Copyright 2001 by Humana Press Inc.

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0163-4984/01/83020103 $12.00

Increased Lipid Peroxidation

in Pregnant Women after Iron
and Vitamin C Supplementation
Brahim Lachili,1 Isabelle Hininger,2 Henri Faure,2
Josiane Arnaud,2 Marie-Jeanne Richard,2
Alain Favier,2 and Anne-Marie Roussel*,2
1

Faculty of Medicine, Batna University, Rue Chahid Boukhlouf

Med El-Hadi, 05000;Batna, Algerie; and 2L.B.S.O.,
Joseph Fourier University, Domaine de la Merci,
38700 La Tronche, France
Received August 31, 2000; Revised October 7, 2000;
Accepted November 11, 2000

ABSTRACT
Iron overload could promote the generation of free radicals and
result in deleterious cellular damages. A physiological increase of oxidative stress has been observed in pregnancy. A routine iron supplement,
especially a combined iron and vitamin C supplementation, without biological justifications (low hemoglobin [Hb] and iron stores) could therefore aggravate this oxidative risk. We investigated the effect of a daily
combined iron supplementation (100 mg/d as fumarate) and vitamin C
(500 mg/d as ascorbate) for the third trimester of pregnancy on lipid
peroxidation (plasma TBARS), antioxidant micronutriments (Zn, Se,
retinol, vitaminE, (-carotene) and antioxidant metalloenzymes (RBC
CuZn SOD and Se-GPX). The iron-supplemented group (n = 27) was
compared to a control group (n = 27), age and number of pregnancies
matched. At delivery, all the women exhibited normal Hb and ferritin
values. In the supplemented group, plasma iron level was higher than in
the control group (26.90 5.52 mmol/L) and TBARs plasma levels were
significantly enhanced (p < 0.05) (3.62 0.36 vs 3.01 0.37 mmol/L). No
significant changes were observed in plasma trace elements and red
blood cell antioxidant metalloenzymes. Furthermore, the -tocopherol
plasma level was lowered in the iron-supplemented groups, suggesting
an increased utilization of vitamin E.
*Author to whom all correspondence and reprint requests should be addressed.
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Lachili et al.
These data show that pharmalogical doses of iron, associated with
high vitamin C intakes, can result in uncontrolled lipid peroxidation.
This is predictive of adverse effects for the mother and the fetus. This
study illustrates the potential harmful effects of iron supplementation
when prescribed only on the assumption of anemia and not on the bases
of biological criteria.
Index Entries: Iron supplementation; pregnancy; lipid peroxidation.

INTRODUCTION
Iron deficiency continues to be one of the most prevalent singlenutrient deficiencies in the world, especially during pregnancy. Thus,
the routine prescription of iron supplements, without biological justifications, is largely used during pregnancy to prevent a possible risk of
anemia. Although this is believed to be desirable for both the health of
the mother and the well-being of the fetus, some scientists disagree.
Routine iron prescription in pregnancy is still controversial since a
report from the US Preventive Services Task Force concluded that evidence was insufficient to recommend for or against routine iron supplementation in pregnancy (1). Indeed, if there is no doubt that the iron
supplementation in pregnancy improves the maternal haematological
index (2), recent work failed to demonstrate a correlation between
maternal hemoglobin and ferritin level and newborn birthweight (3).
Conversely, Thame found an adverse effect of iron overload on birthweight (4). Moreover, iron might have a dual biological role, and could
be pro-oxidant, especially when associated with vitamin C (5). Free iron,
as a transition metal, participates in the generation of free radicals in
vivo, catalyzing the transformation of hydrogen peroxide to the highly
reactive hydroxyl radical via the Fenton and HaberWeiss reactions
(6,7). The hydroxyl radical acts with phospholipid unsaturared fatty
acids to form lipid peroxides, which are a major factor influencing membrane damage and cell injury. Thus, the provision of large doses of iron
might be deleterious in some circumstances in relation with the possible
generation of oxy-free radicals.
Increased oxidative stress was reported to occur in uncomplicated
pregnancy and to be counteracted by a high level of plasma vitamin E
(8,9). However, in this context, iron supplement combined with high
levels of vitamin C could emphasize free-radical generation and lipid
peroxidation, predicting possible adverse effects for the mother and the
fetus.
In this study, our purpose was to investigate the effect of an oral iron
and vitamin C supplementation on lipid peroxidation and antioxidant
defenses (metalloenzymes and micronutriments) in healthy Algerian
pregnant women at delivery.
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SUBJECTS AND METHODS

Subjects
Women were recruited at the Obstetric Departments of Batna, Constantine and Mila Hospitals (Algeria). Two groups of 27 pregnant women,
2037 yr old, number of pregnancies and weeks of gestation matched,
were constituted. The supplemented group (n = 27) received a daily combined supplement (100 mg iron as fumarate and 500 mg of vitamin C as
ascorbate). Iron was prescribed for the last trimester of pregnancy by clinicians as a routine supplementation without biological checking.

Biological Parameters
Plasma iron and hemoglobin level were quantified by colorimetry.
Serum ferritin was determined by the double-antibody immunoenzymatic
assay kit (Boehringer, Meylan, France). Plasma zinc and copper levels
were measured by flame atomic absorption spectrometry and selenium by
atomic absorption spectrometry as previously described (10,11). Plasma
retinol, vitamin E, and -carotene concentrations were simultaneously
determined using high-performance liquid chromatography, in accordance to the method of Arnaud (12). Plasma TBARs (thiobarbituric acid
reactive substances) levels were measured by fluorimetry (13). Erythrocyte
CuZn superoxide dismutase (SOD) activity was measured after hemoglobin precipitation by monitoring the autooxidation of pyrogallol (14).
One unit of SOD is defined as the amount of the enzyme required to
inhibit the rate of pyrogallol autoxidation by 50%. Erythrocyte Se glutathione peroxidase (GPX) activity was measured by the modified method
of Gunzler et al. (15) using terbutyl hydroperoxyde (Sigma Chemical, via
Coger, Paris, France) as the substrate instead of hydrogen peroxide.
Absorbance was monitoring at 366 nm. Results were expressed as millimoles of NADPH (Boehringer-Mannheim, Germany) oxidized per
minute per gram of hemoglobin.
Data are expressed as means SD. Statistical analyses were performed using PCSM statistical software (Deltasoft, Meylan, France). The
students t-test was used to assess differences in means between the
groups. Statistical significance was defined as p < 0.05.

RESULTS
At the beginning of the study, clinical parameters were comparable
between the groups (Table 1). The scores of Apgar to assess the health of
the newborn were similar in the two groups (Table 1). At delivery, all the
women exhibited normal plasma ferritin and hemoglobin levels (Table 2),
but the iron status was significantly higher (p < 0.05) in the supplemented
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Lachili et al.
Table 1
Main Clinical Characteristics of the Patients in the Study

Table 2
Effect of Supplementation on Serum Iron, Serum Ferritin
and Hemoglobin [Hb] Concentrations

* p < 0.05.

group than in the control group (Table 2). The supplementation did not
significantly modify plasma copper, zinc, and selenium levels (Table 3). In
the two groups, the mean plasma zinc level was below the usual cutoff of
deficiency (10.7 mol/L), whereas the plasma selenium level was adequate and the plasma copper level was elevated considering normal values. Erythrocyte GPX and CuZn SOD were not significantly different
between the groups and in the normal range of the laboratory. The plasma
(-carotene concentration was not statistically different between the two
groups, but the vitamin E level (30.51 6.00 vs 32.73 5.44 mol/L) as well
as vitamin E/cholesterol ratio were significantly lowered in supplemented
women (Table 4). Lipid peroxidation (Table 5) monitored by plasma
TBARs/cholesterol ratio was increased significantly in women receiving
iron and vitamin C supplements.
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Table 3
Effect of Iron Supplement on Plasma Trace Elements
and RBC Antioxidant Metalloenzymes in Women at Delivery

Table 4
Effect of Iron Supplementation on Plasma Antioxidant Vitamins
in Women at Delivery

* p < 0.05.

Table 5
Effect of Iron Supplementation on Plasma Lipid Peroxidation
in Women at Delivery

** p < 0.005.
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DISCUSSION
The aim of this work was to measure the potential adverse prooxidant
effects of iron supplementation combined with vitamin C in pregnant
women. The supplemented group received 100 mg of iron and 500 mg of
vitamin C per day for the last trimester of pregnancy.
At delivery, women from the supplemented group exhibited normal
haematological values; the iron plasma level was significantly higher in
the supplemented group. These results agree with previous works reporting an improved serum hemoglobin, ferrous status, and ferritin level after
an oral iron supplement at pharmacological doses. The large iron increase
underlines the efficiency of the iron absorption after oral supplementation
when combined with vitamin C. Indeed, to enhance iron bioavailability,
supplemental ascorbic acid is recommended (16). Ascorbic acid, as an
antioxidant, scavenges singlet oxygen, superoxide, and peroxyl and
hydroxyl radicals. However, in the presence of non-protein-bound iron,
ascorbic acid can also exhibit strong pro-oxidant abilities, probably by the
reduction of ferric iron to the ferrous form (17,18). Several studies showed
that a high iron store could be more dangerous than a moderate iron
depletion (19,20) and be associated with unfavorable outcome (4,21). A
high iron status could result in an increased platelet aggregation and a
higher thrombosis risk (22), suggesting, paradoxically, that iron depletion
during pregnancy might be a physiological process to prevent the adverse
effects of thrombosis. In our study, we observed a significant increase in
TBARs plasma levels and TBARs/cholesterol ratio in the supplemented
group. These data strongly suggest a pro-oxidant role for iron supplementation. Free iron, as a transition metal, catalyzes the formation of OH in
the HaberWeiss cycle. In addition, it is capable of catalyzing the decomposition of lipid hydroperoxydes to form alkoxyl, peroxyl and others radicals. The adverse effects of lipid peroxidation in oxidative diseases have
been widely documented (23). In pregnancy, lipid peroxides could be
harmful (2427). Several reports described a relationship between elevated
lipid peroxides, vitamin E deficiency, and complicated pregnancies. Even
slight increases of lipid peroxides can decrease prostacyclin synthesis (24).
In women with preeclampsia, plasma TBARs and iron levels are significnalty higher relative to normal pregnancy, suggesting that lipid peroxidation might play a role in the etiology of the disease (26). In an
uncomplicated pregnancy, a physiological increase of plasma vitamin E
has been reported (28). This increase, related to hyperlipidemia in the last
trimester of pregnancy, could be a protective balance mechanism against
free radicals. In contrast, in our study, we observed a decreased vitamin E
plasma level and a lower vitamin E/cholesterol ratio in iron-supplemented group, suggesting an increased utilization of vitamin E. Despite
high levels of plasma selenium and antioxidant metalloenzymes, already
reported in the Algerian population (29), the balance between the oxida-

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tive process and antioxidant defences does not appear to be well controlled in iron supplemented women.

CONCLUSION
This study shows that the interest in routine iron supplement, without
biological control at the entry should be considered with caution. In the
case of anemia, it is of interest to give pharmacological doses of iron,
according to expert board recommendations (1), but our results suggest
that when women exhibit a normal status, there are potential harmful
effects of a high iron supplementation during pregnancy. The deleterious
effect of the combined iron/vitamin C supply could be related to its prooxidant properties. Indeed, the present data underline that pharmacological doses of iron, associated with high vitamin C intakes, result in an
increased oxidative stress. Because excess iron might be implicated in the
pathogenesis of several chronic diseases, the provision of therapeutic
doses of iron, even during pregnancy, should be debated regarding its
effectiveness.

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