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Changed Plasma Levels of Zinc and Copper to Zinc Ratio
and Their Possible Associations with Parent- and Teacher-Rated
Symptoms in Children with Attention-Deficit Hyperactivity
Disorder
Alena Viktorinova
1
&Monika Ursinyova
2
&Jana Trebaticka
3
&Iveta Uhnakova
2
&
Zdenka Durackova
1
&Vlasta Masanova
2
Received: 10 March 2015 /Accepted: 1 June 2015
#Springer Science+Business Media New York 2015
Abstract Attention-deficit hyperactivity disorder (ADHD) is
associated with alterations in the metabolism of some trace
elements which may participate in the pathogenesis of this
disorder. The aims of the present study were to investigate
the trace element status (copper (Cu), zinc (Zn), copper to zinc
ratio (Cu/Zn ratio), selenium (Se), and lead (Pb)) of ADHD
children and compare them with the control group. Associa-
tions between examined elements and ratings of ADHD
symptoms were also assessed. Fifty-eight ADHD children
and 50 healthy children (aged 6–14 years) were included in
the study. The concentrations of Cu, Zn, and Se in the plasma
and Pb in the whole blood were measured by atomic absorp-
tion spectrometry. We found lower Zn level (p=0.0005) and
higher Cu/Zn ratio (p=0.015) in ADHD children when com-
pared with the control group. Copper levels in ADHD chil-
dren were higher than those in the control group, but not
significantly (p>0.05). No significant differences in levels of
Se and Pb between both groups were found. Zinc levels cor-
related with parent-rated score for inattention (r=−0.231, p=
0.029) as well as with teacher-rated score for inattention (r=
−0.328, p=0.014). Cu/Zn ratio correlated with teacher-rated
score for inattention (r=0.298, p=0.015). Significant
associations of Se and Pb with parent- and teacher-rated
symptoms were not observed. The results of this study indi-
cate that there are alterations in plasma levels of Cu and Zn as
well as significant relationships to symptoms of ADHD.
Keywords Trace element status .Zinc .Copper .Copper to
zinc ratio .Oxidative stress
Introduction
Alterations in the metabolism of some trace elements may
contribute to the pathogenesis of attention-deficit hyperactiv-
ity disorder (ADHD). It is well known that ADHD is a highly
prevalent neurobehavioral disorder with genetic, environmen-
tal, and biologic etiologies that persists into adolescence and
adulthood in a sizable majority of afflicted children of both
sexes. This disorder is characterized by behavioral symptoms
of inattention, distractibility, hyperactivity, and impulsivity
across the life cycle [1–3].
Several reports have highlighted the participation of oxida-
tive stress in pathogenesis of many pediatric diseases, includ-
ing autism, Down syndrome, and also ADHD [4–6]. In-
creased production of free radicals and oxidative stress may
affect the homeostasis of some trace elements. Abnormal me-
tabolism of some trace elements was detected in the children
with ADHD [7,8]. Trace elements such as Cu, Zn, and Se
play an essential role in the oxidant and antioxidant mecha-
nisms in the organism. Therefore, altered levels of these ele-
ments and their imbalance may lead to increased susceptibility
to oxidative damage of important cellular components, and
this may contribute to the pathogenesis of ADHD. Additional
studies have confirmed that Cu, Se, Pb, Fe, Cd, and Cr may
participate in the mechanisms of free radical produce which
*Alena Viktorinova
alena.viktorinova@fmed.uniba.sk
1
Institute of Medical Chemistry, Biochemistry, and Clinical
Biochemistry, Faculty of Medicine, Comenius University, Sasinkova
2, 811 08 Bratislava, Slovak Republic
2
Laboratory of Toxic and Essential Elements, Department of
Environmental Medicine, Slovak Medical University,
Bratislava, Slovak Republic
3
Department of Pediatric Psychiatry, Faculty of Medicine, Comenius
University, Bratislava, Slovak Republic
Biol Trace Elem Res
DOI 10.1007/s12011-015-0395-3
may result in DNA damage, lipid peroxidation, depletion of
glutathione and protein-bound sulfhydryl groups, and the oth-
er effects [9,10].
The etiology of ADHD is not been clearly identified, al-
though reported evidences support neurobiological and genet-
ic background [6]. Recent structural and functional studies
have suggested that dysfunction in the frontal-subcortical
pathways, especially imbalance in the dopaminergic and nor-
adrenergic systems, may be a contributing factor to the path-
ogenesis of ADHD. Therefore, some symptoms of ADHD
may be caused by dysfunction of catecholamines, in which
copper and zinc especially participate [11,12].
It is common knowledge that copper is necessary for the
catalytic activity of many enzymes which have an essential
role in neurophysiology of this disorder, including Cu/Zn su-
peroxide dismutase (antioxidant protection of cells), tyrosi-
nase and dopamine β-hydroxylase (metabolism of dopamine,
noradrenaline, and epinephrine), monoamine oxidase (degra-
dation of catecholamines), and ceruloplasmin for iron homeo-
stasis in the brain [13]. Several studies have pointed to the role
of copper as a pro-oxidant and its participation in the metal-
catalyzed formation of free radicals. In view of this, excessive
Cu levels and copper-mediated neurotoxicity may be related
to the formation of copper-dopamine complex followed by
oxidation of dopamine. This status may be associated with
physical and mental fatigue, depression, and other mental
problems such as schizophrenia, learning disabilities, hyper-
activity, and general behavioral problems [5].
Zinc is an important cofactor for metabolism of neurotrans-
mitters, prostaglandins, and melatonin and indirectly affects
dopamine metabolism. It is necessary for various
metalloenzymes and metal-protein complexes, particularly in
the central nervous system, and thus contributes to the struc-
ture and function of brain. Moreover, the dopamine transport
system has a zinc-binding site that is essential for transport
mechanisms in the brain [14–16]. Zinc also acts as an antiox-
idant by protecting the sulfhydryl groups of proteins and en-
zymes against free radical attack in the body, particularly in
the brain. This element can also affect cell division, matura-
tion, and growth of the fetus and, later, neurodevelopment and
intellect of children [8]. Therefore, alterations in Zn metabo-
lism during oxidative stress can be important in the develop-
ment of neurological dysfunctions [10,17].
In addition to these functions, zinc plays a substantial role
in the formation and modulation of melatonin. Melatonin is a
hormone that has several functions in the body, including
control of reproductive processes and regulation of dopamine
metabolism as well as the sleep cycle. It has been confirmed
that Zn deficiency may be associated with changes in
neurodevelopment, cognition, emotion, and motor activity in
ADHD children [7,18]. As noted in other studies, zinc sup-
plementation may be a significant contributor to the treatment
of this disorder [19,20].
Selenium has an essential role as a major constituent of
many enzymes, some of which have antioxidant functions.
Protective effects of Se seem to be associated with its presence
in the glutathione peroxidase and thioredoxin reductase,
which are known to protect DNA and other cellular compo-
nents from oxidative damage. It occurs in the tissues mainly in
protein-bound form, as selenoproteins, in which the sulfur is
replaced by selenium, and most of them are expressed in the
brain [9]. The brain represents an organ with high amounts of
Se, which is susceptible on suboptimal Se levels. Therefore,
selenium deficiency may play a substantial role in the patho-
genesis of neurological and psychiatric disorders. The evi-
dence about the effects of selenium on the progress of neuro-
logical and psychiatric disorders in the children is very limited
[4,21].
Some heavy metals, especially lead, have a high affinity for
–SH groups of numerous proteins that are an integral part of
the active site of the enzyme, and this status may lead to
changes in the function of attacked enzymes [22]. Lead is a
neurotoxic element, and the developing brain is vulnerable to
its toxic effects which can be dangerous for fetus and children
during their development and growth. It has been found that
Pb ions readily penetrate the placenta and occur in the same
concentration in both fetal and maternal blood [23]. Binding
of Pb to the biomolecules of placental tissue and its accumu-
lation in the placenta are affected by the state of pregnancy as
well as chemical form of Pb in the maternal blood (diffusible
fraction of Pb better enters into the placenta). The result of the
toxic effects of Pb during pregnancy is fetal damage with
subsequent retardation of growth and development of the cen-
tral nervous system. Moreover, Pb can cause dysfunction of
some neurotransmitters including dopaminergic, glutamater-
gic, and cholinergic systems. These neurotransmitter systems,
especially dopaminergic system, have been related to symp-
toms of ADHD in the children [24,25]. A recent study has
found slightly higher Pb levels in the blood among ADHD
children and also relationships between Pb levels and symp-
toms of hyperactivity and impulsivity. No association between
Pb levels and inattention was observed [26].
The aims of current study were to compare the status of
some trace elements (Cu, Zn, Cu/Zn ratio, Se, and Pb) of
ADHD children with healthy subjects and also to assess the
possibility of their association with parent- and teacher-rated
symptoms of ADHD.
Subjects and Methods
Study Population and Design
Of the 62 outpatients with ADHD treated at the Department of
Pediatric Psychiatry, Faculty of Medicine, Comenius Univer-
sity, Bratislava, 58 children (45 boys and 13 girls), aged 6–
Viktorinova et al.
14 years (mean 9.4±2.1) were enrolled in the study. Four
children did not meet the inclusion criteria and were excluded
from this study.
Inclusion criteria were as follows: early onset of ADHD
(by 6–7 years) and chronicity of the disease (at least 6 months
of symptoms). Exclusion criteria were as designed: situational
hyperactivity, pervasive developmental disorders, schizophre-
nia, mood, anxiety, personality disorder and change due to a
general medical condition, mental retardation, conduct disor-
der, tics, chorea, and other dyskinesia; acute inflammatory
diseases; renal and cardiovascular disorders; and diabetes
mellitus.
The control group consisted of 50 healthy children, aged 6–
14 years, recruited from the general population. Children who
participated in the study did not take nutritional supplements
and any drugs that are known to interfere with metabolism of
studied elements before this study. All parents gave a written
consent for participation of their children in this study. The
study was conducted in accordance with the Helsinki Decla-
ration and was approved by the Ethical Committee of the
Child University Hospital in Bratislava.
Clinical Evaluation of Cognitive and Neurobehavioral
Functions
Children with ADHD were included in the study after evalu-
ation of diagnostic criteria of ADHD as described by
Trebaticka et al. [27]. Clinical symptoms of ADHD children
were investigated by standard questionnaires: Child Attention
Problems (CAP) teacher rating scale, Conner’s Teacher Rat-
ing Scale (CTRS), Conner’s Parent Rating Scale (CPRS), and
Wechsler Intelligence Scale for children.
Sample Collection
Samples of venous blood were taken after overnight fasting
and collected into commercial tubes with sodium citrate (S-
Monovette, Sarstedt, Numbrecht, Germany) for analysis of
basic biochemical parameters. Metal-free tubes for the stan-
dard venipuncture technique (Vacutainer Trace Element
Tubes, Sarstedt, Numbrecht, Germany) were used for analysis
of Cu, Zn, and Se in the plasma and Pb in whole blood. The
first part of syringes with blood for the determination of basic
biochemical parameters was transported to the Biochemical
Clinical Laboratory of the University Hospital, Comenius
University in Bratislava. The second part of syringes with
blood for determination of trace element status was
transported to Institute of Medical Chemistry, Biochemistry,
and Clinical Biochemistry, Faculty of Medicine, Bratislava.
Subsequently, plasma was isolated by centrifugation under
standard conditions and aliquoted into metal-free Eppendorf
test tubes (Eppendorf AG, Hamburg, Germany), frozen, and
stored at −80 °C until further analysis.
Determination of Basic Biochemical Parameters
Plasma levels of basic biochemical parameters (glucose, lipid
profile, uric acid, bilirubin, alkaline phosphatase, aspartate
aminotransferase, alanine aminotransferase, gamma-
glutamyl transferase) were measured by standardized bio-
chemical methods at the Biochemical Clinical Laboratory of
the University Hospital, Comenius University in Bratislava.
The concentrations of measured parameters were compared
with reference values of this laboratory.
Determination of Trace Element Status
Flame technique of atomic absorption spectrometry was used
for determination of Cu and Zn concentrations in the plasma
(FAAS Varian AA240FS, Deuterium background correction,
Techtron Pty., Ltd., Springvale, Australia) [28]. Concentrations
of Se in the plasma and Pb in the whole blood were measured
by electrothermal technique (ETA AAS Varian AA 280Z, Zee-
man background correction, Electrothermic atomizer GTA 120,
Techtron Pty., Ltd., Springvale, Australia) [29,30]. The accura-
cy of determination was evaluated by measuring the metal con-
tents of certificated biological reference materials (Seronorm
™
Trace Elements, Nycomed Pharma, Oslo, Norway).
Statistical Analysis
Shapiro-Wilk Wtest was used to assess the normality or non-
normality distribution of data in investigated groups. Differ-
ences between ADHD group and control group were analyzed
with the Student’sttest for data with a normal distribution.
Results are shown as mean±SD. For comparison of data with
a non-normal distribution, non-parametric Mann-Whitney U
test was used. These results were expressed as median (first
quartile, third quartile). Spearman rank correlation test was used
to evaluate the relationships between measured parameters in
each group and expressed with the Spearman’s rank correlation
coefficient. Statistical significance for all calculations was set at
the level of p<0.05. Data were analyzed using the program
StatsDirect
®
2.3.7 (StatsDirect Sale, Cheshire M33 3UY,
UK). Graphical representation of data was performed by the
Excel 2007 (Microsoft Office Excel Corporation, USA).
Results
The study consisted of 58 ADHD children aged 6–14 years
(mean 9.4±2.1) and 50 healthy children (mean 8.9±2.8). We
did not observe any significant difference between age of
ADHD children and age of the control group (p>0.05).
The plasma levels of Cu, Zn, Cu/Zn ratio, and Se and
whole blood levels of Pb in ADHD children and control group
are shown in Table 1. Plasma levels of biochemical parameters
Zinc and Copper to Zinc Ratio in ADHD Children
were in the range of physiological values in both investigated
groups (data not shown).
We found significantly lower level of Zn (p=0.0005) and
higher Cu/Zn ratio (p=0.015) in ADHD children when com-
pared with control group (Figs. 1and 2). Copper levels in
ADHD children tended to be higher than in the control group,
but this difference was not statistically significant (p>0.05).
We recorded that levels of Se and Pb in ADHD children were
similar to those in healthy children.
Moreover, we evaluated relationships between individual
levels of Cu, Zn, Cu/Zn ratio, Se, and Pb in both study groups.
An imbalance in the levels of Cu and Zn was observed in
ADHD children when compared with healthy children. As
shown in Table 2, there is significant correlation between Cu
and Zn in healthy children (p=0.038), but on the contrary, no
significant relationship between these elements in ADHD
children was found. Statistical analysis showed that there are
strong correlations between Cu/Zn ratio and Cu and also Zn in
both groups of children which were included into our study
(p<0.0001).
Interestingly, the correlation analysis confirmed the exis-
tence of weak correlations between age and Zn (r=0.238), Cu
(r=0.216), and Cu/Zn ratio (r=0.241) in control group
(p<0.05). On the contrary, inverse correlations between age
and Zn levels (r=−0.206, p=0.154) and Cu/Zn ratio (r=
−0.117, p=0.078) were found in ADHD children. The level
of Cu positively correlated with age in this group (r=0.292,
p=0.064). No significant associations between age and Se and
also Pb levels were found in both groups (data not shown).
We also investigated associations between levels of Cu, Zn,
Cu/Zn ratio, Se, and Pb and parent- and teacher-ratedscales of
clinical symptoms of ADHD. A detailed report describing
these ratings was previously published [27]. Decreased Zn
levels in ADHD children weakly inversely correlated (r=
−0.231) with parent-rated score for inattention (CPRS inatten-
tion score) as well as with teacher-rated score for inattention
(CTRS inattention score) (r=−0.328), but statistical signifi-
cantly (p=0.029; p=0.014)asdepictedinFig.3.Correlation
analysis between Cu/Zn ratio and parent- and teacher-rated
symptoms of ADHD showed that increased values of this ratio
significantly correlated (r=0.298, p=0.015) exclusively only
with CTRS inattention (Fig. 4). No significant correlations
between Cu level and ratings of ADHD symptoms and
also between Zn level and Cu/Zn ratio and parent- and
teacher-rated scores for hyperactivity were found (data
not shown).
Despite the fact that there were no differences in the levels
of Se and Pb (p>0.05) between investigated groups of chil-
dren (Table 1), we were also interested in whether there are
any correlations between these elements and the ratings of
Tabl e 1 Trace element status in
the children with ADHD and
control group
Parameter ADHD group (n=58) Control group (n=50) pvalue
Cu (μmol/L) 18.31± 3.34 17.64±2.83 >0.05
Zn (μmol/L) 10.63 (9.37; 11.28) 11.32 (10.56; 12.74) 0.0005
Cu/Zn ratio 1.78 (1.51; 1.99) 1.61 (1.39; 1.78) 0.015
Se (μmol/L) 0.92±0.12 0.93 (0.78; 0.98) >0.05
Pb (μmol/L) 0.116 ±0.08 0.113±0.04 >0.05
Data are given as mean±SD (standard deviation) for parametric variables or as median (first quartile; third
quartile) for nonparametric variables. nis the number of subjects per group. pvalue represents the difference
between measured parameters in ADHD group and control group (p<0.05 was defined as statistically significant)
Fig. 1 Comparison of Zn concentrations between ADHD children and
control group. *p=0.0005
Fig. 2 Comparison of Cu/Zn ratios between ADHD children and control
group. *p=0.015
Viktorinova et al.
ADHD symptoms. Significant associations of Se and Pb with
parent- and teacher-rated symptoms were not observed.
Discussion
In the past decades, a number of studies have disclosed that
the homeostasis of some trace elements is altered in the chil-
dren with ADHD and that these alterations may contribute to
the pathogenesis of this disorder [3]. Although, there has been
an increasing interest about understanding the participation of
trace elements in the pathophysiology of ADHD, but to date,
their role has not yet been elucidated. Several researchers have
studied the occurrence of trace element deficiency among
children with ADHD compared with control subjects. Some
of them found deficiencies of Cu, Zn, Fe, magnesium, and
calcium in ADHD children on the basis of analyses of serum,
red cell, and hair [31,32]. In contrast to the findings of these
studies, we observed that Cu levels in ADHD children were
higher than those in healthy children (Table 1). In addition, we
also found significantly lower levels of Zn and higher Cu/Zn
ratio in ADHD children in comparison to control group
(Figs. 1and 2).
The role of selenium in the pathophysiology of ADHD is
little described in the literature. Some authors have observed
decreased levels of Se in children with specific mental disor-
ders [4,33]. In comparison with these studies, no significant
differences in the levels of Se among both groups of children
included into this study as well as no significant associations
of Se with parent- and teacher-rated symptoms of ADHD were
recorded.
A few studies have described the participation of Pb ions in
the pathogenesis of ADHD and their relation to the symptoms
of this disorder [26,34]. In our study, no differences in Pb
levels between the study groups of children were observed.
Moreover, blood Pb level was not significantly associated
with parent- and teacher-rated symptoms of ADHD.
In view of the knowledge of the present literature, it ap-
pears that there is a link between the metabolisms of copper
and zinc, and imbalance of these elements is still actively
studied [4,16,18]. Reduced levels of Zn often result in ele-
vated levels of Cu due to the dynamic competition between
these metals in the body [35,36]. It has been proven that under
the condition of zinc deficiency, copper tends to accumulate in
the body. This status may be associated with hyperactivity,
learning disabilities, and depression [19,20]. Given the sta-
tistically non-significant trend toward higher levels of Cu
and significantly reduced levels of Zn in children with
ADHD when compared with control group, we assume
that there is a noticeable evidence of an imbalance be-
tween these elements. Our assumption was also support-
ed by correlation analysis (altered association between
these elements is shown in Table 2).
Tabl e 2 Correlation analysis of trace elements in the study groups
Variables ADHD group Control group
rpvalue rpvalue
Zinc
Cu 0.153 0.128 0.264 0.038
Se 0.375 0.002 0.402 0.003
Pb −0.619 0.058 −0.168 >0.05
Cu/Zn ratio
Cu 0.696 <0.0001 0.475 0.0005
Zn −0.536 <0.0001 −0.636 <0.0001
Se −0.226 0.047 −0.077 0.313
Pb 0.357 >0.05 0.206 >0.05
ris Spearman’s rank correlation coefficient. pvalue represents the differ-
ence between correlated parameters in ADHD group and control group
(p<0.05 was defined as statistically significant)
Fig. 3 Associations between Zn concentration and CPRS score for
inattention (r=−0.231, p=0.029) as well as CTRS score for inattention
(r=−0.328, p=0.014) in children with ADHD. CPRS Conner’s Parent
Rating Scale, CTRS Conner’s Teacher Rating Scale
Fig. 4 Association between Cu/Zn ratio and CTRS score for inattention
in children with ADHD (r=0.298, p=0.015). CTRS Conner’s Teacher
Rating Scale
Zinc and Copper to Zinc Ratio in ADHD Children
We considered that Cu/Zn ratio is a more important param-
eter in assessing the relationship between Cu and Zn than the
concentrationofeitherofthesetrace elements. This fact was
confirmed by strong positive correlation between Cu/Zn ratio
and Cu level as well as negative correlation between Cu/Zn
ratio and Zn level (Table 2). An altered ratio of copper and zinc
may be related to an impaired ability of the body to maintain or
regenerate the copper and zinc homeostasis after effect of
destabilizing factors on the metabolism of these elements.
It has been reported that the symptoms of variety psychiat-
ric and neurological disorders may be caused by dysfunction
of dopaminergic, serotonergic, and noradrenergic neurotrans-
mitter systems [11,12]. Optimal levels of copper and zinc are
required for the metabolism and function of neurotransmitters.
Copper is a cofactor for activity of some enzymes in neuro-
transmitter metabolism, such as dopamine hydroxylase and
monoamine oxidase. High levels of Cu can induce damage
of dopaminergic neurons by destroying the antioxidant de-
fense system as has been confirmed in animal experiments
[13]. Zinc may influence the metabolism of melatonin and,
thus, the functioning of the dopaminergic system. Melatonin
participates in the regulation of sleep cycles, and its changed
level can contribute to the symptoms of ADHD. It is known
that Cu metabolism is connected with Zn metabolism and
disturbed relationships between them can lead to delayed de-
velopment, attention deficit disorder, and anti-social behavior,
hyperactivity, autism, and learning difficulties [37].
The present study confirmed previous findings [12]that
decreased Zn levels are inversely associated with parent- and
teacher-rated scores for inattention in ADHD children, but not
with hyperactivity. Moreover, we observed that Cu/Zn ra-
tio positively correlated exclusively only with teacher-
rated score for inattention. Our findings support and
extend the hypothesis, which is based on the argument
that these trace elements can considerably contribute to
the pathogenesis of ADHD [38,39].
Altered levels of Cu, Zn, and the ratio of copper to zinc
may attenuate the antioxidant defense system and create the
conditions for increased oxidative stress in ADHD children.
This assumption is consistent with a recent study [14] focused
on the assessment of the possible role of oxidative stress in the
pathogenesis of ADHD by measuring various biochemical
markers of oxidative metabolism (products of lipid peroxida-
tion, levels of non-esterified fatty acids). Authors concluded
that increased levels of malondialdehyde, decreased Zn levels,
and their correlations with symptoms of inattention support
the oxidative stress theory in the pathogenesis of ADHD.
Conclusion
Based on our findings, we can postulate that an imbalance in
levels of trace elements under the structure of antioxidant
enzyme defense systems may cause damage to some functions
of cell components in the brain depending on the produce of
free radicals. This status may result to changes of behavior in
the children and contribute to the symptoms of ADHD. How-
ever, the precise mechanisms responsible for altered metabo-
lism of trace elements in ADHD children are unclear and
require following studies. Therefore, it is necessary to focus
further research to obtain more information about the relation-
ship between trace minerals, antioxidant defense systems, and
free radical formation in the pathophysiology of ADHD.
Acknowledgments This study was supported by MVTS TW-010002
grant of Ministry of Education, Slovak Republic. Authors wish to thank
all volunteers for their participationin this study and to Mrs. Husekova Z.
and Witkova V. for their technical assistance as well as the nurses for
taking blood samples from children.
Conflict of interest The authors declare that they have no conflict of
interest.
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Zinc and Copper to Zinc Ratio in ADHD Children
