Increased oxidative stress in children with attention deficit hyperactivity disorder

Abstract

Objectives: The purpose of this study was to investigate oxidative stress in children with attention deficit hyperactivity disorder (ADHD).

Methods: Total oxidant status (TOS), total antioxidant status (TAS), paraxonase-1 (PON-1) and arylesterase (ARE) activity were measured in 76 children (44 boys, 32 girls) diagnosed with ADHD according to the DSM-IV and 78 healthy children (46 boys, 32 girls).

Results: Age and sex were similar between the groups (P > 0.05). TOS and the oxidative stress index (OSI) were higher in the patient group than the control group (P < 0.001). PON-1 (P = 0.002), ARE (P = 0.010) activity and TAS (P < 0.001) were lower in the patient group than the control group.

Discussion: We found decreased PON-1, ARE activity and TAS, and increased TOS and OSI in children with ADHD. Our study showed that there is significantly increased oxidative stress in children with ADHD.

Keywords:

• Attention deficit hyperactivity disorder
• Oxidative stress
• Paraxonase-1
• Arylesterase

Introduction

It is known that attention deficit hyperactivity disorder (ADHD) is one of the most common neurobehavioral disorders in children.¹ The prevalence of ADHD is 6–7% in children and it is more common in boys than girls.^2,3 Difficulty concentrating, hyperactivity, and impulsivity are the main symptoms of the disorder.⁴ Symptoms frequently affect patients’ school achievements and social interaction with the environment. Home life may also be very difficult, as the constant activity of children with ADHD tends to cause conflict in their families.^5–7 The first-line treatment for patients with severe ADHD is considered pharmacological therapy, such as methylphenidate.⁸ Curative therapy is not recommended for this disorder because the precise cause is not known.

Although easily recognized clinically, the reasons and pathophysiology of ADHD are not fully understood. It is thought that many factors may be conducive to the etiopathogenesis of ADHD, including genetic and neurochemical factors.^9–11 There is evidence that oxidative stress could also contribute to the pathogenesis. It has long been known that oxidative stress is an important factor in cell destruction, damage, and death. Many recent studies on adults have shown that it also plays an important role in the pathogenesis of many psychiatric disorders, such as schizophrenia, obsessive compulsive disorder, bipolar disorder, and autism.^12–17 Furthermore, it has been demonstrated that oxidative stress contributes to the development of some psychiatric disorders in children, particularly autism, developmental disorders, and attention deficit hyperactivity disorder.^16–19 Other studies have shown that oxidative stress decreases in patients with ADHD. Oztop et al. found low levels of some oxidants and no difference in the antioxidant parameters of ADHD children, and they concluded that there may not be a direct relationship between oxidative stress and ADHD.²⁰ Similarly, Dvoráková et al. determined that there are low total antioxidant levels in children with ADHD.²¹ Low levels of oxidant nitric oxide (NO) have also been reported in children with ADHD.²² Furthermore, it has been shown that malondialdehyde (MDA) is statistically significantly low in children with ADHD.²³ In addition, in some studies oxidative stress was found to be increased in patients with ADHD. For example, increased levels of NO and the lipid peroxidation product MDA as well as decreased antioxidant superoxide dismutase activity were evident in adult ADHD patients.^23,24

Serum paraoxonase-1 (PON-1) is an antioxidant enzyme transported along with HDL-C in the plasma.²⁵ It is a 45-kDa glycoprotein that is expressed in the liver, and it has paraoxonase, arylesterase, and dyazoxonase activities.²⁵ In particular, it reduces low density lipoprotein cholesterol oxidation and atherosclerosis.^25,26 Additionally, serum PON-1 expression is also down-regulated by oxidative stress.²⁷ It is related to many neurological diseases, such as epilepsy and schizophrenia.^28,29 Oztop et al. showed that PON-1 activity has been found to be similar in ADHD patients and controls.²⁰ Oxidative status may be useful in understanding the pathophysiology of the disorder and its treatment. A few studies have been conducted on oxidative stress in people with ADHD, but the results are conflicting. Furthermore, the oxidative parameters used in these studies did not reflect the total oxidant and antioxidant status. We therefore opted to investigate the total oxidant status (TOS) and total antioxidant status (TAS) of children with ADHD. The aim of this study is to reveal the TAS, TOS, and oxidative balance in children with ADHD.

Materials and methods

Subjects

Between August 2012 and August 2014, 80 children (48 boys and 32 girls; 8–17 years of age) were brought to the Children's Mental Health Outpatient Clinic by their parents and diagnosed with ADHD in accordance with the Turkish version of the DSM-IV criteria.³⁰ We also recruited 80 healthy children and adolescents (46 boys and 34 girls; 8–18 years) of similar age and sex, who had attended a pediatric outpatient clinic for other reasons, such as vaccination, growth follow-up and did not meet the criteria for removal from study. Four patients and two healthy controls were excluded from the study due to reasons including drug use in the previous 6 months. The local ethics committee approved the study (date: 21 February 2012 and Harran University Ethics Committee approval number: 020.05.00.050.01.04-79). All the participants were informed about the study and gave their voluntary signed consent.

Patients with a history of acute or chronic systemic disease, such as epilepsy and diabetes mellitus, and who had co-morbid psychiatric disorders and mental retardation were excluded. Patients who had used psychotropic drugs in the previous 6 months and those who had received any antioxidant agents (e.g. vitamins E and C) were also excluded. None of the controls had chronic inflammatory or infectious diseases that could affect the oxidative stress parameters.

Blood samples

Blood samples were obtained following an overnight fast. The samples were withdrawn from the cubital vein into blood tubes and kept for 30 minutes at room temperature. Later, the serum was separated from the cells by centrifugation at 3000 rpm for 10 minutes and stored at −80°C until analysis.

Measurement of paraoxonase and arylesterase activity

The paraoxonase activity measurements were performed in the absence (basal activity) and presence of NaCl (salt-stimulated activity). The rate of paraoxon hydrolysis (diethyl-p-nitrophenylphosphate) was measured by monitoring the increase in absorbance at 412 nm at 25°C. The amount of generated p-nitrophenol was calculated from the molar absorptivity coefficient at pH 10.5, which was 18.290 M⁻¹ cm⁻¹.³¹ Paraoxonase activity was expressed as U/l serum.

To measure the arylesterase enzyme activity, phenylacetate was used as a substrate. Enzymatic activity was calculated from the molar absorptivity coefficient of the produced phenol, which was 1.310 M⁻¹ cm⁻¹. One unit of arylesterase activity was defined as 1 μmol phenol generated/min under the aforementioned conditions and expressed as U/l serum.^31,32 Arylesterase activity was expressed as U/l serum.

Measurement of TAS

Serum TAS levels were determined using Erel's method, a novel automated measurement method.³³ In this method, hydroxyl radical, the most potent radical, was created via Fenton reaction. In this reaction, the hydroxyl radical is produced by mixing a hydrogen peroxide solution with a ferrous ion. In the assay, the ferrous ion solution present in Reagent 1 is mixed with hydrogen peroxide, which is present in Reagent 2. The resultant radicals, such as brown-colored dianisidinyl radical cation, which is produced by the hydroxyl radical, are also potent radicals.

In this assay, the antioxidative effect of the sample against the potent free radical reactions initiated by the produced hydroxyl radical was measured. The assay gave excellent precision values, which were lower than 3% (the coefficient of variation). The results were expressed as mmol Trolox equiv./l.

Measurement of TOS

Serum TOS levels were determined using a novel automated measurement method of Erel.³⁴ The oxidants present in the sample oxidized the ferrous ion-o-dianisidine complex to form ferric ions. The oxidation reaction was enhanced by the glycerol molecules present in the reaction medium. In an acidic medium, ferric ions make a colorful complex of xylenol orange. The intensity of the color, which was measured spectrophotometrically, was related to the total amount of oxidant molecules present in the sample. The assay was calibrated by hydrogen peroxide. The results were expressed as μmol H₂O₂ equiv./l.

Oxidative stress index

The percentage ratio of the TOS to the TAS level was accepted as the oxidative stress index (OSI). The aim of this calculation is to determine the direction of the oxidative balance in the body. If this value is 1, it is in equilibrium with the total antioxidant effect and total oxidant effect. The OSI value was calculated according to the formula: OSI = (TOS/TAS) × 100.³⁵ This formula can be applied after synchronizing the TOS and TAS units; TAS levels were multiplied by 1000, to synchronize with the units of TOS, for calculation of OSI. The OSI value was expressed as an arbitrary unit.

Statistical analysis

The Statistics Program for Social Science for Windows version 11.5 (SPSS, Inc., Chicago, IL) was used for statistical analysis. To determine the distribution of parameters, the one-sample Kolmogorov–Smirnov test was used, and the distribution was evaluated as normal. For a comparison of the parameters between the groups, independent samples t-tests and χ² tests were used. The results of numeric parameters were expressed as mean ± standard deviation (SD) and the results of categorical parameters were expressed as percentages. Values of P < 0.05 were considered statistically significant. The relationships of TAS, TOS, OSI, PON-1, and ARE with the other parameters were investigated using Pearson's correlation test. The direct effect of the related parameters on these parameters was investigated by linear regression analysis.

Results

The demographic and biochemical data of the patients and controls are summarized in Table 1. Age and sex were similar between the groups (P = 0.230 and χ² 0.005, respectively). The mean age of the ADHD patients was 11.222 ± 2.08 (8–17) years compared with 12.15 ± 1.04 (8–18) years in the control group.

Table 1 Demographic and biochemical data on the group

	Patient group (n = 76)	Control group (n = 78)	t and x^2 value	P value
Age, year	11.22 ± 2.08	12.15 ± 1.04	3.2	0.230
Sex, female (%)	45	49	0.005*	0.123
TAS (mequiv. Troloks/l)	0.73 ± 0.17	0.95 ± 0.17	−8.10	<0.001
TOS (mmol H2O2/l)	37.29 ± 8.51	27.34 ± 7.11	7.75	<0.001
OSI, arbitrary unit	5.47 ± 1.52	2.97 ± 0.96	11.84	<0.001
Paraoxonase activity (U/l)	142.30 ± 47.16	167.55 ± 49.50	−3.17	0.002
Arylesterase activity (U/l)	94.59 ± 19.14	102.51 ± 18.59	−2.61	0.010

Table obtained from the data of independent t-test and chi-square. P < 0.05 was considered significant.

*χ² values.

OSI, oxidative stress index; TOS, total oxidant status; TAS, total antioxidant status.

TOS and OSI were higher in the patient group than the control group (P < 0.001; t = 7.75 and 11.84, respectively) (Table 1), while TAS was lower in the patient group (P < 0.001; t = −8.10). The activity of PON-1 and ARE were lower in the patient group than the controls (P = 0.002 and 0.01; t = −3.17 and −2.61, respectively) (Table 1).

OSI negatively correlated with PON-1, ARE, and TAS and correlated positively with TOS. Regression analysis showed that TAS and TOS independently influenced OSI. The Pearson correlation analysis showed that TAS positively correlated with PON-1 and ARE and correlated negatively with TOS. The regression analysis showed that only TAS was influenced by TOS; TOS negatively correlated with TAS. The regression analysis also showed that TOS was influenced by TAS independently (Table 2).

Table 2 TAS, TOS, and OSI related and effected parameters

	Beta correlation coefficients	P value	Beta regression coefficients	P value
Oxidative stress index
Paraoxonase activity	−0.203	0.015	−0.005	0.848
Arylesterase activity	−0.271	0.001	−0.009	0.734
Total oxidant status	0.790	<0.001	0.624	<0.001
Total antioxidant status	−0.751	<0.001	−0.579	<0.001
Total antioxidant status
Paraoxonase activity	0.181	0.028	0.073	0.437
Arylesterase activity	0.159	0.049	0.159	0.093
Total oxidant status	−0.276	0.001	−0.240	0.004
Total oxidant status
Total antioxidant status	−0.276	0.001	−0.247	0.004

Table obtained from the data from the Pearson correlation test and linear regression test. P < 0.05 was considered significant.

Discussion

To the best of our knowledge, this is the first study to evaluate PON-1 and ARE levels in conjunction with the oxidative stress parameters TOS and TAS. We have shown that markedly increased oxidative stress is determined by increased TOS and a decreased TAS antioxidant enzyme, such as PON-1 and ARE, in children with ADHD.

Changes in oxidative parameters may be related to ADHD, although studies on this subject show inadequate and contradictory results. Selek et al. showed that ADHD is related to higher oxidative stress in adult patients.²⁴ In their study, the TAS, TOS, and OSI of the ADHD patients were significantly higher than those of the controls. There were similar results in our study; however, we found that TAS was lower in the patient group. In addition, PON-1 and ARE were lower in patients with ADHD than those in the controls in a previous adult study.²³ Nevertheless, many parameters may increase oxidative stress in adults. There are a few conflicting studies on ADHD in children. Some studies have shown that there is decreased oxidative stress in children with ADHD. Oztop et al. demonstrated that the levels of the oxidant parameters MDA and 8-OHDG were statistically significantly lower in children with ADHD compared to the controls in their study, and there was no difference between the groups with respect to advanced oxidation protein products and PON-1 and thiol levels.²⁰ In another study, Archana et al. found that protein thiols were increased in children with ADHD compared to controls, but ceruloplasmin, an antioxidant enzyme, was similar between the two groups.^19,36 Another study showed low MDA in ADHD patients,¹⁹ where the presence of the disorder and the use of methylphenidate increased oxidative stress in experimental models.^37,38 For example, Martins et al. showed that chronic exposure to methylphenidate induces oxidative damage in the brains of young rats, while Kawatani et al. found that the urinary levels of acrolein-lysine, an oxidant molecule, were increased in children with ADHD compared to the controls in their study.³⁹ In the study of Ceylan et al., the activity of glutathione peroxidise, which is an antioxidant enzyme, was found to be significantly lower in patients with ADHD than the controls.⁴⁰ Another study performed by Dvoráková et al. revealed that pycnogenol – an extract from the pine bark – administration decreased oxidized glutathione and TAS levels and increased reduced glutathione in children with ADHD.²¹ Some studies have shown that oxidant levels are neither decreased nor increased in patients with ADHD. For instance, the activity of superoxide dismutase and cathalase, which are antioxidant enzymes, did not show significant differences between ADHD and control groups, but these studies did not clearly show the total oxidative balance, and they provided conflicting results.⁴²

We investigated the parameters of total oxidative status indicators, such as TOS, TAS, and the oxidative stress index. We saw increased TOS and OSI and decreased TAS at the end of the study, with OSI indicating that the oxidative balance was drawn to the side of oxidative stress. Some oxidants and antioxidants, which were added in to the medium, may have a rapid effect on oxidative stress. However, a change in the antioxidants with a protein and enzyme structure is hard and needs much more time because of the need for an effect at the genetic level. Our study showed that there is also a reduction in antioxidant enzymes, such as PON-1 and ARE. Our study also differed from the previously published studies that evaluated oxidative stress parameters, including TAS and TOS, and the PON-1 and ARE levels.

In patients with ADHD, increased oxidative stress may occur for many reasons. For example, it may be the result of chronic inflammation. Data have provided evidence of increased inflammation in patients with ADHD.⁴¹ Genetics may also play a role. For example, genetics has been shown to increase oxidative stress in mice lacking the dopamine D5 receptor.⁴² Previously, it was demonstrated that ADHD may be associated with these receptor disorders.⁴³ It is not known if increased oxidative stress is the cause or the outcome of disorder. In the presence of ADHD in children, restlessness and motion are present, and increased oxidative stress is apparent after increased physical activity.⁴⁴

There were some limitations to our study in that it was a cross-sectional design. Without longitudinal studies, it is difficult to interpret the precise effect of oxidative stress on pediatric patients with ADHD. Nevertheless, to the best of our knowledge, this is the first study to evaluate the overall oxidative balance in pediatric ADHD patients. According to our findings, we suggest an association between oxidative stress and ADHD in children. In addition to conventional therapy, antioxidant treatment and/or foods that are high in antioxidant content are therefore appropriate in this group of patients.

Along with other etiological factors, increased oxidative stress and decreased antioxidants in ADHD patients may suggest an increased risk of the disorder. We were not able to describe the nature of this association due to the cross-sectional design of our study. Without longitudinal studies, it is difficult to interpret the trait effect of oxidative stress in pediatric ADHD. To prove this association, correlations between some psychiatric-biochemical markers and oxidative stress parameters should have been analyzed in the present study. Therefore, prospective multicenter studies are needed to understand more precisely the effect of oxidative status in patients with ADHD and its role in the pathophysiology of the disorder. In the light of these results, it has been suggested that the use of drugs containing antioxidants, together with conventional pharmacotherapy, may be beneficial for the prevention and treatment of ADHD.

Disclaimer statement

Contributors Authors H.S., H.K., and S.B.K. designed the study. H.K. and F.K. collected the samples. H.S., H.B., and E.S. conducted the biochemical analysis. N.A. and H.K. performed the statistical analysis. N.A. edited the writing of the manuscript. Finally, all the authors revised the paper and gave their consent for publication.

Funding None.

Conflict of interest None.

Ethics approval For our study, ethics approval was obtained from the Harran University Medical Faculty Ethics Committee.