Introduction

Alterations in the metabolism of some trace elements may contribute to the pathogenesis of attention-deficit hyperactivity disorder (ADHD). It is well known that ADHD is a highly prevalent neurobehavioral disorder with genetic, environmental, 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 oxidative stress in pathogenesis of many pediatric diseases, including autism, Down syndrome, and also ADHD [4–6]. Increased production of free radicals and oxidative stress may affect the homeostasis of some trace elements. Abnormal metabolism 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 mechanisms in the organism. Therefore, altered levels of these elements 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 may result in DNA damage, lipid peroxidation, depletion of glutathione and protein-bound sulfhydryl groups, and the other effects [9, 10].

The etiology of ADHD is not been clearly identified, although reported evidences support neurobiological and genetic background [6]. Recent structural and functional studies have suggested that dysfunction in the frontal-subcortical pathways, especially imbalance in the dopaminergic and noradrenergic systems, may be a contributing factor to the pathogenesis 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 superoxide dismutase (antioxidant protection of cells), tyrosinase and dopamine β-hydroxylase (metabolism of dopamine, noradrenaline, and epinephrine), monoamine oxidase (degradation of catecholamines), and ceruloplasmin for iron homeostasis 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, hyperactivity, and general behavioral problems [5].

Zinc is an important cofactor for metabolism of neurotransmitters, 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 structure 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 antioxidant by protecting the sulfhydryl groups of proteins and enzymes against free radical attack in the body, particularly in the brain. This element can also affect cell division, maturation, and growth of the fetus and, later, neurodevelopment and intellect of children [8]. Therefore, alterations in Zn metabolism during oxidative stress can be important in the development 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 supplementation 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 components 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 pathogenesis of neurological and psychiatric disorders. The evidence about the effects of selenium on the progress of neurological 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 accumulation 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 central nervous system. Moreover, Pb can cause dysfunction of some neurotransmitters including dopaminergic, glutamatergic, and cholinergic systems. These neurotransmitter systems, especially dopaminergic system, have been related to symptoms 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 symptoms 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 University, Bratislava, 58 children (45 boys and 13 girls), aged 6–14 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, schizophrenia, mood, anxiety, personality disorder and change due to a general medical condition, mental retardation, conduct disorder, 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 Declaration 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 evaluation 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 Rating 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 standard 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 biochemical 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, Zeeman background correction, Electrothermic atomizer GTA 120, Techtron Pty., Ltd., Springvale, Australia) [29, 30]. The accuracy of determination was evaluated by measuring the metal contents of certificated biological reference materials (Seronorm™ Trace Elements, Nycomed Pharma, Oslo, Norway).

Statistical Analysis

Shapiro-Wilk W test was used to assess the normality or non-normality distribution of data in investigated groups. Differences between ADHD group and control group were analyzed with the Student’s t test 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 were in the range of physiological values in both investigated groups (data not shown).

Table 1 Trace element status in the children with ADHD and control group
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We found significantly lower level of Zn (p = 0.0005) and higher Cu/Zn ratio (p = 0.015) in ADHD children when compared with control group (Figs. 1 and 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.

Fig. 1
figure 1

Comparison of Zn concentrations between ADHD children and control group. *p = 0.0005

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Fig. 2
figure 2

Comparison of Cu/Zn ratios between ADHD children and control group. *p = 0.015

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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).

Table 2 Correlation analysis of trace elements in the study groups
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Interestingly, the correlation analysis confirmed the existence 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-rated scales 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 inattention score) as well as with teacher-rated score for inattention (CTRS inattention score) (r = −0.328), but statistical significantly (p = 0.029; p = 0.014) as depicted in Fig. 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).

Fig. 3
figure 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

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Fig. 4
figure 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

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Despite the fact that there were no differences in the levels of Se and Pb (p > 0.05) between investigated groups of children (Table 1), we were also interested in whether there are any correlations between these elements and the ratings of 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 children 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. 1 and 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 disorders [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 appears 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 elevated 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 statistically 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 between these elements. Our assumption was also supported by correlation analysis (altered association between these elements is shown in Table 2).

We considered that Cu/Zn ratio is a more important parameter in assessing the relationship between Cu and Zn than the concentration of either of these trace 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 psychiatric and neurological disorders may be caused by dysfunction of dopaminergic, serotonergic, and noradrenergic neurotransmitter 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 neurotransmitter metabolism, such as dopamine hydroxylase and monoamine oxidase. High levels of Cu can induce damage of dopaminergic neurons by destroying the antioxidant defense 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 development, 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 ratio 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 peroxidation, 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. However, the precise mechanisms responsible for altered metabolism 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 relationship between trace minerals, antioxidant defense systems, and free radical formation in the pathophysiology of ADHD.