Reproductive Toxicology 19 (2004) 149–154

Serum lead, cadmium, and zinc levels in newborns with neural tube
defects from a polluted zone in Mexico
Marı́a de Lourdes Carrillo-Poncea , Verónica Araceli Martı́nez-Ordazb,∗ ,
Vı́ctor Manuel Velasco-Rodrı́guezb , Andrés Hernández-Garcı́aa ,
Marı́a Concepción Hernández-Serranoa , Francisca Sanmiguela
a Centro de Investigación Biomédica de la Universidad Autónoma de Coahuila, Biomedical Research Center
at the Autonomous University of Coahuila, Mexico
b Clinical Epidemiology Research Unit at the Mexican Institute of Social Security of the Nacional Medical Center in Torreón,
Instituto Mexicano del Seguro Social del Centro Médico Nacional, Unidad de Investigación en Epidemiologı́a Clı́nica,
Calle Dalias 356 Coahuila Torreón, Jardı́n, CP 27200, Torreón, Coahuila, México

Received 4 April 2003; received in revised form 16 July 2004; accepted 23 July 2004
Available online 15 September 2004

Abstract

Serum lead, cadmium and zinc levels from 31 newborns with neural tube defects (NTD), and 54 healthy controls living in a polluted area in
Mexico were estimated using atomic absorption spectrophotometry (AAS). NTD family history was found to be of greater importance in the
case group (OR 6.95, 95% CI 1.51–36.3, p = 0.002). In 25% of the children, serum lead concentrations were above the admissible maximum
level (AML) of 10 ␮g/dL within 24 h of extra-uterine life. Cadmium concentrations were below the AML. Zinc deficiency was found in nine
(29%) of the cases and four (9.3%) of the controls (p = 0.04). The logistic regression multivariate analysis showed no correlation between
NTD and high levels of any of these metals; however, a positive correlation was found to zinc deficiency (OR 5.0, 95% CI 1.07–23.00, p =
0.04). These results focus attention to the surrounding nutritional and maternal health factors of major importance in disease etiology.
© 2004 Elsevier Inc. All rights reserved.

Keywords: Lead; Cadmium; Zinc; Neural tube defects; Newborn babies; Congenital malformations

1. Introduction The city of Torreón in the state of Coahuila, Mexico is

Neural tube defects (NTD) comprise a group of congenital
malformations that include spina bifida, anencephaly and en-
cephalocele. NTDs are an important cause of perinatal mor-
bidity and mortality; however, the etiology of these defects
is not completely understood and is often been considered
multifactorial. Genetic, environmental as well as nutritional
factors may play a role in predisposition. Among the potential
environmental agents, industrial contaminants such as metals
have been previously implicated in clinical studies [1–3].
∗ Corresponding author. Tel.: +52 871 7 29 08 00;
fax: +52 871 7 21 15 15.
E-mail address: veromart@internetual.com.mx
(V.A. Martı́nez-Ordaz).
located in the North central region of the Mexican Republic.
Regional activities of great importance include mineral ex-
traction and mining. A metallurgic mining complex founded
in 1901 is located within the city. This complex is consid-
ered the most important mining producer of zinc, lead, cad-
mium, gold, silver, copper, antimony, and sulfuric acid in
Latin America. The region also has an important history of
heavy metal pollution, mainly lead, cadmium, and arsenic,
whose quantities in dust, air, water, and several organic tis-
sues exceed the admissible maximum levels (AML) for hu-
mans [4–6]. Of these metals, lead and cadmium have been
shown to cause teratogenic effects in mammals; studies in
humans are inconclusive to date. On the other hand, zinc is
a trace element required by the organism for multiple bi-
ological functions, among them cell division, development

0890-6238/$ – see front matter © 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.reprotox.2004.07.003
150 M.d.L. Carrillo-Ponce et al. / Reproductive Toxicology 19 (2004) 149–154
and differentiation. As a co-factor for many enzymes in var-
ious metabolic pathways the participation of zinc in nucleic
acid synthesis and absorption of folic acid may be protective
against certain congenital malformations. On the other hand,
at high concentrations, zinc has been demonstrated to have
cytotoxic properties in some animal species. Just as with lead
and cadmium any evidence in humans is scarce and incon-
sistent [7–12].
Faced with the high prevalence of NTD in our region (3.5
per 1000 live newborn babies) [13], according to the Epi-
demiological Registry and Surveillance for External Congen-
tial Malformations (Registro y Vigilancia Epidemiológica de
Malformaciones Congénitas Externas, RYVEMCE) [14], we
conducted a study to compare the serum lead, cadmium and
zinc levels in newborns with NTD and controls.
2. Materials and methods
A case-control study was conducted at the Mexican Insti-
tute of Social Security (Instituto Mexicano del Seguro Social,
IMSS) in the city of Torreón, Coahuila Mexico. As cases,
live newborns with NTD were consecutively included in the
study. As controls, we included the subsequent two new-
borns after each case, paired by gender, and without appar-
ent congenital malformations. Direct interviews were carried
out with the patient’s mother and a questionnaire was given
to the parents on family history (birth place, age, school-
ing, occupation, place of residency, chronic diseases and ad-
dictions), as well as on the mother’s gynecological and ob-
stetrical history (pregnancies, births, abortions, stillbirths).
Any history of congenital malformations (in the previous
two generations) was also recorded. The gestational char-
acteristics related to the newborn babies under study (ma-
ternal age, length of time between pregnancies, exposure to
physical, chemical and biological factors, type of birth and
somatometry) were recorded. Children of mothers with dia-
betes mellitus or epilepsy, or those with febrile syndrome or
exposure to radiation during the first trimester of the preg-
nancy were excluded from the study. After a detailed expla-
nation of the aim of the study, consent was obtained from
the parents of each baby. A peripheral blood sample us-
ing a heparinized syringe was collected from all newborns
within the first 24 h of extra-uterine life. Samples were pre-
viously treated as described by Smith et al. [15], and then the
serum samples were analyzed for zinc, cadmium and lead
levels.
Formeasuring lead, a diphasic ammonium phosphate ma-
trix modifier in the Ultrex and Triton X-100 grade was added
to an aliquot of total blood. A Perkin-Elmer Model 373
atomic absorption spectrophotometer with a Perkin-Elmer
HGA 2200 graphite furnace was used together with a hol-
low cathode lamp and a Perkin-Elmer deuterium corrector.
Cadmium was analyzed using a total blood aliquot digested
with HNO3 and H2 O2 until a pale crystalline yellow solution
was obtained which was supplemented to a volume of 10 ml
with 1% HNO3 . For digestion of the samples, the traditional
hot plate method was used. Standard and addition curves
were drawn to estimate the percentage of metal recovered
(cadmium or lead). Calibration curves were estimated using
aqueous standards for establishing a straight-line equation.
For quantification in the graphite furnace, it was necessary to
optimize each of the stages that constituted the atomization
cycle (drying, ashes, atomization), including time and tem-
perature using a Perkin-Elmer cadmium discharge lamp with-
out an electrode. Zinc was quantified using a plasma aliquot
diluted in distilled water and de-ionized. An air/acetylene
flame burner was used for interpolating the concentration of
zinc in the calibration curve developed with standards in a
glycerol–water mixture.
In order to measure whether serum levels were high or not,
the following values were considered as elevated: cadmium
>5 ␮g/L, zinc >160 ␮g/dL, lead >10 ␮g/dL. Zinc deficiency
was suspected if <75 ␮g/dL [16]. The abbreviation ␮g/dL
refers to micrograms of lead per deciliter of whole blood.
Descriptive statistics were performed as well as a Stu-
dent’s t-test, chi square tests, and simple linear regression.
Odds ratios and 95% confidence intervals were calculated
as part of logistic regression analysis using the SPSS 10.0
statistical computer package, in a saturated model using for-
ward stepwise procedures modeling according to likelihood
ratio.
3. Results
Thirty-one live newborn babies with NTD and 62 healthy
newborns were studied. Eight children were excluded from
the control group since their blood samples were not us-
able for metal content analysis. A final total of 31 cases
and 54 controls were included in the study. The most fre-
quent type of NTD was spina bifida 28 (90.3%), followed by
encephalocele 2 (6.5%) and anencephaly 1 (3.2%). The fe-
male gender predominated (2:1) in both the case and control
group.
Ages for the case-mothers fluctuated between 16 and 41
years old, while the ages of the mothers for the controls
ranged between 15 and 35 years. The ages for the case-fathers
varied between 19 and 43 years old, and those for the con-
trols ranged between 17 and 49. No statistically significant
differences were found between the age groups. The majority
of the patients resided in urban and suburban areas. Approx-
imately 45% of the case-mothers and 42.6% of the control
mothers had some elementary schooling, while only 3.5%
of the mothers and 8% of the fathers from both groups had
a university education or higher. Multiparity (four or more
pregnancies) was reported in 22.6% of the mothers of the
cases, and in 26% of the mothers of the control group mem-
bers. No risk tendency to NTD was found to be related to the
number of pregnancies (X2 tendency = 0.02, p = 0.89). The
only significant difference between the groups corresponded
to the family history (in two previous generations) of other
 M.d.L. Carrillo-Ponce et al. / Reproductive Toxicology 19 (2004) 149–154 151

Table 1 Table 2
Newborn family history Perinatal history and newborn characteristics
Cases Controls p Cases Controls p
(n = 31) (n = 54) (n = 31) (n = 54)
Maternal age (years ± S.D.) 24.1 ± 6.4 23.8 ± 5.3 0.59 Newborn weight (kg) 3.1 ± 0.59 3.2 ± 0.44 0.20
Paternal age (years ± S.D.) 27.8 ± 6.3 26.9 ± 6.8 0.73 Newborn length (cm) 48.0 ± 1.67 50.1 ± 1.67 0.99
Low maternal schoolinga (n) 14 (45.2%) 23 (42.6.%) 0.62 Gestational age (weeks) 37.9 ± 2.6 39.3 ± 1.51 0.005∗
Rural residency (n) 3 6 0.57 Length of time between 4.5 ± 2.7 3.8 ± 2.0 <0.03∗
Multiparityb (n) 7 (22%) 14 (26%) 0.73 pregnancies (years)
Previous abortions (n) 5 7 0.39 Preceding abortion (n) 1 3 0.56
Previous use of hormonesc (n) 12 22 0.99 Preceding stillborn (n) 1 0 0.34
NTD historyd (n) 6 0 0.001∗
Maternal exposure during 1st trimester to
History of other congenital 3 3 0.78
Drugs (n)a 4 10 0.71
malformationsd (n)
Infections (n)b 5 9 0.81
Maternal exposure at work (n) 1 2 0.70
Hormones (n)c 1 1 0.61
Paternal exposure at work (n) 3 6 0.69
Maternal trauma (n) 2 0 0.64
a Schooling equal or less than an elementary school. Active maternal smoking 2 1 0.55
b Parity equal to four or more pregnancies. (n)
c Use of hormones as birth control. a Drugs: analgesics, antihistamines, antibiotics.
d Family history in first and second generation. b Infections: urinary tract and typhoid fever.
∗ Statistically significant. c Hormonal birth control.
∗ Statistically significant.
children with NTD. All mothers (both for cases and con-
Table 3
trols) took zinc and folic acid free multivitamins between the Lead, cadmium, zinc serum levels in newborns
second and third trimester of pregnancy through a pre-natal
Metal Cases (n = 31) Controls (n = 54) p
control program, but none were given supplemental zinc or
Lead (␮g/dL) 6.18 ± 5.9 7.03 ± 5.4 0.6 NS
folic acid. Work exposure to heavy metals, pesticides, and
Cadmium (␮g/L) 6.18 ± 5.9 0.16 ± 0.4 0.8 NS
other chemical products was more frequent in fathers than Zinc (␮g/dL) 115 ± 45.7 143 ± 53.6 0.03∗
in mothers, with no differences between cases and controls Values expressed as mean concentrations ± S.D. NS: not significant.
(Table 1). ∗ Statistically significant.

The average gestational length for the cases was 37.9 ±

2.7 weeks (ranging from 30 to 41 weeks), and for controls 1.07–23, p = 0.04) and the family history of NTD (OR 6.95,
39.3 ± 1.5 weeks (ranging 36–42 weeks), p = 0.005. The 95% CI 1.51–36.3, p = 0.002) (Table 5).
shortest length of time between pregnancies was 10 months
in one of the controls, and the rest ranged between 1 and 8
years, with a longer period of time since the last pregnancy 4. Discussion
in the case group (p < 0.03). The stratified analysis by age
showed no association between the length of time between The serum lead concentration levels within the first 24 h of
pregnancies with the risk of NTD (X2 tendency = 0.04, p extra-uterine life in our cases the work were within the toxic
= 0.83). A history of abortions and/or stillbirths, smoking, range for a high percentage of the children. In 7 (23%) of the
maternal exposure during the first trimester of pregnancy to cases and 15 (28%) of the controls, serum lead concentrations
infections, drugs, hormones, and trauma were low in both exceeded the AML of 10 ␮g/dL established by the Mexican
study groups (Table 2). Official Norm and the World Health Organization, reflecting
Serum lead concentrations in newborns ranged between an important maternal exposure to these metals. The average
0 and 22.5 ␮g/dL, while for cadmium, concentrations were lead concentration in the case group was 6.18 ± 5.9, and it was
between 0 and 0.99 ␮g/L. The differences for both metals
between groups were not significant. Zinc concentrations in Table 4
children with NTD (51–208 ␮g/dL, X = 115 ± 45.7) was Correlation between serum lead, cadmium, and zinc levels in newborns with
NTD
lower than in the control group (64–296.5 ␮g/dL, X = 143.6
± 53.6), p = 0.03 (Table 3). Twenty-nine percent of the cases Cadmium (␮g/L) Lead (␮g/dL) Zinc (␮g/dL)
and 9.3% of the controls had zinc deficiency (p = 0.04), and Cadmium r = 1.00 r = 0.0494 r = −0.2742
in 16% of the cases and 29.6% of the controls, zinc concen- p = 0.728 p = 0.049∗
trations were high (p = 0.27). The simple linear regression Lead r = 0.0494 r = 1.00 r = 0.0716
analysis between serum concentrations of the three metals p = 0.728 p = 0.614
showed a weak inverse relationship between cadmium and Zinc r = −0.2742 r = 0.0716 r = 1.00
zinc concentrations (r = 0.02742, p = 0.049) (Table 4). The p = 0.049∗ p = 0.614
logistic regression analysis showed an association between r: Linear regression value.
∗ Significant at p < 0.05.
the presence of NTD and zinc deficiency (OR 5, 95% CI
152 M.d.L. Carrillo-Ponce et al. / Reproductive Toxicology 19 (2004) 149–154
Table 5
Logistic regression analysis of the variables included in the study
Risk factor OR 95% CI
Low levels of zinc 5.0 1.07–23.3
High levels of zinc 0.49 0.11–2.01
High levels of lead 0.73 0.19–2.84
High levels of cadmium 0.43 0.06–3.18
NTD history 6.95 1.51–36.3
Low maternal schooling 1.26 0.46–3.43
Rural residency 0.86 0.15–4.31
Multiparity 0.83 0.26–2.62
History of abortions 1.48 0.33–6.70
OR: odds ratio, 95% CI: 95% confidence interval, NS: not significant.
∗ Significant at p < 0.05.
7.03 ± 5.4 ␮g/dL for the control group. Studies carried out
in other countries such as Poland, report average concentra-
tions of 2.01 ␮g/dL and 3.83 ␮g/dL [17], in Czechoslovakia
4.82 ␮g/dL [18] and in Taiwan 4.09 ␮g/dL in umbilical cord
samples from healthy children [19]. In a study carried out
in Greece with 47 children, average lead levels were 13.1
± 6.0 ␮g/dL, much higher than those reported here [20].
Nonetheless, we found no association between high serum
lead levels and NTDs. With respect to this, there is some con-
troversy as to the teratogenic effects of lead on humans. Al-
though this effect has been demonstrated in mammals [8] and
in diverse epidemiologic reports in humans [21], certain stud-
ies on the maternal-fetal transfer of lead postulate that the pas-
sage of this metal across the placenta starts after the 12th week
of gestation when neural tube development has concluded [7],
weakening the biological plausibility of this association.
In relation to cadmium, concentrations were low and did
not surpass the AML in any of the cases or controls within
our study population in spite of the fact that high levels of
cadmium air and water pollution levels have been confirmed
in our city [5]. It is possible that the lace of an effect can be
explained by the of cadmium inability cross the placenta, as
has been documented in multiple studies [9,10,22]. Although
certain experiments in animals have reported teratogenic ef-
fects of this metal when administered during early stages of
pregnancy, studies in humans are scarce, and the only exist-
ing evidence is a study by Jiang in 1991 where a relationship
is shown between excess cadmium and NTDs [23]. Our find-
ings do not confirm this association.
With respect to zinc, we found that 38.3% (29% of the
cases and 9.3% of the controls) of the newborn babies stud-
ied had zinc deficiency. Although we could not find the cause
for this deficiency because we did not measure zinc in moth-
ers, we believe that an important contributing factor might
have been the fact that the population taking part in the study
came from low socioeconomic levels, with a diet based on
grain, cereals and vegetables, rich in phytates, calcium and
fiber. The tortilla is a food that is consumed daily in our coun-
try and in many marginal areas of Latin America. This food
contains a high amount of calcium, derived from the wash-
ing and processing of corn. The elements found in their diet
are considered as potential inhibitors for zinc absorption, and
one of the main causes of its deficiency in underdeveloped
countries [11,24–26].
p Our results showed an association between NTD and zinc
0.04∗ deficiency (OR 5.0, 95% CI 1.07–23.3, p = 0.04). Although
0.42 (NS) this association has been previously mentioned in other stud-
0.65 (NS) ies [11,12,24,27–29], the lack of consistency with the neg-
0.41(NS)
0.002**
ative results from other reports [30–32] has motivated con-
0.62 (NS) troversy on the real causal relationship of this association.
0.83 (NS) One of the factors implied in this diversity of findings has
0.73 (NS) been without a doubt, the heterogeneity of the methodology
0.56 (NS) used in the studies, the time relationship and the different
sources and methods to assess zinc concentrations (mater-
nal, fetal and/or newborn). The majority of the studies that
report an association measure zinc levels in maternal tissue.
Studies using serum concentrations from newborns, as in our
case, are scarce. Among those we can cite are Srinivas and
colleagues who recently conducted a study in 80 children
with NTD and 80 healthy controls, measuring zinc concen-
trations in their sera, as well as in hair samples from the
mothers and newborns. Their results show an association be-
tween NTD and low zinc levels in the hair both from mothers
and children [33]. No differences were found in the serum
levels, nonetheless, the sera values (80.1 ± 12.9 ␮g/dL for
cases and 77.8 ± 5.3 ␮g/dL for controls) are below those
in our series (115 ± 45.7 ␮g/dL and 143 ± 53.6 ␮g/dL), a
situation that may be due to differences in the chemical pro-
cedures for analyzing zinc, as well as the statistical method-
ology used, since the variation of zinc levels in their report is
very small and they only analyzed the difference between the
mean concentrations, not assessing the odds ratios as in our
analysis.
The diversity of zinc functions in the organism has given
rise to diverse hypotheses to explain the association between
zinc deficiency and NTD, such as abnormal synthesis of nu-
cleic acids and proteins, abnormal polymerization of tubulin,
chromosomal defects, excessive cell death and an increase of
cell membrane lipid peroxidation [25]. Recently, a promising
finding for the prevention of this disease has been the discov-
ery of the protective effect of folic acid [34–37]. This has lead
to the establishment of supplemental programs for pregnant
women. In spite of these actions, NTD continue to be one of
the most frequent congenital malformations worldwide. This
leads us to think that not all NTDs are related to folic acid,
and participation of other genetic and nutritional factors play
an important role in this entity. According to that, MRC Vi-
tamin Study Research Group report only 70% of NTDs were
prevented by supplemental folic acid, zinc deficiency could
play a role also [11,12,24,27,28]. Recently, Velie et al. stud-
ied the association of the periconceptional use of vitamins,
minerals, and food supplements in mothers of 430 newborn
babies with NTD and 429 controls through a questionnaire
[11]. Their results show a protective effect by zinc for NTD.
Animal studies have suggested an interaction between zinc
and folate. In a recent study, Hong et al. found low homo-
cysteine and folate plasma concentrations in rats with zinc
deficiency, probably secondary to an increase in the activ-
 M.d.L. Carrillo-Ponce et al. / Reproductive Toxicology 19 (2004) 149–154
ity of liver methionine synthetase [38], although this has not
been studied in humans.
Taking into account the multifactorial etiology of the
NTD, we compared our study groups in relation to the family
histories of the newborns that have been considered at risk
for this pathology (Table 1). The only significant difference
between the cases and controls was the family history of NTD
in the case group, a finding that agrees with those by other au-
thors. We found that NTD in the affected siblings (3–8%) are
considerably more frequent than in the general population,
a situation that supports the genetic contribution, widely ac-
cepted in the majority of congenital malformations [1,2,39].
We also analyzed the characteristics of newborns and their
gestational length (Table 2), observing that in those cases with
a shorter gestational length than in controls, a finding that is
related to the greater frequency of cesarean section births in
this group due to the presence of malformations that alter the
dynamics of birth. It was also observed that the length of time
between pregnancies of the cases was longer than that of the
controls, but no association was found with the presence of
NTD, and we had no cases with very short length of time be-
tween pregnancies (less than 6 months) that have been related
to the greater frequency of congenital malformations.
5. Conclusions
This study did not find an association between high lead,
cadmium or zinc serum levels in newborns and the pres-
ence of NTD, although an association was found between
these defects and low zinc levels. These findings focus atten-
tion once again to the importance of nutritional and maternal
health factors in this disease’s etiology. Further studies in
humans must be carried out to clarify the metabolic interre-
lations between folic acid and zinc to provide a more solid
support for nutritional supplementation programs during ges-
tation.
Acknowledgment
We are indebted to Marı́a Elena Cortez López for her col-
laboration concerning the technical aspects of this work.
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