Changes in oxidative stress and cellular immunity serum markers in attention-deficit/hyperactivity disorder
Mehmet Fatih Ceylan, MD, Department of Child and Adolescent Psychiatry, Dr Sami Ulus Children's Hospital, Ankara 90 38010, Turkey. Email: firstname.lastname@example.org
Aims: Attention-deficit/hyperactivity disorder (ADHD) is a developmental disorder with an etiopathogeny not fully understood. According to the prevailing view, the main factors contributing to the disorder are prefrontal dopamine deficiency and central dopaminergic dysfunction, but the factors/mechanisms involved in the brain dysfunction and its consequences are not well known. We suggest that changes in oxidative metabolism and cellular immunity may be involved. In this study, we aimed to investigate whether there are associations between ADHD and changes in serum levels of nitric oxide synthase (NOS), xanthine oxidase (XO), glutathione S-transferase (GST) and paraoxonase-1 (PON-1) activities, which are important markers of oxidative stress, and adenosine deaminase (ADA) activity, marker of cellular immunity.
Methods: The study sample consisted of 35 child or adolescent patients diagnosed with ADHD according to DSM-IV-TR criteria. Thirty-five healthy subjects were also included in the study as controls. Venous blood samples were collected, and NOS, XO, GST, PON-1 and ADA activities were measured.
Results: NOS, XO and ADA activities of the patients were significantly higher than those of the controls. GST and PON-1 activities of the patients were significantly lower than those of the controls.
Conclusions: Changes in oxidative metabolism and cellular immunity may have a role in the etiopathogenesis of ADHD.
ATTENTION-DEFICIT/HYPERACTIVITY DISORDER (ADHD) consists of a persistent pattern of inattention, hyperactivity and impulsivity.1 ADHD is relatively common, affecting an estimated 5–10% of school-aged children, depending on the definition and study.2 ADHD is usually identified in childhood and persists into adulthood in about 60% of individuals with childhood onset.3
The cause and pathophysiology of ADHD is incompletely understood. Research into the neurobiology and genetics of ADHD is robust and continues to break new ground regarding both methodological approaches and substantive findings.4 A number of reviews that have addressed the neurobiology of ADHD have focused on imaging and genetics. Relatively little attention has been given to factors/mechanisms involved in the brain dysfunction and its consequences,5 such as the oxidative metabolism and the immune system response, very important in cellular pathology. Free radicals, such as the superoxide anion and hydroxyl radicals, are reactive chemical species generated during the normal metabolic processes and, in excess, can damage lipids, proteins and DNA of neuronal and non-neuronal cells.6 Cellular immunity may contribute to the occurrence of the disorder by injuring neuronal cells, similar to oxidative metabolism.
Associations between brain cells and behavior may be direct; for example, brain cells may play a role in the cause of psychiatric disorder as serving in neurotransmitter synthesis. There are numerous studies indicating that oxidant-mediated neuronal damage plays a role in the pathophysiology of schizophrenia, autism, bipolar mood disorders and obsessive–compulsive disorder.7–10 A few studies have evaluated oxidative metabolism in ADHD,11–13 but to our knowledge, no study has evaluated nitric oxide synthase (NOS), xanthine oxidase (XO), glutathione S-transferases (GST), paraoxonase-1 (PON-1), and adenosine deaminase (ADA) in child and adolescence patients with ADHD.
Nitric oxide (NO) is known to be both a reactive oxygen species and a neurotransmitter in the central nervous system (CNS) and peripheral nervous system. The generation of NO following N-methyl-D-aspartate or norepinephrine receptor activation seems to be important in the context of CNS pathology.14 NO is produced from L-arginine by NOS. During the catalytic cycle of many enzymes, free radicals appear. One of the most important enzymatic sources of the superoxide anion radical is XO. This enzyme is located in all nucleated cells and catalyzes the conversion of hypoxanthine and xanthine to uric acid, the rate-limiting step in purine nucleotide catabolism.15 PON-1 has antioxidant properties. PON-1 circulates in the bloodstream associated with high-density lipoproteins (HDL). Paraoxonases are detoxifying enzymes involved in the metabolism of organophosphates. Their very important role consists of preventing the formation of lipoproteins.16 The GST is an important enzymatic system of the cellular mechanism of detoxification that protects cells against reactive oxygen metabolites due to the conjugation of glutathione with electrophilic compounds. GST enzymes are involved in the metabolism of xenobiotics that include environmental carcinogens and reactive oxygen species.17 The great majority of catalyzing reactions between glutathione and electrophilic compounds results in detoxification products, although in a few cases, the metabolic product can be more reactive than the original one. ADA has been accepted as an important enzyme in the maturation and function of T lymphocytes. Its main physiological activity is related to lymphocytic proliferation and differentiation. It is, therefore, known that ADA activity is higher in T cells than B-lymphocytes. As an indicator of cellular immunity, plasma activity of this enzyme has been suggested to be increased in inflammatory diseases, which causes a cell-mediated immune response.18
According to the prevailing view, the main factors contributing to the disorder are prefrontal dopamine deficiency and central dopaminergic dysfunction19 but the factors/mechanisms involved in the brain dysfunction and its consequences are not well known. We suggest that changes in oxidative metabolism and cellular immunity may be involved. In this study, we aimed to investigate whether there are associations between ADHD and changes in serum levels of NOS, XO, GST and PON-1 activities, which are important markers of oxidative stress, and ADA activity, marker of cellular immunity.
Thirty-five ADHD patients and 35 healthy volunteer controls from the Gazi University Faculty of Medicine, Child and Adolescent Psychiatry Clinic who participated in a previously conducted11 study were included. Age, sex and weight of the patients and controls showed homogeneity, and there were no significant differences between the groups (P > 0.05). The demographic data of the subjects are summarized in Table 1. Regarding ADHD subtype, 57% (n = 20) of the patients were diagnosed with ADHD-combined type, 40% (n = 14) were diagnosed with ADHD-inattentive type, and 3% (n = 1) of the patients were diagnosed with ADHD-hyperactive/impulsive type.
Table 1. Demographic data of the participants in two groups
|Sex (n)|| || || |
| Male||25||23||P = 0.607|
| Female||10||12||(X2 = 0.256)|
|Age: mean ± SD (year)||10.0 ± 2.4||10.2 ± 2.9||P = 0.757 (t = 0.311)|
|Age range (year)||7–15||7–15|| |
All subjects were screened for psychiatric disorders according to DSM-IV criteria and using the Schedule for Affective Disorders and Schizophrenia for School Aged Children, Present and Lifetime Version (K-SADS-PL),20 by a licensed child and adolescent psychiatrist. K-SADS-PL is a semi-structured interview form used for determining psychopathology in children and adolescents. A Turkish adaptation of this form was made by Gökler and colleagues.21 The Conners' Teacher Rating Scale was obtained from all patients.22 As the research focused on the disease entity, attention deficit, hyperactivity and impulsivity subscale scores were taken into consideration. A Turkish adaptation of this scale was made by Dereboy and colleagues.23 Participants underwent IQ evaluation using the Wechsler Intelligence Scale for Children-Revised (WISC-R).24 WISC-R was administered to the children by an experienced psychologist, and the children who had a full-scale IQ > 80 and fulfilled the inclusion criteria were enrolled.
The patients with comorbid psychiatric disorders and those who were mentally retarded were excluded. Case and control groups had similar distributions of age and sex. Case and control groups with a history of chronic systemic diseases, such as diabetes mellitus, epilepsy and severe head injury, were also excluded. Patients who had used psychotropic drugs in the previous 6 months were excluded.
After a complete description of the study to the subjects, written informed consent was obtained from all subjects. The ethics committee of the Gazi University Faculty of Medicine approved the trial. Also, a semi-structured form was used to detect several sociodemographic and clinical variables of the patients. The medical records of the patients were reviewed as well.
Venous blood samples from the left forearm vein were collected into 10-mL vacutainer tubes between 07.00 and 08.00 hours after overnight fasting. The blood samples were centrifuged at 3000 rpm for 10 min to obtain sera. Samples were stored frozen at −80°C before analysis. The biochemical analysis was performed after all of the blood samples were collected.
NOS activity was measured using the method of Durak et al.25 NOS activity was also measured at the same time, which is known to produce NO by catalyzing a five-electron oxidation of guanidino nitrogen of L-arginine. Measurement of the NO pool (mainly consisting of NO·+ NO2-) is based on the Griess reaction, in which to a greater extent, nitric oxide (NO·), and to a lesser extent nitrite anion (NO2-), undergo a diazotization reaction with sulfanilic acid. The absorbance of complex one formed with N-(1-napthyl-ethylene diamine) reflects the sum of NO· and NO2- levels in the reaction medium, which is termed the NO pool in this study. In this method, sodium nitroprusside is used as the chemical standard and the reaction scheme given for the NOS activity measurement, except for the incubation of the sample with arginine, is the same as NO pool determination. Results are expressed in mIU/L.
XO (EC 220.127.116.11) activity was measured spectrophotometrically by the formation of uric acid from xanthine through the increase in absorbance at 293 nm.26 One unit of activity was defined as 1 µmol uric acid formed per min at 37°C, pH 7.5. Results are expressed in mIU/L.
GST activity was measured using the method of Habig et al.27 1-chloro-2,4-dinitrobenzene (CDNB) is a synthetic GST substrate. Briefly, CDNB was added to buffer containing GSH and an aliquot of sample to be tested. Upon the addition of CDNB, the change in absorbance at 340 nm was measured as a function of time. Results are expressed in mIU/mL.
PON-1 activities were measured according to the method of Furlong et al. on 1-mL aliquots of fasting lithium-heparin-plasma that had been stored at −80°C.28 Purified paraoxon was obtained from ICN K&K and chlorpyrifos oxon from Dow Chemical Co. Paraoxonase activity was measured spectrophotometrically by monitoring the formation of p-nitrophenol at 405 nm. Results are expressed in mIU/mL.
Serum ADA activity was estimated spectrophotometrically by the method of Giuisti, which is based on the indirect measurements of the formation ammonia, produced when ADA acts in excess of adenosine.29 Results are expressed as IU/L and were calculated as mean ± standard deviation. Assays were conducted blind to clinical information. The biochemist was blinded to blood samples.
spss for Windows 11.5 (spss, Chicago, IL, USA) was used to analyze the data statistically. In the case of normally distributed and homogenous variables, the significant differences between groups were estimated using two-tailed t-tests. The Mann–Whitney U-test was used to investigate the non-parametric hypotheses when comparing two independent samples. Bivariate comparisons were examined via Spearman correlation coefficients; values were corrected for ties. In addition, χ2-tests were used to evaluate categorical data. Differences were accepted as significant when P < 0.05 and all analyses were two-tailed.
NOS, XO and ADA activities of the patients were significantly higher than those of the controls (Table 2). GST and PON-1 activities of the patients were significantly lower than those of the controls (Table 2).
Table 2. Serum measures of subjects
|NOS (mIU/L)||4.89 ± 1.80||3.27 ± 1.64||P < 0.001; t = 3.9|
|XO (mIU/L)||3.2 ± 1.3||1.8 ± 0.9||P < 0.001; t = 3.9|
|ADA (IU/L)||22.7 ± 6.1||18.0 ± 6.3||P = 0.002; t = 3.2|
|GST (mIU/mL)||2.76 ± 0.40||3.08 ± 0.36||P = 0.001; t = 3.5|
|PON-1 (mIU/mL)||11.0 ± 4.0||15.9 ± 3.2||P < 0.001; t = 5.6|
Sociodemographic features, such as sex, age, and weight, were not correlated with biochemical parameters (P > 0.05). There was no significant difference in biochemical parameters among the combined and predominantly inattentive subtypes of ADHD (P > 0.05).
It was found that increasing of the hyperactivity score in the Conners' Teacher Rating Scale was correlated with increases in NOS enzyme activity (r = 0.36; P < 0.05). Except for this correlation, scores were not correlated with any of the mentioned biochemical parameters (P > 0.05).
The first finding of this study is that NOS activities are higher in ADHD patients than in controls. Increases in NOS activity may cause an increase in the output of NO radicals. In recent studies, NO and MDA were found to be increased in adult-ADHD.12,13 In our other study, remarkably high levels of NO and MDA oxidants as well as low GSH-Px activities suggested an oxidative imbalance in pediatric patients with ADHD.11 The second finding is the higher XO activities in patients. XO acts on xanthine and hypoxanthine with the resultant production of oxygen free radicals. XO is an important source of free radical generation.30 Patients with panic disorder were found to have higher XO and ADA activities than control patients, and after 8 weeks of antidepressant treatment, XO and ADA activities decreased significantly.31
Oxidative stress represents a common etiopathological factor of diverse psychiatric conditions but cannot be used as a specific diagnostic requirement for any exclusive disorder. Both the disorder of the brain and the specific brain dysfunctions or lesions (which are the basis of the disease and central or systemic side-effects consecutive to them) may influence changes of the serum markers. But, the causal correlation between psychiatric disorders and increased oxidants is still unclear. It is not known whether the increased oxidants cause psychiatric disorders or psychiatric disorders lead to increases in oxidants.32 However, oxidative stress has been suggested to cause DNA33 and RNA34 damage in some psychiatric disorders. Bilici et al. suggested that free-radical-mediated neuronal damage has a role in the pathophysiology of depression.35 Additionally, the reduction in oxidant levels with treatment in some psychiatric disorders14,31 suggests that oxidants may contribute to the occurrence of psychiatric disorders.
The third finding of this study is that the enzymatic antioxidants, GST and PON-1, have lower activities in the ADHD patients than in the controls. When oxidants are produced in excessive amounts or antioxidant defense systems are inefficient, chain reactions can cause cellular injury. For instance, the oxidants are related with the membrane-associated pathologies in the central nervous system and may have an important role in neuropsychiatric disorders.12,32 Membrane-associated pathologies can affect the neurotransmitter functioning.36,37 Additionally, dopamine is very susceptible to auto-oxidation when antioxidant defense is weak.13 This condition may contribute to the occurrence of ADHD.
The fourth result of our study is the increased ADA in patients with ADHD. ADA plays a crucial role in lymphocyte proliferation and differentiation and shows its highest activity in T-lymphocytes. Because a correlation exists between adenosine deaminase and cell-mediated immunity, we suggest that cell-mediated immunity may play an important role in the immunopathogenesis of ADHD. Waldrep reported two children with explosive onsets of ADHD following streptococcal infections.38 Streptococcal infections during childhood are very common, and the diseases attributed to streptococs are usually self-limited. However, a small part of them result in neurologic and/or psychiatric symptoms after infection.39 There is epidemiologic evidence that some pediatric-onset neuropsychiatric disorders, including obsessive–compulsive disorder, tic disorders, ADHD, and major depressive disorder, may be temporally related to prior streptococcal infections. Whether this correlation is the result of a non-specific stress response or secondary to an activation of the immune system remains to be determined.40 Additionally, ADA is an important enzyme involved in purine and DNA metabolism. XO catalyzes the conversion reaction of hypoxanthine and xanthine to uric acid, which is the last reaction in purine metabolism.41 Thus, purine and DNA metabolism of the patients seems to be altered.
In brain disorders, problems with inhibitory brain processes that are important in task mastery are common and may be linked to inattention and hyperactivity.42 Many studies support the hypothesis that dysfunctions in prefrontal cortex, basal ganglia, and the related neurotransmitter systems underlie inhibitory deficits in ADHD.43 We suggest that oxidative metabolism and cellular immunity may contribute to the formation of ADHD by affecting neuronal structure, which results in corruption of dopamine synthesis and neurotransmission.
ADHD and autism are both highly heritable neurodevelopmental disorders. Evidence indicates that ADHD and autism co-occur with a high frequency.44 There is mounting evidence of immune dysregulation in autistic individuals, and recent research has revealed the link between the immune system and nervous system.45 Additionally, several studies have shown increased levels of oxidants in individuals with autism.8,46 Like autism, immune dysregulation and free radical-mediated neuronal damage may play an important role in the immunopathogenesis of ADHD.
Fifth, we found that there is not a statistically significant difference between the combined type and inattentive type in activities of biochemical parameters. This result coordinates with the DSM classification for integrity of the disease.
Studied serum markers in peripheral blood may have therapeutic and/or diagnosis value. In particular, we found that increases in the hyperactivity point in the Conners' Teacher Rating Scale were correlated with increases in NOS enzyme activity. Increases in the NOS enzyme activity can result in the overproduction of NO radicals. Thus, increasing the oxidant levels might affect the disease in a negative way. Dopamine-mediated cytotoxicity is also mediated through N-methyl-D-aspartate receptors, in which some oxidants, such as NO, are involved.47 Therefore, high NO may have progressively damaged the vulnerable pathways of attention and physical activity.13 An Italian study found improvement in a rat model of hyperactivity and attention deficit with NOS inhibitor treatment.48 Further medication designs should focus on approved high NOS enzyme activities in patients and efficient NOS inhibitor treatment.
Multiple early genetic and environmental influences of small effect act together to create a spectrum of neurobiological risk by altering brain structure and function that mediate the emergence of ADHD. The evidence is strongest for maternal smoking, for which a dose–response correlation with ADHD appears to exist.49 Nicotine's action on the production and function of neurotransmitters makes it a prime suspect in the pathology of ADHD.50 Smoking is associated with both an increase in oxidative stress and inflammation.51 In light of these findings, it may be hypothesized that inflammation and oxidative stress play an important role in the cause of ADHD, and our findings support this hypothesis. This mechanism may be an essential factor that elucidates the pathogenesis of ADHD due to maternal smoking.
In most epidemiological studies, the combined and inattentive subtypes of ADHD are found to be the prevalent subtypes and the hyperactive/impulsive subtype is found to be the least frequent subtype.52 Our ADHD group was consistent with the literature in terms of the distribution of subtypes. Furthermore, exclusion of comorbid psychiatric disorders and mental retardation contributes to the reliability of the present study.
Changes in oxidative metabolism and cellular immunity may have a role in the etiopathogenesis of ADHD. Additionally, these changes in peripheral blood may have therapeutic and/or diagnosis value. Our findings may pioneer further clinical enzymology and biochemical studies on ADHD.
This study was supported by a grant from the Gazi University Research Foundation.