Brain-Derived Neurotrophic Factor as a Biomarker in Children with Attention Deficit-Hyperactivity Disorder 1* 2 2 2

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1 JKIMSU, Vol. 4, No. 4, Ot-De 2015 ISSN ORIGINAL ARTICLE Brain-Derived Neurotrophi Fator as a Biomarker in Children with Attention Defiit-Hyperativity Disorder 1* Farshid Saadat, Maryam Kosha, Ali Amiry, Gholamreza Torabi 1 Department of Immunology, Shool of Mediine, Guilan University of Medial Sienes, Rasht Iran, Department of Psyhiatry, Shafa Hospital, Rasht, Iran Abstrat : Bakground: Evidene suggests that Brain-Derived Neurotrophi Fator (BDNF) is involved in the pathogenesis of Attention-Defiit Hyperativity Disorder (ADHD), although experimental data regarding the ontribution of BDNF onentration to this psyhiatri disorder are ontroversial. Aim: To evaluate the plasma levels of BDNF in patients with ADHD. Material and Methods: In this ross setional study, ADHD and ontrols were reruited from the outpatient lini of the Shafa Hospital, Rasht; between Marh 2012 and April Clinial data onerning ADHD diagnosis and blood samples for patients were olleted before treatment. Medial, neurologial and psyhiatri o-morbidities were exluded. The mean of BDNF onentration measured and ompared with healthy ontrols. BDNF assay was determined using ELISA kits aording to manufaturer's instrutions. Desriptive statistial analysis was used with analysis of variane to find the signifiane of data. Results: Statistial analyses showed that the mean BDNF levels were signifiantly lower in ADHD patients and its subgroups as ompared with normal ontrol subjets (p<0.001). Conlusion: This study showed a dramatially lower BDNF plasma levels in untreated patients with ADHD, whih might be useful adjunt method for diagnosis of ADHD in soiety. Keywords: Brain-Derived Neurotrophi Fator (BDNF), Attention-Defiit Hyperativity Disorder (ADHD), BDNF Blood Level Introdution: Attention defiit-hyperativity disorder (ADHD) is a mental and neurobehavioral disorder haraterized by inattention, impulsivity and hyperativity. Diagnosing ADHD is based on its s y m p t o m s t o i n a t t e n t i o n ( A D H D - I ), hyperativity-impulsiveness (ADHD-H) or a ombination of inattention and hyperativity (ADHD-C) [1]. ADHD affets hildren globally and is diagnosed about twelve perent of Iranian kindergartens and shool-aged hildren [2]. Moreover, its symptoms an be diffiult to differentiate from other disorders, inreasing the likelihood that the diagnosis of ADHD would be missed. Although, the definite auses of ADHD are ambiguous, some fators suh as genetis, dietary and the soial environmental fators might be important to ontributors in this disorder [3, 4]. Reently, there is evidene, whih suggests that brain-derived neurotrophi fator, is involved in the pathogenesis of ADHD [5]. Brain-derived neurotrophi fator (BDNF) is a 25-kDa member of the neurotrophin family and highly expressed in ortial and hippoampal strutures. It enhanes the growth and maintenane of several neuronal systems as well Journal of Krishna Institute of Medial Sienes University 10

2 JKIMSU, Vol. 4, No. 4, Ot-De 2015 as partiipates in mehanisms of neuronal plastiity. Aumulating evidene suggests BDNF as a andidate moleule involved in the pathophysiology of mental disorders [6]. In the absene of normal levels of BDNF, mie exhibit enhaned aggressiveness, hyperativity [7]. In addition, Tsai et al suggest that redution of BDNF ativity, espeially in the midbrain region, might play a role in the pathogenesis of ADHD [8]. The experimental data regarding the ontribution of BDNF levels to this psyhiatri disorder are ontroversial. The purpose of this study was to evaluate the plasma levels of BDNF in patients with ADHD. Material and Methods: Study Population: In this ross setional study, partiipants were reruited from the outpatient lini of the Shafa Hospital, Rasht; between Marh 2012 and April Sample size was alulated on n = 29 per group based on previous measurements of BDNF, assuming a minimum differene of pg/ml with a SD of 53.5 pg/ml and 32.3 pg/ml, and the detetion of this differene at a signifiane level of 5%. The total studied sample onsisted of 58 partiipants divided into ADHD and ontrols mathed by age and gender. Twenty-nine healthy hildren, living in the same area with similar demographi harateristis and without any aute or hroni medial problem, were inluded in the study as a ontrol group. They were hosen from hildren that met no riteria for a psyhiatri diagnosis after a areful evaluation by a speialized hild and adolesent psyhiatrist. The Raven's progressive matries were applied to the ontrol group and hildren with IQ below 70 were exluded. The ethial guidelines for study protool were followed in aordane with Guilan University of Medial Sienes ethial ommittee ats. All partiipants and their parents provided their informed onsents. Exlusion Criteria : Partiipants with previous history of major Axis I disorders, ondut disorder, pervasive developmental disorders, ti disorders, impulse ontrol disorders, elimination disorders, eating disorders as well as individuals with an IQ<70 were exluded. In addition, subjets with history of neurologial, metaboli, ardia, liver, kidney and respiratory disease as well as Axis III disorders were not inluded in the present study. Instruments: The diagnosis of ADHD was made by DSM-IV- TR strutured linial interview. Kiddie-Shedule for Affetive Disorders and Shizophrenia for Shool-Age Children-Present and the Lifetime- Persian Version (K-SADS-PL-P) were onduted for partiipants. The IQ was measured by the Raven's progressive matries. The severity of ADHD symptoms were assessed using Conner's Parents Rating Sale (CPRS). The CPRS is a welldesigned instrument that promises to be instrumental in the evaluation of hildren with ADHD behavior problems suh as hyperativity, impulsivity, and anxiety. All the (K-SADS-PL-P) interviews and IQ tests were made by one person. Journal of Krishna Institute of Medial Sienes University 11

3 JKIMSU, Vol. 4, No. 4, Ot-De 2015 Clinial Assessments: Clinially two experiened psyhiatrists performed omplete medial history, physial examination and the K-SADS-PL-P interviews with all ADHD and ontrol hildren and their parents. Their agreement on ADHD and its subtypes was aeptable for all diagnoses. Disagreements that ould not be resolved by disussion would lead to exlusion, but in this study agreement ould be ahieved and all subjet ould retained. In addition, the onfirmatory evaluation was made by one board-ertified hild psyhiatrist (M. Kosha) with a ombination of review of the ompleted questionnaire and a short interview. Blood Samples and BDNF Measurement: Blood samples were drawn from anteubital vein between 8 a.m. and 12 a.m. The samples were olleted into tubes ontaining heparin. After sampling, tubes were entrifuged at 1000 g for 10 min immediately and aliquots of plasma were stored at?20?c. After blood sampling, routine hematology tests were done within a few hours. BDNF levels were determined using the human BDNF Quantikine immuno assay Kit (R&D Systems, Minneapolis, USA), aording to the manufaturer's instrutions. Briefly, the BDNF in the samples was bound by the immobilized antibody in eah well. An enzyme linked monolonal antibody speifi for BDNF was added to the wells. Following a washing, a substrate was added and olor hanged in proportion to the amount of BDNF. The reation was stopped with sulfuri aid and the absorbane was measured at 450 nm. Statistial Analyses: The differenes in plasma levels of BDNF between two mentioned groups were ompared using the Student's t test. BDNF levels were tested for normality of distribution by means of the Kolmogorov-Smirnov test. Comparison between ADHD subgroups for parametri data was performed using Analysis of Variane (ANOVA). The analyses were performed using the SPSS statistial pakage, version 16.0 and P <0.001 were onsidered signifiant. Results: In this study, 29 ADHD patients and ontrols mathed by age and gender were enrolled. Some demographi and psyhometri data of ADHD patients and ontrol subjets are shown in Table 1. Table 1: Results of ADHD Patients and Control Subjets is Stratified by Demographi and Psyhometri Data Parameters ADHD Subjets (N=29) Control subjets (N=29) P Sex (female/male) 5/24 6/ Age (years) 7.59 ± ± CPRS ± ± 6.84 <0.001 Age and CRPS are shown as mean ± SD. Journal of Krishna Institute of Medial Sienes University 12

4 JKIMSU, Vol. 4, No. 4, Ot-De 2015 The age of patient varied from years (Mean 7.59 ± 2.01). Control subjets and ADHD subjets showed no differenes between age ranges and sex, but they showed statistial differenes between symptoms (*p<0.001). There were no signifiant differene in the Mean body weight and IQ sores of the two groups. Based on DSM-IV-TR strutured linial interview and K-SADS-PL-P, all partiipants with attention defiit hyperativity disorder were ategorized inattentive ADHD (13.8%), hyperativity-impulsiveness ADHD (34.5%) and ombined ADHD- (51.7%) subtypes. Aording to data obtained by Conner's' Parent Rating Sales, there were signifiant statistial differenes between different diagnosti subtypes of ADHD patients regarding, ondut problems (F= , P< 0.001), impulsive (F= , P< 0.001), anxiety (F= 7.405, P= 0.001) and hyperativity (F= , P< 0.001). Although there is no signifiant differenes in ADHD subtypes, severity of learning problems being the highest in the hyperative type (Table 2). Table 2: Psyhometri Variables of Different Subtypes of ADHD Assessed Using Analysis of Variane (N=29) Psyhometri variables ADHD-C Mean BDNF ± S.D (N=15) ADHD-I Mean BDNF ± S.D (N=5) ADHD-H Mean BDNF ± S.D (N=9) F P value Condut problems ± ± ± <0.001* Learning problems ± ± ± Psyhosomati ± ± ± * Impulsivity ± ± ± <0.001* Anxiety ± ± ± * Hyperativity ± ± ± <0.001* Total ± ± ± Plasma levels of BDNF in ADHD group and healthy group were ± pg/ml and ± pg/ml, respetively. This differene was statistially signifiant (P <0.001). The onentration of BDNF in patients with various ADHD groups is depited in Fig. 1. Journal of Krishna Institute of Medial Sienes University 13

5 JKIMSU, Vol. 4, No. 4, Ot-De 2015 ADHD = attention defiit hyperativity disorder; BDNF = Brain-derived neurotrophi fator = No signifiant differene among subtypes The differene of BDNF onentration between ADHD subtypes and ontrol was signifiant (P < 0.001*) Aording to our results the differenes in plasma BDNF levels between ADHD subtypes were not statistially signifiant (P=0.550). However, the mean plasma BDNF levels in different subtypes of ADHD groups (ADHD-I, ADHD-H, ADHD-C) were statistially signifiant as ompared with normal (P<0.001). Moreover, aording to Pearson's orrelation test, there was no orrelation between the BDNF titer and ondut problems, learning problems, psyhosomati, impulsivehyperative and anxiety of ADHD symptoms assessed using CPRS. Disussion: Fig 1: The Means Plasma Levels of BDNF in Different ADHD Subtypes and Control Obtained from Immunoassay Test The finding of this study reveals a signifiant derease in plasma levels of BDNF in hildren with all subtypes of ADHD ompared with normal ontrols. Additionally, we ould not demonstrate any assoiation between plasma BDNF levels and psyhometri symptoms aording to Conner's Adult Rating Subsales. As we exluded all o-morbidities and attendane of drug naive ADHD hildren in the survey, lower level of BDNF annot be attributed to omorbidities or previous mediation. Nowadays, the diagnosis of ADHD has been based on history taking and diagnosti interview. It means that no simple objetive test suh as blood test, an aid in making diagnosis. Thus, aording to the powerful P value between plasma levels of BDNF of the two groups, it seems to be a sensitive maker for this disorder. Dereased in BDNF levels in ADHD hildren Journal of Krishna Institute of Medial Sienes University 14

6 JKIMSU, Vol. 4, No. 4, Ot-De 2015 may be primary, as an underlying neurodevelopmental defiit in ADHD or seondary to dysregulation of neural system in this disorder. Sine, plasma levels of BDNF are a marker of entral levels of this protein, our finding support the hypothesis of Tsai about the role of BDNF in the pathogenesis of ADHD [8]. The hypothesis of BDNF as primary underlying defiit in ADHD is supported by inreasing evidene about the role of BDNF in the development of dopamine system [5, 8]. However, more studies with moleular and biologial methodology either are neessary to larify the exat role of BDNF in neurobiologial pathology of ADHD or may provide a new diretion for treatment of this disorder. Lower levels of BDNF in untreated ADHD hildren is onsistent with animal studies [9-11] and Roso's study in adult ADHD [5]. Roso et al have reported a signifiant derease in serum BDNF in adult ADHD ompared to ontrol. They also have found no signifiant orrelations between serum BDNF and different subtypes of ADHD or sores on CPRS [5]. These findings are ongruent with our results. As, adult ADHD is in the ontinuum of hildhood disorder and the etiologi fators are similar, it seems the role of BDNF may also persist for the disorder at all ages [12]. Our ADHD hildren were drug naive, so previous treatment ould not affet on the results. As like as ADHD hildren here, adult patients with ADHD also have shown dereased levels of BDNF. It may be explained by the fat that BDNF is ating as an imperative mediator in the omplex neural system dysfuntion. On the other hand, our result was not onsistent with the results of Shim et al, who have reported higher BDNF levels in ADHD hildren ompared to ontrols. They have suggested a ompensatory mehanism by upregulation of BDNF in response to dopaminergi and serotonergi dysregulaition for their results [13]. Though we believe the ompensation mehanism, the age ranges of our sample were lower than Shim's study; it may be aeptable that the smaller ADHD hildren do not fulfill this mehanism. Moreover, derease in serum levels of BDNF have also been reported in adults with ADHD ompared to healthy ontrols [5]. These results suggest a role for BDNF in ADHD, at least in those patients whose disorder persists throughout life. Therefore, the ompensation mehanism might not ontribute in this proess. Although higher BDNF levels in ADHD hildren have been reported by Shim et al they have not onsidered the influene of physial ativity on BDNF levels in their samples. Some researhers studied the role of exerise on serum brainderived neurotrophi fator onentrations in human subjets. BDNF levels in humans are signifiantly elevated in response to exerise, and the magnitude of inrease is exerise intensity dependent [14, 15]. This phenomenon an be expanded to ADHD hildren, as they are naturally hyperative. There are some lues on the assoiation of BDNF polymorphism with different subtypes of ADHD [16]. A geneti study revealed that there is an assoiation between the met allele of BDNF val66met SNP and ADHD symptoms of hyperativity and impulsivity in European populations [17]. Based on these finding, the role of speifi geneti subtype in our region should Journal of Krishna Institute of Medial Sienes University 15

7 JKIMSU, Vol. 4, No. 4, Ot-De 2015 onsidered. Reently, some researhers report no alteration of serum BDNF levels in untreated patients with ADHD [18, 19]. These ontraditory results may attribute to heterogeniity of ADHD syndrome, multi fatorial pathophysiology of this disorder, different blood sampling methods, geneti patterns and laboratories differenes. Although, we made an effort to hek all the known reasons affeting on BDNF levels, some limitations ould be noted in our study. These limitations are the small sample size and few females inluded in the study. Cho et al, 2010 provided a preliminary evidene for a genderspeifi assoiation between BDNF and ADHD [20]. Aording to our findings, there is no obvious differene between male and female in both healthy and ADHD groups. Due to a limited age range and sex, we annot expand our results to all ADHD patients. Another limitation of this study was that it did not measure BDNF levels of platelets. As Lommatzsh et al [21] have found, BDNF levels in plasma to some extent orrelated with BDNF levels in platelets. Sine, no signifiant differenes between platelet ounts of our samples are seen; platelet ount does not seem to onfound our results. Conlusion: Data obtained from this study showed a dramatially detrimental plasma level of BDNF in untreated patients with attention defiithyperativity disorder. It may onsider as a valuable adjunt method for diagnosis of ADHD in soiety. Further studies are required to eluidate the role of this neurotrophin in ADHD. 1. MCarthy S, Wilton L, Murray ML, Hodgkins P, Asherson P, Wong IC. The epidemiology of pharmaologially treated attention defiit hyperativity disorder [ADHD] in hildren, adolesents and adults in UK primary are. BMC Pediatr 2012; 12: Abdekhodaie Z, Tabatabaei SM, Gholizadeh M. The investigation of ADHD prevalene in kindergarten hildren in northeast Iran and a determination of the riterion validity of Conners' questionnaire via linial interview. Res Dev Disabil 2012; 33: Millihap JG, Yee MM. The diet fator in attentiondefiit/hyperativity disorder. Pediatris 2012; 129: Arher T, Osar-Berman M, Blum K. Epigenetis in Developmental Disorder: ADHD and Endophenotypes. J Genet Syndr Gene Ther 2011; 2: Corominas-Roso M, Ramos-Quiroga JA, Ribases M, Sanhez-Mora C, Palomar G, Valero S, Bosh R, Casas Referenes M. Dereased serum levels of brain-derived neurotrophi fator in adults with attention-defiit hyperativity disorder. Int J Neuropsyhopharmaol 2013; 30: Binder DK, Sharfman HE. Brain-derived neurotrophi fator. Growth Fators 2004; 22: Kernie SG, Liebl DJ, Parada LF. BDNF regulates eating behavior and loomotor ativity in mie. EMBO J 2000; 19: Tsai SJ. Attention-defiit hyperativity disorder may be assoiated with dereased entral brain-derived neurotrophi fator ativity: linial and therapeuti impliations. Med Hypotheses 2007; 68: Lee TH, Lee CH, Kim IH, Yan BC, Park JH, Kwon SH et al. Effets of ADHD therapeuti agents, methylphenidate and atomoxetine, on hippoampal neurogenesis in the adolesent mouse dentate gyrus. Neurosiene Lett 2012; 524: Kim H, Heo HI, Kim DH, Ko IG, Lee SS, Kim SE, et al. Journal of Krishna Institute of Medial Sienes University 16

8 JKIMSU, Vol. 4, No. 4, Ot-De 2015 Treadmill exerise and methylphenidate ameliorate symptoms of attention defiit/hyperativity disorder through enhaning dopamine synthesis and brain derived neurotrophi fator expression in spontaneous hypertensive rats. Neurosiene Lett 2011; 504: Kernie SG, Liebl DJ, Parada LF. BDNF regulates eating behavior and loomotor ativity in mie. EMBO J 2000; 19: Spetie L, Arnold EL. Attention-Defiit/Hyperativity Disorder. In:Martin A, Volkmar FR, editors. Lewis's Child and Adolesent Psyhiatry a Comprehensive Textbook. 4th ed. Philadelphia; Lippinott Williams & Wilkins; 2007: Shim SH, Hwangbo Y, Kwon YJ, Jeong HY, Lee BH, Lee HJ, et al. Inreased levels of plasma brain derived neurotrophi fator [BDNF] in hildren with attention defiit hyperativity disorder [ADHD]. Progress in Neuro Psyhopharmaology & Biologial Psyhiatry 2008; 32: Ferris LT, Williams JS, Shen CL. The effet of aute exerise on serum brain-derived neurotrophi fator levels and ognitive funtion. Med Si Sports Exer 2007; 39: Tang SW, Chu E, Hui T, Helmeste D, Law C. Influene of exerise on serum brain-derived neurotrophi fator onentrations in healthy human subjets. Neurosi Lett 2008; 431: Bergman O, Westberg L, Lihtenstein P, Eriksson E, Larsson H. Study on the possible assoiation of brainderived neurotrophi fator polymorphism with the developmental ourse of symptoms of attention defiit and hyperativity. Int J Neuropsyhopharmaol 2011; 14: Sanhez-Mora C, Ribases M, Ramos-Quiroga JA, Casas M, Bosh R, Boreatti-Hummer A,et al. Metaanalysis of brain-derived neurotrophi fator p.val66met in adult ADHD in four European populations. Am J Med Genet B Neuropsyhiatr Genet 2010; 153B: Sassellati C, Zanardini R, Tiberti A, Pezzani M, Valenti V, Effedri P, et al. Serum brain-derived neurotrophi fator [BDNF] levels in attention defiit-hyperativity disorder [ADHD]. Eur Child Adoles Psyhiatry 2014; 23: Sargini E, Pekanlar akay A, Resmi H, Cengizhan SA, Ozek H, Ellidokuz H,et al. Evaluation of Serum Brain- Derived Neurotrophi Fator Levels in Children with Attention Defiit Hyperativity Disorder: Preliminary Data. Arhives of Neuropsyhiatry 2012; 49: Cho SC, Kim HW, Kim BN, Kim JW, Shin MS, Chung S, et al. Gender-speifi assoiation of the brainderived neurotrophi fator gene with attentiondefiit/hyperativity disorder. Psyhiatry Invest 2010; 7: Lommatzsh M, Zingler D, Shuhbaek K, Shloetke K, Zingler C, Shuff-Werner P, et al. The impat of age, weight and gender on BDNF levels in human platelets and plasma. Neurobiol Aging 2005; 26: *Author for Correspondene: Dr. Farshid Saadat, Department of Immunology, Shool of Mediine, Guilan University of Medial Sienes, P.O. Box: 3477, Rasht, Iran fsaadat@alumnus.tums.a.ir Cell: Journal of Krishna Institute of Medial Sienes University 17