hyperactivity disorder combined presentation with and without comorbid conduct disorder

Author’s Accepted Manuscript Serum levels of cortisol, dehydroepiandrosterone, and oxytocin in children with attentiondeficit/hyperactivity disorder combined presentation with and without comorbid conduct disorder Ümit Işık, Ayhan Bilgiç, Aysun Toker, Ibrahim Kılınç www.elsevier.com/locate/psychres

PII: DOI: Reference:

S0165-1781(17)31063-6 https://doi.org/10.1016/j.psychres.2017.12.076 PSY11112

To appear in: Psychiatry Research Received date: 11 June 2017 Revised date: 10 December 2017 Accepted date: 31 December 2017 Cite this article as: Ümit Işık, Ayhan Bilgiç, Aysun Toker and Ibrahim Kılınç, Serum levels of cortisol, dehydroepiandrosterone, and oxytocin in children with attention-deficit/hyperactivity disorder combined presentation with and without comorbid conduct disorder, Psychiatry Research, https://doi.org/10.1016/j.psychres.2017.12.076 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Serum levels of cortisol, dehydroepiandrosterone, and oxytocin in children with attentiondeficit/hyperactivity disorder combined presentation with and without comorbid conduct disorder Ümit Işıka, Ayhan Bilgiçb, Aysun Tokerc, İbrahim Kılınçd a

Department of Child and Adolescent Psychiatry, Yozgat City Hospital, Yozgat, Turkey,

b

Department of Child and Adolescent Psychiatry, Meram School of Medicine, Necmettin

Erbakan University, Konya, Turkey, c

Private physician,

d

Department of Biochemistry, Meram School of Medicine, Necmettin Erbakan University,

Konya, Turkey

Short title: Neuroendocrine hormones in ADHD and conduct disorder Corresponding Author: Ümit Işık, M.D., Department of Child and Adolescent Psychiatry, Yozgat City Hospital, Yozgat, Turkey. E-mail: [email protected] Tel: +90 354 219 00 10

Abstract The present study aimed to investigate serum cortisol, dehydroepiandrosterone (DHEA), and oxytocin levels of children with attention-deficit/hyperactivity disorder (ADHD) combined presentation and those diagnosed with ADHD combined presentation and coexisting conduct disorder. A total of 74 drug-naive children with ADHD combined presentation alone, 32 children with ADHD combined presentation + conduct disorder, and 42 healthy controls were included. The severities of ADHD and conduct disorder symptoms were assessed via parentand teacher-rated questionnaires. The severity of aggression, anxiety, and depression symptoms of the children were assessed by the self-report inventories. Independent of potential confounders, including age, sex, pubertal stage, and severity of depression and anxiety, serum oxytocin levels of the ADHD combined presentation + conduct disorder group

2

were significantly lower than those of both the ADHD combined presentation alone and control groups. There was also a trend for the ADHD combined presentation + conduct disorder group to show lower serum DHEA levels than that of the ADHD combined presentation alone group. However, serum cortisol levels did not show significant alterations among the groups. These findings suggest that oxytocin and DHEA may play a role in the pathophysiology of conduct disorder, at least in the presence of ADHD combined presentation. Keywords:

attention-deficit/hyperactivity

disorder,

conduct

disorder,

cortisol,

dehydroepiandrosterone, oxytocin

1. Introduction Attention-deficit/hyperactivity

disorder

(ADHD)

is

a

frequent

childhood-onset

neurodevelopmental condition defined by persistent symptoms of inattention, hyperactivity, and/or impulsivity that cause impairments in two or more settings. Although the complex interactions of neuroanatomical and neurochemical systems are suspected in the etiology of ADHD, the exact neurobiological mechanisms underlying the disorder are currently unknown (Spencer et al., 2007). In recent years, researchers have raised concerns regarding the potential roles of neuroendocrine hormones in the etiopathogenesis of ADHD (Wang and Chen, 2013). Studies concerning the relationship between ADHD and these hormones have focused mostly on cortisol, dehydroepiandrosterone (DHEA), and oxytocin (Wang et al., 2011; Isaksson et al., 2012; Scassellati et al., 2012; Wang and Chen, 2013; Faraone et al., 2014; Taurines et al., 2014; Sasaki et al., 2015). However, to date, no sufficient data has confirmed a relationship between ADHD and these hormones. Conduct disorder is another frequent childhood psychiatric condition characterized by a repetitive and persistent pattern of behavior that violates major rules, societal norms, and laws. The most important risk factors for the development of conduct disorder include the presence of ADHD, child abuse, poor parental supervision, low school achievement, low intelligence quotient (IQ), and living in high-crime neighborhoods (Latimer et al., 2012). However, convincing evidence of causal linkages has remained elusive. Similar to ADHD, recent studies have suggested that neuroendocrine hormones might be related to the

3

etiopathogenesis of conduct disorder (Alink et al., 2008; Dorn et al., 2009; Freitag et al., 2009; Golubchik et al., 2009; Dadds et al., 2014; Levy et al., 2015; Northover et al., 2016). Unfortunately, the findings of these studies are conflicting and more effort is needed to achieve conclusive data. Cortisol plays an important role in regulating central nervous system neurotransmitters and behaviors, such as attention, learning, memory, and emotion, which are all closely related with ADHD (Talge et al., 2007). Thus, a number of studies have investigated the potential link between cortisol and ADHD. Several authors have reported reduced cortisol levels in ADHD patients and suggested different explanations for their findings (Blomqvist et al., 2007; Ma et al., 2011; Isaksson et al., 2012, 2015; Scassellati et al., 2012; Palma et al., 2015). Accordingly, reduced levels can stem from a displaced diurnal cortisol curve in ADHD, or they can reflect a lower reactivity of the hypothalamic-pituitary-adrenal (HPA) axis in the disorder, or they can simply be the result of genetic influences. However, findings are not consistent across studies and other authors have failed to demonstrate significant differences in cortisol levels between ADHD and controls (Sondeijker et al., 2007; Pesonen et al., 2011; Wang et al., 2011; Kuppili et al., 2017). Despite its potential importance, it remains unclear whether the changes in circulating cortisol levels are related to ADHD or coexisting behavioral problems. Only a minority of studies on cortisol levels in children with ADHD have controlled comorbid behavioral problems, and these have yielded controversial results (Blomqvist et al., 2007). Some studies have found lower cortisol levels in children with ADHD and comorbid oppositional defiant disorder/conduct disorder, but not in children with ADHD alone (Cakaloz et al., 2005; Freitag et al., 2009). However, another study detected no effect of coexisting oppositional defiant disorder/conduct disorder on cortisol levels in an ADHD sample (Isaksson et al., 2012). Similarly, a recent study reported no differences between patients with ADHD alone and patients with ADHD coexisting with conduct disorder with respect to baseline cortisol levels in male adolescents (Northover et al., 2016). Irrespective of the presence of ADHD, there are studies that have focused on the link between cortisol and conduct disorder. These studies have generally reported reduced cortisol levels in individuals with conduct disorder, and a recent meta-analysis of 82 studies with child and adolescent samples found a significant but small (d = 0.10) relationship between low cortisol levels and antisocial behavior (Alink et al., 2008).

4

DHEA is a major circulating neurosteroid in humans that plays several vital neurophysiological roles (Golubchik et al., 2007). Its functions include guiding cortical projections to appropriate targets; hence, it is crucial for the regulation of neurodevelopment (Wang et al., 2011). Wang et al. (2011) compared DHEA levels in subjects with ADHD and healthy controls and showed that DHEA levels were significantly lower in the ADHD subjects. In another study, Strous et al. (2001) examined the relationship between clinical symptomatology and DHEA levels in male subjects with ADHD combined subtype and found a correlation between higher blood levels of DHEA and fewer ADHD symptoms. So far, no study on DHEA levels in children with ADHD have controlled for the presence of comorbid conduct disorder. To our knowledge, only three previous studies have evaluated the association between conduct disorder and DHEA, all of which failed to find a relationship (Pajer et al., 2006; Dorn et al., 2009; Golubchik et al., 2009). Pajer et al. (2006) examined DHEA levels in girls 15–17 years of age with conduct disorder and failed to demonstrate any relationship between DHEA levels and conduct disorder. Golubchik et al. (2009) examined neurosteroid blood levels in adolescents with conduct disorder as compared to normal controls and showed that DHEA levels did not differ between the two groups. Another group assessed DHEA levels in children and adolescents with oppositional defiant disorder/conduct disorder and normal controls, and this group also did not find a difference between groups (Dorn et al., 2009). Oxytocin is classically known for its role as a hormone involved in parturition and lactation. However, recent studies have shown that it also has crucial effects on attachment, trust, stress management, social cognition, mood and memory (Ishak et al., 2011; Martinetz and Neumann, 2015; Sasaki et al., 2016). To date, few studies have investigated the role of oxytocin in ADHD. Taurines et al. (2014) compared plasma oxytocin levels in subjects with ADHD, autism spectrum disorder (ASD), and normally developing controls. They demonstrated that plasma oxytocin concentrations were significantly lower in ADHD subjects than in healthy controls and those with ASD. Later reports also detected low oxytocin levels in the ADHD group compared with controls and suggested that oxytocin may play a role in the pathogenesis of ADHD (Sasaki et al., 2015; Demirci et al., 2016). Only a few studies have investigated the putative role of oxytocin in conduct disorder, and these have proposed that oxytocinergic transmission may be involved in conduct disorder and psychopathy. Levy et al. (2015) assessed the relationship between salivary oxytocin levels and conduct disorder and found an inverse correlation between salivary oxytocin levels

5

and the severity of conduct problems. Another study by Fetissov et al. (2006) evaluated oxytocin-reactive autoantibody in children with conduct disorder and reported higher autoantibody levels in the conduct disorder group compared to the non-conduct disorder group. Moreover, Dadds et al. (2014) demonstrated that serum oxytocin levels in youth were correlated with callous-unemotional trait severity. Two different genetic studies have also evaluated the involvement of oxytocin in conduct problems (Beitchman et al., 2012; Sakai et al., 2012). Beitchman et al. (2012) detected a link among certain oxytocin receptor genotypes and higher callous-unemotional scores in children. However, another study failed to show a link between oxytocin receptor genotypes and conduct disorder (Sakai et al., 2012). There are no studies that have compared ADHD and conduct disorder in any of the abovementioned studies related to oxytocin. Furthermore, to date, no studies have compared children with ADHD alone and children with ADHD and coexisting behavioral disorders with respect to circulating oxytocin levels. Neurohormones can easily cross the blood-brain barrier and research has demonstrated that peripheral neuroactive steroids, such as cortisol and DHEA, and peripheral oxytocin concentrations, can be used as a valid proxy for steroids and oxytocin concentrations in the cerebrospinal fluid (Ross and Young, 2009; Kancheva et al., 2010; Carson et al., 2015). According to the previously cited research, few studies have investigated the links among ADHD and conduct disorder with circulating levels of cortisol, DHEA, and oxytocin. Moreover, most of these studies have not considered any potential confounders, such as coexisting psychiatric disorders, pubertal status, age, and sex. The present study aimed to compare serum cortisol, DHEA, and oxytocin levels of children with ADHD combined presentation alone, those with ADHD combined presentation and coexisting conduct disorder (ADHD + conduct disorder), and healthy controls. This study also examined the relationships between the clinical symptom severity of ADHD and conduct disorder and neurohormones levels. We hypothesized that children with ADHD combined presentation alone would show reduced cortisol, DHEA, and oxytocin levels relative to controls, and children with ADHD combined presentation + conduct disorder would show reduced cortisol, DHEA, and oxytocin levels relative to both those with ADHD combined presentation alone and control subjects. Furthermore, we hypothesized an inverse relationship between serum neurohormone levels and the severity of ADHD, conduct disorder, and aggression.

2. Experimental procedures 2.1. Participants

6

Participants were recruited from the Outpatient Clinic for Child and Adolescent Psychiatry at the Meram Faculty of Medicine, Necmettin Erbakan University (NEU). The study included never-treated children and adolescents aged 8 to 16 years who were diagnosed with ADHD combined presentation and those diagnosed with ADHD combined presentation and coexisting conduct disorder based on the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria. To identify homogeneous groups of patients with ADHD, only children with ADHD combined presentation were included in the study. Exclusion criteria were the presence of major physical, neurological, or endocrinological diseases, ASD, bipolar disorder, psychotic disorder, substance use disorder, head trauma, and intellectual disability. Participants who had a history of using a psychotropic drug, corticosteroid medication, or any hormonal treatment before were also excluded from the investigation. The control group consisted of healthy volunteer children. The same inclusion and exclusion criteria applied to the control group. The majority of participants were included in both the present study and our recently published study that was related to the evaluation of serum neurotrophin levels in ADHD (Bilgiç et al., 2016). However, unlike the previous study, children over 16 years of age were not enrolled in the present study because the Wechsler Intelligence Scale for Children-Revised (WISC-R) cannot be used for children older than 16. Additionally, some children, or their parents, wanted to participate in only one study. This study was approved by the NEU Ethical Committee and all the procedures were in accordance with the Declaration of Helsinki and local laws and regulations. The parents gave their written informed consent after investigators explained the purpose and course of the study. Oral assent was also obtained from all subjects. In the patient groups, a total of 133 children were approached, however, 4 of them refused to participate and 23 were excluded based on the exclusion criteria. In the control group, 52 children were approached and 10 were excluded. The distribution of coexisting psychiatric disorders in the ADHD combined presentation and ADHD combined presentation + conduct disorder groups, according to KSADS-PL, are given in Table 1. None of the patients was treated with a psychotropic drug for their comorbid psychiatric conditions. 2.2. Diagnostic and symptom assessment The Schedule for Affective Disorders and Schizophrenia for School-Aged Children, Present and Lifetime Version (K-SADS-PL) was applied for patient and control subjects by a child and adolescent psychiatrist (ÜI) (Gökler et al., 2004). The diagnoses of ADHD combined presentation and conduct disorder were made according to the DSM-5 criteria. Because the

7

K-SADS-PL has not been adapted for the DSM-5, changes in the diagnostic criteria for ADHD in the DSM-5 (e.g., age of onset criteria, multiple settings requirement) were applied to the K-SADS-PL by the researchers. To determine the children’s depression and anxiety levels, participants completed the Children’s Depression Inventory (CDI) and the Screen for Child Anxiety-Related Emotional Disorders (SCARED), respectively (Öy, 1991; Karaceylan, 2005). Children were also administered the Reactive-Proactive Aggression Questionnaire (RPAQ) for measuring the severity of aggression (Uz Bas and Yurdabakan, 2012). Both parents and teachers of the participants filled out psychological questionnaires for the assessment of ADHD and levels of disruptive behavior symptoms of the children. Parents completed the Turgay DSM-IV-Based Child and Adolescent Behavioral Disorders Screening and Rating Scale (T-DSM-IV-S) (Turgay, 1994; Ercan et al., 2001) and the Conners’ Parent Rating Scale-Revised Short (CPRS-RS) (Kaner et al., 2013a), and teachers completed the T-DSM-IV-S and the Conners’ Teacher Rating Scale-Revised Short (CTRS-RS) (Kaner et al., 2013b). For each scale, higher scores indicate higher levels of symptoms. The Wechsler Intelligence Scale for ChildrenRevised (WISC-R) (Savaşır and Şahin, 1995) was applied to the participants by certified psychologists to determine IQ scores. The reliability and validity of all questionnaires have been previously established for the Turkish population. The pubertal stage and body mass index (BMI) percentile were also assessed. The pubertal development stage was assessed during the mother or main caregiver interview using schematic drawings of secondary sex characteristics related to the five standard Tanner stages of pubertal development. The five pubertal stages can be discriminated from 1 (infantile) to 5 (complete puberty). These ratings have been widely used and have demonstrated good reliability and validity (Morris and Udry, 1980). Height and weight were measured in the outpatient unit on the day the participant gave a blood sample. Height was measured in meters and weight in kilograms. 2.3. Blood samples Peripheral venous blood samples for cortisol, DHEA, and oxytocin were drawn from an antecubital vein between 8:00 and 10:00 a.m. after a 12-hour overnight fast. In this way, possible changes induced by circadian variations or by previous meals were minimized. All participants were instructed to avoid unusual physical activity for 24 hours prior to the blood collection. Participants were also asked to not perform any heavy exercise, smoke, or drink anything prior to the blood sampling. Blood samples from the controls were acquired in a

8

similar manner at the same time of day. Blood samples of patients and controls were extracted following centrifugation. The tubes were centrifuged at 4000 rpm for 5 minutes at 4 °C and the serum was stored at –80 °C until analysis. Serum levels of DHEA, cortisol, and oxytocin were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits following the manufacturers’ protocols. ELISA kits were used for the serum analyses of DHEA, cortisol (DRG Diagnostic: DHEA [serum]: EIA-3415; cortisol [serum]: EIA - 1887), and oxytocin (CUSABIO: oxytocin [serum]: E08994h). Patient and control samples were run together in the same plates. 2.4. Statistical analysis Statistical analyses were performed with SPSS 21.0 statistical software (SPSS Inc., Chicago, IL, USA). All variables were inspected for normality using the Kolmogorov-Smirnov test of normality. Chi-square or one-way analysis of variance (ANOVA) tests were used to assess group differences in variables, as appropriate. Variables are presented as either a number (n), percentage (%), mean ± standard deviation (SD), or frequency. Distributions of hormones and log-transformed hormones were examined by histogram plots and descriptive statistics. Any variables not displaying a normal distribution were log-transformed, including serum DHEA and oxytocin levels. Because correlations among circulating neurohormone levels have been reported in previous studies (Cardoso et al., 2013), multivariate analysis of covariance (MANCOVA) was performed for possible confounding variables. These analyses were performed with adjustments for age, sex, pubertal stage, and the severity of anxiety and depression, which were defined as those variables associated with a p value < 0.1 or of theoretical relevance. In these analyses, cortisol, DHEA, and oxytocin were defined as dependent variables and combined as one factor. Because DHEA and oxytocin values were positively skewed, they were transformed to the natural logarithm scale to render the distributions more symmetrical and normally distributed as tested with Kolmogorov-Smirnov analyses. Cortisol values were normally distributed. Since groups differed significantly in these variables, we then performed three separate one-way analysis of covariance (ANCOVA) for all three markers to detect domains with significant between-group differences controlling for the same confounders. Effect sizes are reported as partial eta-squared (η2; small ≥ .01, medium ≥ .06, large ≥ .14) for ANCOVA for all other group comparisons. Correlational analyses were used to determine associations between the clinical and biological variables. Non-parametric or parametric correlation tests were performed using Spearman’s rho or Pearson’s r. A value of p < 0.05

9

(two-tailed) was considered to indicate significance. Where indicated, Bonferroni tests were conducted for post hoc tests with corrections for multiple testing.

3. Results The final study population consisted of 74 (57 boys and 17 girls) children with ADHD combined presentation alone, 32 (27 boys and 5 girls) children with ADHD combined presentation + conduct disorder, and 42 (29 boys and 13 girls) children who were healthy controls. Table 2 presents and compares the descriptive data and clinical variables of the study groups. As shown in Table 2, there was no significant between-group differences for age, sex, BMI percentile, and Tanner stage. Verbal performance and total intelligence scores, based on the WISC-R, were higher in the healthy control group than in the ADHD combined presentation alone and ADHD combined presentation + conduct disorder groups. No significant differences were found between the ADHD combined presentation alone group and ADHD combined presentation + conduct disorder group with respect to intelligence scores. Table 2 also presents the scores on the CTRS-RS, CPRS-RS, T-DSM-IV-S, RPAQ, CDI, and SCARED. Since depression and anxiety symptoms are a part of ADHD, a one-way MANCOVA was conducted with three diagnostic groups as the independent variable, while only age, sex, and pubertal stage were used as covariates. The MANCOVA showed significant overall group differences among study groups on serum neurohormone levels [V (Pillai’s trace) = 0.100, F(6, 282) = 2.470, p = 0.024, hp2 = 0.050]. Separate univariate ANCOVAs on the outcome variables revealed significant differences among the study groups with respect to serum levels of oxytocin [F(2, 142) = 5.573, p = 0.005, hp2 = 0.073] and DHEA [F(2, 142) = 3.085, p = 0.049, hp2 = 0.042]. However, only a trend for a significant difference was found among the study groups in terms of serum cortisol levels [F(2, 142) = 2.497, p = 0.086, hp2 = 0.034]. Post hoc univariate analyses indicated serum oxytocin levels were significantly higher in the ADHD combined presentation and control groups than the ADHD combined presentation + conduct disorder group; and serum DHEA levels were significantly higher in the ADHD combined presentation group than the ADHD combined presentation + conduct disorder group (Table 3). One-way MANCOVA tests were then conducted with the diagnostic groups as the independent variable, while, as well as age, sex, and pubertal stage, the severity of depression and anxiety were also used as covariates. The analyses showed significant overall group

10

differences among the study groups for serum neurohormone levels [V (Pillai’s trace) = 0.093, F(6, 278) = 2.250, p = 0.039, hp2 = 0.046]. Similar to the first analysis, separate univariate ANCOVAs on the outcome variables revealed significant differences among the study groups with respect to serum levels of oxytocin [F(2, 140) = 5.053, p = 0.008, hp2 = 0.067]. However, we detected only a trend for a significant difference among the study groups for serum DHEA levels [F(2, 140) = 2.712, p = 0.070, hp2 = 0.037], and no significant difference was detected among the study groups for serum cortisol levels [F(2, 140) = 1.964, p = 0.144, hp2 = 0.027]. Post hoc univariate analyses indicated serum oxytocin levels were significantly higher in the ADHD combined presentation and control groups than the ADHD combined presentation + conduct disorder group; and serum DHEA levels were significantly higher in the ADHD combined presentation group than the ADHD combined presentation + conduct disorder group. Post hoc analyses and pairwise comparisons are reported in Table 3 and Figure 1. Correlations among serum neurohormone levels were assessed in the entire patient sample (ADHD combined presentation alone group and the ADHD combined presentation + conduct disorder group). Cortisol levels were positively correlated with DHEA and oxytocin levels (rs = 0.335, p < 0.001; rs = 0.499, p < 0.001, respectively). However, no correlation was found between DHEA and oxytocin levels (rs = 0.145, p = 0.134). The correlations between the CTRS-RS and CPRS-RS subscale scores and parent and teacher rated T-DSM-IV-S

subscale scores were also evaluated. All subscales of parent- and teacher-rated subscale scores showed significant correlations with each other and correlation coefficients were among 0.220 to 0.437. The correlations between serum DHEA, oxytocin, and cortisol levels and the parentand teacher-rated T-DSM-IV-S, CPRS-RS, CTRS-RS, RPAQ, CDI, and SCARED scores were also evaluated for the entire patient sample. There were negative correlations between the serum DHEA levels and the teacher-rated T-DSM-IV-S attention-deficit and hyperactivity-impulsivity scores (r = -0.185, p = 0.038; r = -0.202, p = 0.023, respectively), the parent-rated T-DSM-IV-S hyperactivity-impulsivity scores (r = -0.180, p = 0.028), and the CPRS-RS hyperactivity scores (r = -0.201, p = 0.015). A negative correlation between the serum cortisol levels and the teacher-rated T-DSM-IV-S oppositional defiant behavior scores was also detected (r = -0.183, p = 0.042) (Figure 2). No other associations were found for serum neurohormone levels and questionnaire scores.

4. Discussion

11

This study investigated serum DHEA, oxytocin, and cortisol levels in children with ADHD combined presentation alone, children with ADHD combined presentation + conduct disorder, and healthy controls to elucidate possible roles of these neuroendocrine hormones in the neurobiological groundwork of these disorders. The analyses demonstrated that the serum oxytocin levels of the ADHD combined presentation + conduct disorder group were significantly lower than those of the ADHD combined presentation alone and control groups and the serum DHEA levels of the ADHD combined presentation + conduct disorder group were significantly lower than those of the ADHD combined presentation alone group independent of potential confounders, including age, sex, and pubertal stage. Afterwards, as well as age, sex, and pubertal stage, the effects of the severity of depressive and anxiety symptoms were also controlled and the analyses showed the same results for the relationship between serum oxytocin levels and the study groups. However, there was only a trend for a lower serum DHEA level in the ADHD combined presentation + conduct disorder group than the ADHD combined presentation alone group. No significant differences were found between the ADHD combined presentation alone group and controls with respect to serum DHEA and oxytocin levels, and serum cortisol levels did not show significant alterations among the groups in any of the analyses. The present study found no significant difference between the children with ADHD combined presentation alone and controls for serum oxytocin levels. In recent years, several teams have investigated the potential role of oxytocin in ADHD, and all of these studies reported lower circulating oxytocin levels in subjects with ADHD (Taurines et al., 2014; Sasaki et al., 2015; Demirci et al., 2016). Our findings are not consistent with these studies and failed to support the role of oxytocin in the etiology of ADHD. Except for the study by Demirci et al. (2016) studies have not considered the presence of coexisting conduct disorder, and none of the studies controlled the effects of potential confounders. Moreover, while the samples of these studies included all subtypes of ADHD subjects, we only recruited children with ADHD combined subtype. Therefore, the inconsistencies among findings might be related to these methodological differences. Our findings showed that children with ADHD combined presentation + conduct disorder have lower serum oxytocin levels than both children with ADHD combined presentation alone and the controls. To our knowledge, no previous study has investigated the circulating oxytocin levels in patients with ADHD + conduct disorder. However, two different correlational studies found an inverse correlation between salivary and plasma oxytocin levels

12

and conduct problems and callous unemotional traits, respectively (Dadds et al., 2014; Levy et al., 2015). Our results suggest a link between lower circulating oxytocin levels and conduct disorder. The underlying mechanisms of how oxytocin can be related to conduct disorder in the presence of ADHD have not been elucidated. However, it is well known that some unfavorable psychiatric conditions, such as impaired attachment and poor empathy, are related to both lower circulating oxytocin levels and conduct disorder (Buchheim et al., 2009; Feeser et al., 2015). Therefore, we suggest that lower oxytocin levels simplify the development of conduct disorder by producing impairing effects on these variables. Our study found no significant difference between the children with ADHD combined presentation alone and controls for serum DHEA levels. However, we also investigated the correlation between the severity of ADHD symptoms and serum DHEA levels and detected negative associations between increased attention deficit and hyperactivity exhibited by all ADHD patients with lower serum DHEA levels. To our knowledge, only one case-control study has investigated the role of DHEA in ADHD and found lower saliva DHEA levels in the ADHD group than in the healthy controls (Wang et al., 2011). These authors proposed that DHEA might exert its positive effects in ADHD patients through stimulatory or antagonist effects at gamma-aminobutyric acid (GABA)-A receptors and facilitation of Nmethyl-D-aspartate (NMDA) activity. In line with our results, an inverse relationship between DHEA blood levels and the severity of hyperactivity/impulsivity symptoms has also been reported (Strous et al., 2001). When controlling all confounders, we found a trend for lower serum DHEA levels in children with ADHD combined presentation + conduct disorder compared to those with ADHD combined presentation alone. So far, few studies have investigated the involvement of DHEA in conduct disorder and these could not find an association between DHEA and the disorder (Pajer et al., 2006; Dorn et al., 2009; Golubchik et al., 2009). However, to our knowledge, no previous studies have specifically scrutinized the circulating DHEA levels in patients with ADHD + conduct disorder. It is well known that patients with ADHD + conduct disorder have, at least partially, distinct neurobiological and genetic characteristics that are seen in both patients with ADHD and conduct disorder alone (Caspi et al., 2008). For instance, a genetic variant of catechol-0-methyl transferase only shows an association with ADHD + conduct disorder, but not with either ADHD or conduct disorder alone (Caspi et al., 2008). Therefore, we suggest that lower circulating DHEA levels could reflect another unique biological characteristic of patients with ADHD + conduct disorder. DHEA is an anabolic

13

hormone and has been shown to have neuroprotective, antioxidative, anti-inflammatory, and antiglucocorticoid effects, and is suggested to play a significant role in the protection against the negative consequences of stress (Maninger et al., 2009). It is well known that exposure to, for example, perinatal or psychosocial stresses, is related to the emergence of conduct disorder (Latimer et al., 2012). Therefore, it may be proposed that low DHEA levels may not protect against the negative consequences of stress and may trigger the development of conduct disorder in children with ADHD. Our study showed no significant differences in serum cortisol levels between patients with ADHD combined presentation alone and the controls. Though some studies have failed to demonstrate any relationship between cortisol levels and ADHD (Sondeijker et al., 2007; Pesonen et al., 2011; Wang et al., 2011; Kuppili et al., 2017), which are in line with our findings, others have reported low cortisol levels in children with ADHD (Blomqvist et al., 2007; Ma et al., 2011; Isaksson et al., 2012, 2015; Scassellati et al., 2012; Palma et al., 2015). For instance, Wang et al. (2011) examined salivary cortisol levels in 50 ADHD patients and 50 age- and sex-matched controls and found no significant differences in salivary cortisol levels. Similarly, in a small-sample study,

Cakaloz et al. (2005) found no significant

difference between the ADHD and control groups for plasma cortisol levels. The present study also found no significant alteration in serum cortisol levels in children with ADHD combined presentation + conduct disorder compared to the other two groups. However, we found a negative correlation between the serum cortisol levels and oppositional defiant behavior scores. A majority of studies have reported a significant relationship between low cortisol levels and antisocial behavior (Alink et al., 2008). However, similar to ADHD, data regarding a link between circulating cortisol levels and conduct disorder is not unique. Some studies have found no significant differences in cortisol levels between patients with conduct disorder and healthy controls (Van Goozen et al., 2000; Azar et al., 2004), whereas one study has reported increased cortisol levels in these patients (Van Bokhoven et al., 2005). Cortisol secretion has a diurnal variation and is characterized by high levels during awakening, a further increase during the morning, and a gradual decrease throughout the day until midnight (Tsigos and Chrousos, 2002). Therefore, the sampling time may be responsible for the different results among studies.

Furthermore, oxytocin

administration reduces cortisol levels and baseline oxytocin levels are inversely associated with circulating cortisol ( Linnen et al., 2012; Cardoso et al., 2013). However, previous

14

studies did not take into account the role of other substances that modulate cortisol levels, such as oxytocin (Maldonado et al., 2009; Ma et al., 2011; Wang et al., 2011). There are some limitations in our study. First, there were only three groups, an ADHD combined presentation group, an ADHD combined presentation + conduct disorder group, and healthy controls. However, there was not a conduct disorder alone group, and the recruitment of a conduct disorder alone group might be helpful to disentangle what is related specifically to ADHD, specifically to conduct disorder, or to their combination. Second, although we took into account the confounding effect of the coexisting depression and anxiety symptoms, our findings might be affected by comorbid psychiatric disorders. Third, though we do not know the most reliable and accurate method for measuring neuroendocrine hormones, we measured only the serum levels of these substances from blood samples of children and adolescents. Fourth, the time window between 8:00 and 10:00 a.m. may be large from the viewpoint of the cortisol circadian rhythm, and the measurement of neuroendocrine hormones at a single time point might not be adequate. Fifth, preterm birth and low birth weight were not exclusion criteria. Sixth, when evaluating the correlations between serum neurohormone levels and psychiatric symptoms, our data based on self-report, parent-report or teacher-report questionnaires; therefore, it might cause inconsistencies among findings. Finally, this study was limited by the cross-sectional nature of the report. In conclusion, we found a decrease in serum oxytocin levels in treatment-naive children with ADHD combined presentation + conduct disorder compared with those with ADHD combined presentation alone and healthy controls. Our findings also showed a trend for lower serum DHEA levels in children with ADHD combined presentation + conduct disorder than in those with ADHD combined presentation alone. These findings suggest that these neuroendocrine hormones may play a role in the pathophysiology of conduct disorder, at least in the presence of ADHD. However, these hormones do not appear to have relationships with ADHD alone. To corroborate our findings, replications of this research with larger groups using heterogeneous populations that address the limitations of our study are needed. Additionally, future studies involving subjects with conduct disorder alone could be useful in elucidating whether these neuroendocrine hormones have a general association with conduct disorder or if they are only associated with the disorder in the presence of ADHD.

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Fig. 1 Box plots representing the distribution of serum a DHEA, b Oxytocin, and c Cortisol levels in children with ADHD, ADHD + CD and controls. DHEA dehydroepiandrosterone, ADHD attention-deficit/hyperactivity disorder, CD Conduct disorder. Analysis of covariance (ANCOVA) was used after adjusted for age, sex, pubertal stage and the severity of depression and anxiety for comparisons between three groups

23

Fig. 2 Scatter plots of the correlations between serum serum neurohormone levels and the clinical symptom severity of ADHD and conduct disorder. a Illustrates the correlations of the serum DHEA levels and teacher-rated T-DSM-IV-S attention-deficit. b Illustrates the correlations of the serum DHEA levels and teacher-rated T-DSM-IV-S hyperactivity impulsivity scores. c Illustrates the correlations of the serum DHEA levels and the parent-rated T-DSM-IV-S hyperactivityimpulsivity scores. d Illustrates the correlations of the serum DHEA levels and the the CPRS-RS hyperactivity scores. e Illustrates the correlations of the serum cortisol levels and the the CPRS-RS the teacher-rated T-DSM-IV-S oppositional defiant behavior scores. DHEA dehydroepiandrosterone, T-DSM-IV-S Turgay DSM-IV-Based Child and Adolescent Behavioral Disorders Screening and Rating Scale, CPRS-RS Conners’ Parent Rating Scale-Revised Short.

Table 1. The distribution of coexisting psychiatric disorders in the ADHD combined presentation and ADHD + conduct disorder groups Comorbidities

ADHD

ADHD + CD

(n = 74)

(n = 32)

%

n

%

n

Oppositional Defiant Disorder

62

48

90

29

Specific Phobias

25

20

25

8

Enuresis

15

12

25

8

Separation Anxiety Disorder

15

12

9

3

Generalized Anxiety Disorder

14

11

3

1

Major Depression

7

6

6

2

Encopresis

5

4

9

3

Tic Disorders

3

3

9

3

Social Phobia

3

3

3

1

24 Obsessive Compulsive Disorder

1

1

0

0

ADHD Attention-deficit/hyperactivity disorder, CD Conduct Disorder

Table 2. Demographic characteristics of the groups ADHD

ADHD + CD

Controls

(N = 74)

(N = 32)

(N = 42)

(Group 1)

(Group 2)

(Group 3)

N

%

N

%

N

%

X2

df

p

57/17

77/23

27/5

84/16

29/13

69/31

2.401

2

.301

Mean

SD

Mean

SD

Mean

SD

F

df

p

10.2

2.1

10.1

1.8

10.9

2.8

1.651

2

.195

-

Verbal

94.6

14.6

93.4

14.5

108.7

14.3

14.951

2

<0.001

3>1, 3>2

Performance

99.7

15.4

97

13.1

109.8

16.9

7.965

2

0.001

3>1, 3>2

Total

96.9

14.8

94.7

13.7

110.3

16.2

13.523

2

<0.001

3>1, 3>2

BMI Percentile

57.2

28.7

60.3

27.7

58.5

32.4

.130

2

.878

-

Tanner stage

1.6

1

1.7

1.1

2

1

1.063

2

.348

-

Boys/Girls

Age (in years)

Statistical analysis

Post-hoc comparisonsa

-

WISC-R

Teacher T-DSM-IV-S AD

14.8

6

16

4.6

2.7

3.7

77.269

2

<0.001

1>3, 2>3

HA/I

14.4

6.9

13.7

5.6

1.7

3.3

59.830

2

<0.001

1>3, 2>3

OD

8.9

5.5

13

5.1

1.2

1.6

55.771

2

<0.001

2>1>3

CD

2.4

2.5

6.5

7.1

.0

.3

23.206

2

<0.001

2>1>3

AD

15.1

5.7

17

4.5

4.4

4.5

72.452

2

<0.001

1>3, 2>3

HA/I

17.2

5.1

20

5.1

3.4

3.3

149.207

2

<0.001

2>1>3

OD

10.6

4.1

16.3

4.5

3.8

2.5

97.085

2

<0.001

2>1>3

CD

2

1.8

8.3

2.8

.5

.8

173.911

2

<0.001

2>1>3

OD

4.4

4

7.6

3.7

.5

1

36.142

2

<0.001

2>1>3

CP-I

6.4

3.8

7.3

3.4

.7

1.1

47.616

2

<0.001

1>3, 2>3

HA

10.9

5.4

11.8

5

1.2

2.3

60.324

2

<0.001

1>3, 2>3

OD

8.9

3.9

13.4

3.3

3.1

2.6

79.808

2

<0.001

2>1>3

CP-I

10.9

4.6

13.6

3.8

2.6

3.3

77.486

2

<0.001

2>1>3

HA

9.2

4.1

12.2

3.8

1.5

2.4

89.967

2

<0.001

2>1>3

Reactive

9.1

4.8

11.2

4.9

5.1

3.6

17.707

2

<0.001

1>3, 2>3

Proactive

2.7

3.1

4.7

3.7

.5

.8

19.645

2

<0.001

2>1>3

Total

11.8

7.3

16

8

5.7

4.1

21.766

2

<0.001

2>1>3

CDI Total

13.8

6.3

15.3

6.9

9.7

4.2

9.492

2

<0.001

1>3, 2>3

SCARED Total

30.4

12.1

26.6

11.4

22.4

11.5

6.274

2

0.002

1>3

Parent T-DSM-IV-S

CTRS-RS

CPRS-RS

RPAQ

25

BMI Body Mass İndex, AD Attention-Deficit, HA/I Hyperactivity–Impulsivity, OD Oppositional Defiant Behavior, CD Conduct Disorder, CTRS-RS Conners Teacher Rating Scale-Revised Short, CPRS-RS Conners Parent Rating Scale-Revised Short, CP-I Cognitive ProblemsInattention, HA Hyperactivity, CDI Children’s Depression Inventory, SCARED Screen for Child Anxiety-Related Emotional Disorders, WISC-R The Wechsler Intelligence Scale for Children-Revised. a

Bonferroni, p <0.05

Table 3. Serum DHEA, Oxytocin and Cortisol Levels for ADHD combined presentation, ADHD + CD and Control Groups

Measu re

ADHD

ADHD +

Controls

ANCOVAa

ANOVA

ANCOVAb

Post Hoc

Post Hoc

(N:74)

CD

(N:42)

Comparis

Comparis

(Group 1)

(N:32)

(Group 3)

onsa

onsb

1>2

-

3>2, 1>2

3>2, 1>2

-

-

(Group 2)

Me

SD

an

DHEA

Me

SD

an

.67

.26

.55

Me

SD

an

.23

.71

.26

*

F

p

F

p

hp2

F

(2.14

(2.14

(2.14

5)

2)

0)

p

hp2

3.48

.03

3.08

.04

.04

2.71

.07

.03

3

3

5

9

2

2

0

7

6.07

.00

5.57

.00

.07

5.05

.00

.06

9

3

3

5

3

3

8

7

(ng/mL ) Oxytoc

1.74

.36

1.49

.42

1.78

.36

in* (μIU/m L) Cortiso l

194.

112

140.

114

198.

136

2.69

.07

2.49

.08

.03

1.96

.14

.02

7

.6

3

.6

3

.7

5

1

7

6

4

4

4

7

(ng/mL ) *Log-transformed variables a Covariates: age, sex, pubertal stage b Covariates: age, sex, and pubertal stage and the severity of depression and anxiety Post Hoc Comparisonsa,b Bonferroni

Highlights  Serum cortisol, DHEA and oxytocin levels were compared among 74 children with ADHD combined presentation (ADHD alone group), 32 children with ADHD combined presentation and coexisting conduct disorder (ADHD + conduct disorder group) and 42 controls.

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 When controlling the potential effects of age, sex, and pubertal stage, serum oxytocin levels of the ADHD combined presentation + conduct disorder group were significantly lower than both those of the ADHD combined presentation alone and control groups, and serum DHEA levels of the ADHD combined presentation + conduct disorder group were significantly lower than ADHD combined presentation alone group.

 When controlling the potential effects of age, sex, pubertal stage, anxiety and depression, serum oxytocin levels of the ADHD combined presentation + conduct disorder group were significantly lower than both those of the ADHD combined presentation alone and control groups. However, there was only a trend for lower serum DHEA levels in the ADHD combined presentation + conduct disorder group than those of the ADHD combined presentation alone group.

 Negative correlations between the serum DHEA levels and the severity of both inattention and hyperactivity–impulsivity scores; and a negative correlation between the serum cortisol levels and oppositional defiant behavior score were noted in the entire patient group.