthy pregnant
women (n = 37) and non-pregnant women (n = 30) as
control groups expecting common differences between all
pregnant women and controls and between preeclampsia and
the other pregnancies indicating specific disbalance of the
oxidative stress and analyzed trace elements.
Methods Serum Fe was determined by spectrophotometric
method, Cu and Zn were determined by atomic absorp-
tion spectrometry, TOC was determined by Enzymatic
ANTIOX-CAP assay and TAC by Peroxide-activity assay.
Results Serum Cu and TOC were significantly higher
while Zn was lower in all pregnant groups regardless of
hypertensive disorders. Serum Fe and TAC concentrations
were found to be significantly higher in pregnant women
with preeclampsia compared to pregnant controls.
Conclusion Increase of TOC in all pregnant women our
study points to latent oxidative stress in pregnancy. Fe
might have a role in etiopathogenesis of preeclampsia
while the increase of TAC in the very beginning of pre-
eclampsia might represent a stressdefence mechanism of
the body. It has still to be revealed whether significantly
higher serum Fe levels are associated with preeclampsia as
a cause or as a consequence of this disorder.
Keywords Hypertensive disorders of pregnancy

Total oxidant capacity
Total antioxidant capacity

Iron
Copper
Zinc
Introduction
Hypertensive disorders in pregnancy are one of most
important causes of maternal morbidity and mortality as
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well as concomitant complications for the fetus and new-
born children. Epidemiological studies confirm that high
blood pressure disorders occur in 12–22 % of all preg-
nancies [1]. They cause relative deficiency in placental
blood flow that limits fetal supply of nutrients and oxygen
and consequently it can induce intrauterine growth
restriction or even oxygen deprivation of the fetus.
Preeclampsia and gestational hypertension are exclu-
sively pregnancy-related hypertensive disorders with or
without proteinuria, respectively, that appear after 20th
week of gestation [2]. The cause of these two disorders is
still unknown and ongoing investigations are in search of
predicting and risk factors. An early suboptimal placenta-
tion associated with insufficient remodeling of spiral
arteries is possible cause of preeclampsia [3–9]. This leads
to endothelial dysfunction, vasoconstriction and placental
hypoperfusion with ischemia, and finally to the symptoms
and complications in pregnancy attributed to this specific
disorder [6, 7, 9]. In spite of the fact that the cause of
impaired trofoblast invasion is unknown it is likely that
oxidative stress following deprived placental perfusion and
ischemia could be the subsequent mechanism of this dis-
order [7, 8, 10]. Of particular importance for the onset of
oxidative stress might be Fe and Cu, which are known as
transition metals with strongest pro-oxidant capacities, as
well as Cu and Zn, which are required for the activity of the
first-step antioxidant enzymes (superoxide dismutase).
Essential trace elements are involved in various bio-
chemical pathways [11, 12]. Their specific and the most
important functions for health are catalytic role in chemical
reactions and in structural function in large molecules such
as enzymes and hormones [13]. Alterations in concentra-
tions and homeostasis of each of these micronutrients in
body are well-known contributors in pathophysiology of
various disorders and diseases [12, 14]. Micronutrients
body supply can have important impact on increased
metabolic demands of changed physiology of pregnant
women and requirements of fetus. There is considerable
evidence concerning trace elements serum level changes
associated with hypertensive disorders of pregnancy
[13, 15, 16]. However, the relevance of Fe, Cu or Zn that is
of high importance in growth regulation and in pro/anti-
oxidant homeostasis was not yet compared in relation to
the onset of possible oxidative stress in normal or patho-
logical pregnancies.
The aim of the conducted study was to determine con-
centrations of Fe, Cu and Zn, to calculate their ratios and to
determine total oxidative and antioxidative capacities in
sera of pregnant women with and without hypertensive
disorders and in women of reproductive age. We hypoth-
esized that common differences between all pregnant
women and controls could be revealed, while preeclampsia
and the other pregnancies would further differ indicating
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Arch Gynecol Obstet (2013) 287:19–24
specific disbalance of the oxidative stress and evaluated
trace elements in preeclampsia.
Materials and methods
Study population
This prospective study was conducted in University Hos-
pital ‘‘Merkur’’ among pregnant women with hypertensive
disorders that developed after 20th gestational week,
healthy pregnant women after 20th gestational week and
healthy women without pregnancy in their reproductive
age. The study was approved by Ethical committees of the
University of Zagreb and Clinical Hospital ‘‘Merkur’’. All
participants were recruited from Obstetrics and Gynecol-
ogy Clinic of Hospital ‘‘Merkur’’. Informed consents were
obtained from all women in the study. Four study groups
were determined: (1) study group: 30 pregnant women with
preeclampsia (P), (2) study group: 30 pregnant women with
gestational hypertension (GH), (3) control group: 37
pregnant women with undisturbed pregnancies, pregnant
controls (CP) and (4) control group: 30 healthy women out
of pregnancy in their reproductive age, controls (C). At the
moment of initial hypertensive disorder in study groups, in
pregnant controls, and in the follicular phase of menstrual
cycle in control group of women, blood and urine samples
were obtained. Blood was taken from the peripheral vein.
Trace elements
Serum Fe, Cu and Zn were determined at Institute of
Clinical Chemistry and Laboratory Medicine, University
Hospital ‘‘Merkur’’, accredited according to ISO 15189,
Medical laboratories—particular requirements for quality
and competence since 2007 [17]. Blood samples for
determination of trace elements were collected after an
overnight fast and under controlled pre-analytical condi-
tions into Vacutainer Trace elements Tubes (Becton
Dickinson, Kat. No. 368380, Oxford, UK). The blood was
left to clot at room temperature for 30 min and then cen-
trifuged at 3,500 rpm for 10 min at 4 °C. Serum Fe was
determined on fresh sera on the day of blood collection.
Sera for the determination of Cu and Zn were frozen in
polypropylene tubes at -70 °C until analysis. Serum Fe
was measured spectrophotometrically using TPTZ method
(Olympus Diagnostic GmbH, Hamburg, Germany) on the
Olympus AU 600 analyzer (Olympus Mishima Co., Ltd.,
Shizuoka, Japan). Serum Cu and Zn concentrations were
determined by flame atomic absorption spectrometry
(AAS) after tenfold dilution with deionized water on a AA-
6300 atomic absorption spectrophotometer (Shimadzu
Corp., Kyoto, Japan) equipped with ASC 6100 autosampler
Arch Gynecol Obstet (2013) 287:19–24
for AAS (Shimadzu Corp.). Wavelength of 324.8 nm, slit
width of 0.7 nm and lamp current of 6 mA was employed
for Cu. The standard addition method for calibration was
used. Stock Cu atomic absorption standard solution
(Sigma-Aldrich, Kat. No. 207071) containing 986 lg/ml
Cu in 1 wt.% HNO3 was diluted with deionized water to
obtain Cu working standard. Cu working standard was
added to pooled human serum for calibration to produce
concentrations 0, 12.4, 24.8 and 31.0 lmol/L of Cu,
respectively. Zn was measured at wavelength of 213.9 nm,
using a slit width of 0.7 nm and lamp current of 8 mA. Zn
Atomic Absorption Standard Solution (Sigma-Aldrich,
Kat. No. Z2750) containing 1,000 lg/ml Zn in 1 wt.% HCl
was diluted for calibration with deionized water to obtain
Zn working standard. The Zn working standard was added
in pooled human serum to produce concentrations 0, 9.4,
15.6 and 24.4 lmol/L of Zn, respectively. Traceability of
analytical methods is achieved through a manufacturer’s
reference materials (calibrators) or reference methods.
Analyzer-based calibrations are routinely performed for
compensation of systematic effects. Estimates of within-
laboratory precision are provided by internal quality con-
trol data using pool serum samples and commercial control
sera. Trueness estimates are based on the long-term results
of external quality assessment (EQA) obtained by partici-
pation of the Institute of Clinical Chemistry and Laboratory
Medicine University Hospital Merkur in the National EQA
Scheme organized by Croatian Society of Medical Bio-
chemists and International EQA schemes for general and
special medical biochemistry organized by Labquality—
WHO Collaborating Centre for Education and Training in
Laboratory Quality Assurance, 00520, Helsinki, Finland.
Estimation of measurement uncertainties is done on the
basis of the ‘‘Guide to the Expression of Uncertainty in
Measurement’’ [18]. The uncertainty components that we
use are uncertainties related to calibrator, within-laboratory
precision and trueness estimates based on the results of
EQA. The obtained expanded measurement uncertainties
(k = 2) for Fe, Cu and Zn as well as Fe concentrations in
the normal concentration range were 8.2, 12.2 and 15.1 %,
respectively.
Total serum antioxidant capacity (TAC) and total serum
oxidant capacity (total serum peroxide measurement)
(TOC)
Total serum antioxidant capacity and TOC were deter-
mined from the sera that were frozen at -70 °C and then
analyzed in Laboratory for oxidative stress, Division of
Molecular Medicine at Rud̄er Bošković Institute. Enzy-
matic ANTIOX-CAP assay (AOC-Dr Tatzber KEG,
Vienna, Austria) was used to test reactive oxygen species
(ROS) scavenging properties of serum antioxidants using
21
uric acid standards [19]. Briefly, 10 ll of sera were added
into each well of the 96-microwell plate in duplicates and
mixed with 100 ll of Reagent 2 containing 0.0003 % (v/v)
hydrogen peroxide. The absorbance was measured at
450 nm (Easy-Reader 400 FW, SLT Lab Instruments
GmbH, Austria). Afterwards, 100 ll of Reagent 1 con-
taining 1.25 mU horse radish peroxidase (HRP) followed
by tetramethylbenzidine (TMB) was added. After 15 min
of incubation and addition of 50 ll of the Stop Reagent, the
absorbance was measured at 450 nm. Results were pre-
sented as a difference between the second and the first
absorbance values in relation to the uric standards. Serum
peroxide concentrations (POX-Dr Tatzber, KEG, Vienna,
Austria) were determined by rapid in vitro diagnostic assay
(Peroxide-activity assay; LDN, Germany) as previously
described [20]. The assay is based on a peroxide/peroxi-
dase reaction using TMB as chromogenic substrate. Per-
oxide levels are expressed as ‘‘lM H2O2 equivalents’’
based on ranging values of fresh peroxide solutions as
standards. Ten microlitres of serum samples of unknown
peroxide content were placed into 96-microwell plate in
duplicates and incubated at 37 °C with a mixture of defined
HRP activity, TMB chromogene and phosphate buffer for
30 min and absorbance was determined in a multiwall plate
reader (easy-Reader 400 FW, SLT Lab Instruments GmbH,
Austria) at 450 nm.
Statistics
The results are presented descriptively; mean ± standard
deviation for normally distributed data or as median
(minimum - maximum) for variables that did not show
normal distribution. Statistical analysis was performed
using the Kolmogorov–Smirnov normality test, Kruskal–
Wallis test with appropriate post hoc tests as well as Mann–
Whitney test and Spearman correlation coefficient.
A P \ 0.05 was considered statistically significant.
Results
The difference and anthropological characteristics of the
groups are presented in Table 1. There were no significant
differences between groups in age, parity and gestation at
the moment of blood sampling. Women with preeclampsia
had significantly higher systolic blood pressure then
women with gestational hypertension and pregnant controls
had significantly lower BMI then pregnant women with
hypertensive disorders (P \ 0.05).
Trace elements serum concentrations and their ratios are
presented in Table 2. Serum Fe concentrations were sig-
nificantly higher in women with preeclampsia in compar-
ison to healthy pregnant women. In all groups of pregnant
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Table 1 Anthropometric characteristics (in mean) of the groups at Discussion

the moment of sampling
Attribute P GH CP C Hypertensive disorder of pregnancy named preeclampsia
(n = 30) (n = 30) (n = 37) (n = 30) can be related to an oxidative stress disorder [21]. There is
enough relevant evidence that excessive imbalance
Age (year) 31.2 30.8 30.8 29.68
between ROS and body antioxidant defense forces found in
Primipara 73.33 56.67 64.00
(%) this pathological condition of pregnancy lead to damaging
SBP 166.38 154.00 121.22 modifications of cell functions and intracellular compart-
DBP 98.1 100.5 76.22 ments caused by oxidative stress [21]. The precise cause of
GA (days) 255.83 261.57 261.92
preeclampsia is not known. Published evidence of oxida-
BMI 31.28 30.07 27.67
tive stress presence in normal pregnancies suggests that
redox imbalance serves beneficial events in pregnancy
SBP systolic blood pressure, DBP diastolic blood pressure, GA ges- [22]. Moreover, detectable antioxidant system activity in
tational age, BMI body mass index, P preeclampsia, GH gestational
hypertension, CP pregnant controls, C controls pregnancy is stimulated by increased oxidant activity
comparing to women out of pregnancy [22]. Inadequate
trophoblast invasion, placental suboptimal perfusion and
ischemia present in preeclampsia lead to initiating changes
women, serum Cu concentrations were significantly higher, that can be documented in maternal circulation as inten-
Zn concentrations were significantly lower and Cu/Fe sifying oxidative stress [7, 23, 24]. In light of the results in
ratios were significantly higher than in control group of this study regarding TOC and TAC values, we can con-
women out of pregnancy. In women with preeclampsia, clude the following. In normal pregnancies, TOC was
Cu/Fe and Zn/Fe ratios were significantly lower than in significantly higher than in women out of pregnancy. At the
healthy pregnant women. Cu/Zn ratio in women out of very beginning of pregnancies with hypertensive disorders
pregnancy was significantly lower than in all other groups. that were evaluated, there were no significant differences in
Total oxidant and antioxidant capacities are presented in TOC between them and healthy pregnancies indicating
Table 2. TAC in group with preeclampsia and in control physiological increase of the oxidative status in pregnancy
group of women out of pregnancy was significantly higher in general, as was recently described for the process of
in comparison to healthy pregnant women and to those birth in general, in particular for mothers with the babies
with gestational hypertension. TOC in control group was small for gestational age [25]. On the other hand, TAC in
significantly lower than in all groups of pregnant women. group with preeclampsia showed statistically significant
Correlations between trace elements and TOC and TAC higher values in comparison to normal pregnancies. This
were presented in Table 3. Positive correlations were found reveals that certain redox imbalance compared to normal
in group with preeclampsia between Fe and TAC, in all pregnancies is present even at the very beginning of the
groups between Cu and TOC and in control group between disorder and that protecting antioxidant capacities of the
Cu and TAC. body are additionally activated. Nevertheless, although

Table 2 Serum concentrations of copper, zinc, iron, their ratios, total oxidative and total antioxidative capacities
C CP GH P

Cu (lmol/L) 15.3 (13.97–17.45)* 32.04 ± 7.33 33.32 ± 5.59 33.91 ± 8.19

Zn (lmol/L) 10.85 ± 1.72* 8.85 ± 1.43 9.16 ± 1.27 9.23 ± 1.43
Fe (lmol/L) 16.10 ± 6.56 13.57 ± 7.16 16.77 ± 10.58 18.87 ± 8.04**
Cu/Zn 1.57 ± 0.62* 3.69 ± 0.97 3.68 ± 0.72 3.73 ± 0.93
Cu/Fe 1.34 ± 1.01* 3.39 ± 2.81 3.24 ± 3.53 2.33 ± 1.92**
Zn/Fe 0.83 ± 0.45 0.90 ± 0.60 0.85 ± 0.93 0.61 ± 0.38**
TOC (mmol/L) 31.90 ± 59.72* 151.41 ± 69.86 195,48 ± 104.61 164.37 ± 79.76
TAC (mmol/L) 3.08 ± 1.11*** 2.48 ± 0.96 2.35 ± 1.27 3.00 ± 1.03***
Values are given as mean ± SD or median (minimum - maximum)
P preeclampsia, GH gestational hypertension, CP pregnant controls, C controls
* p \ 0.05 in comparison to CP, GH and P, Mann–Whitney test
** p \ 0.05 in comparison to CP, Mann–Whitney test
*** p \ 0.05 in comparison to CP and GH, Mann–Whitney test

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Table 3 Correlation: total oxidative and antioxidative capacities and mostly a standard care. Ideally, low Fe status should be
trace elements determined before or at the beginning of the pregnancy.
TOC TAC However, Fe excess can mediate formation of highly
reactive-free radical OH
in the presence of oxygen
r P r P
according to Fenton and Haber-Weiss reactions [33–35].
P Cu 0.72 \0.001 -0.21 0.268 Thus, induced oxidative stress confirms that Fe as a catalyst
Zn 0.35 0.057 0.11 0.551 of free radical reactions can be toxic [32, 33]. It has been
Fe -0.11 0.559 0.38 0.04 reported that Fe supplementation leads to increased levels
GH Cu 0.75 \0.001 0.07 0.733 of oxidative stress markers in pregnant rats and women in
Zn 0.14 0.462 -0.09 0.651 comparison to controls [36–38]. In addition, previous
Fe -0.35 0.056 -0.07 0.7 studies came to conclusion that important source of cir-
CP Cu 0.72 \0.001 -0.11 0.535 culating Fe can be placental thrombotic and necrotic tissue
Zn -0.13 0.439 0 0.989 of women with preeclampsia where certain amounts of red
Fe -0.19 0.271 0.15 0.364 blood cells are defragmented [31]. The fact that in this
C Cu 0.37 0.045 -0.39 0.033 study significantly higher serum concentrations of Fe were
Zn 0.23 0.219 0.07 0.706 found in women with preeclampsia compared to uncom-
Fe -0.09 0.651 0.24 0.211 plicated pregnancies implements that Fe may be involved
in pathogenesis of preeclampsia.
r Spearman correlation coefficient
Positive correlation was found between Fe and TAC in
P preeclampsia, GH gestational hypertension, CP pregnant controls,
group with preeclampsia that connects the potential Fe-
C controls
induced oxidant formation or oxidative stress with activa-
tion of body antioxidant protection mechanism in pre-
these serum capacities may not represent oxidant or anti- eclampsia. There was significant positive correlation
oxidant activities in maternal circulation they are sensitive between Cu and TOC in all groups and negative correlation
and reliable detectors of oxidative stress in vivo [26]. between Cu and TAC in controls. In light of that, this study
Evaluated trace elements as antioxidant nutrients or, on showed that Cu may be the related to circulating oxidant
the other hand, as participants in redox processes involved production forces but not exclusively to the development of
as components of metaloenzymes or as cofactors in two evaluated hypertensive disorders. Differences of ratios
chemical reactions, they could be easily manipulated by between trace elements in groups were consistent with their
diet or by supplementation therapy [13]. Significant chan- absolute values, with highlight to Cu and Fe values.
ges in serum concentrations of Cu and Zn in normal
pregnancies were already observed [27–30]. Serum Cu
levels in normal pregnancy increase accordingly to the Conclusion
rising concentrations of its main transport protein cerulo-
plasmin induced by high estrogen levels of pregnancy The results obtained support our initial hypothesis that
[27, 31]. Zn levels tend to get lower in normal pregnancies common differences between all pregnant women and con-
as a result of higher demands of mother and fetus and trols could be revealed, while preeclampsia and the other
pregnancy correlated hemodilution [28]. There are some pregnancies would further differ indicating specific disbal-
published studies that show statistically significant higher ance of the oxidative stress in preeclampsia. Namely, the
levels of Cu and lower levels of Zn in circulation of women normal pregnancies were characterized by increased levels
with preeclampsia in comparison to healthy pregnant of Cu and ROS production and enhanced antioxidant pro-
women [29–31]. This study did not show difference tection, while preeclampsia is associated with increase of Fe
between groups of pregnant women regarding serum con- and imbalance in oxidative homeostasis. Such an imbalance
centrations of Cu and Zn. Cu serum levels were signifi- can be detected even in the beginning of the preeclamptic
cantly higher and Zn levels significantly lower in all groups disorder inducing a significant increase of total serum anti-
of pregnant women equally without differences between oxidant capacities. It has still to be revealed whether sig-
healthy and pathological pregnancies. nificantly higher serum Fe levels are associated with
It is well known that Fe deficiency is a risk factor for preeclampsia as a cause or as a consequence of this disorder.
premature delivery, stillbirth and low birth-weight of In both cases, the results of this study indicate that the Fe
children [28, 32]. Increased Fe requirements and sub- supplementation for non-anemic pregnant women might
sequent Fe deficiency in pregnancy can hardly be have harmful effects as a risk of developing preeclampsia.
overcome by changes in diet [32]. In practice, Fe supple-
mentation during pregnancy is recommended and has been Conflict of interest None.

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