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Altered tryptophan and alanine transport in fibroblasts from boys with attention-deficit/hyperactivity disorder (ADHD): an in vitro study

Altered tryptophan and alanine transport in fibroblasts from boys with... Background: The catecholaminergic and serotonergic neurotransmitter systems are implicated in the pathophysiology of attention-deficit/hyperactivity disorder (ADHD). The amino acid tyrosine is the precursor for synthesis of the catecholamines dopamine and norepinephrine, while tryptophan is the precursor of serotonin. A disturbed transport of tyrosine, as well as other amino acids, has been found in a number of other psychiatric disorders, such as schizophrenia, bipolar disorder and autism, when using the fibroblast cell model. Hence, the aim of this study was to explore whether children with ADHD may have disturbed amino acid transport. Methods: Fibroblast cells were cultured from skin biopsies obtained from 14 boys diagnosed with ADHD and from 13 matching boys without a diagnosis of a developmental disorder. Transport of the amino acids tyrosine, tryptophan and alanine across the cell membrane was measured by the cluster tray method. The kinetic parameters, maximal transport capacity (V ) and affinity constant (K ) were determined. Any difference between max m the two groups was analyzed by Student’s unpaired t-test or the Mann Whitney U test. Results: The ADHD group had significantly decreased V (p = 0.039) and K (increased affinity) (p = 0.010) of max m tryptophan transport in comparison to controls. They also had a significantly higher V of alanine transport (p = max 0.031), but the Km of alanine transport did not differ significantly. There were no significant differences in any of the kinetic parameters regarding tyrosine transport in fibroblasts for the ADHD group. Conclusions: Tryptophan uses the same transport systems in both fibroblasts and at the blood brain barrier (BBB). Hence, a decreased transport capacity of tryptophan implies that less tryptophan is being transported across the BBB in the ADHD group. This could lead to deficient serotonin access in the brain that might cause disturbances in both the serotonergic and the catecholaminergic neurotransmitter systems, since these systems are highly interconnected. The physiological importance of an elevated transport capacity of alanine to the brain is not known to date. Background As for many other neurodevelopmental disorders no Attention-deficit/hyperactivity disorder (ADHD) is a single etiology of ADHD has been provided, but a num- neurodevelopmental disorder with a prevalence in chil- ber of underlying theories exist. The disorder is highly dren of about 5-7% worldwide [1,2]. It is clinically char- heritable, based on family, twin and adoption studies [5], but the genetic architecture of ADHD is suspected to be acterized by a persistent pattern of inattention and/or hyperactivity-impulsivity that affect cognitive, beha- complex. However, a number of candidate genes have vioural, emotional and social functioning and symptoms been identified and among these are several genes asso- may also persist into adulthood [1,3,4]. ciated with the catecholaminergic system [6]. Also, a recent study shows that children with ADHD have an increased rate of large copy number variants (CNVs) * Correspondence: nikolaos.venizelos@oru.se especially at chromosome 16 [7], which further support Department of Clinical Medicine, School of Health and Medical Sciences, ADHD as a genetic disorder. Örebro University, 701 82 Örebro, Sweden Full list of author information is available at the end of the article © 2011 Johansson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Johansson et al. Behavioral and Brain Functions 2011, 7:40 Page 2 of 7 http://www.behavioralandbrainfunctions.com/content/7/1/40 Many neuro-imaging studies have identified abnormal- was found mainly to be transported by system L, with ities of brain structure and function in ADHD and in LAT1 as the main transporter [15], in accordance with particular, a dysfunction of the fronto-subcortical path- the transport of tyrosine through human brain micro- ways in the brain has been implicated [8]. These path- vascular endothelial cells [18,20,21]. Tyrosine is also, to ways control attention and motor behaviour, and it is a less extent, transported through system A, by its iso- known that the catecholamines; dopamine and norepi- form ATA2 [12]. System A is known to transport short- chain amino acids, such as alanine, but in a study by nephrine, are vital for their function [8]. Moreover, sti- Vumma et al in 2008 [15] it was demonstrated that mulant agents, such as methylphenidate, amphetamine approximately 50% of alanine was transported through and atomoxetine, which currently are the most common drugs used for treatment of ADHD, act primarily on the the LAT1 isoform of system L, which denotes a compe- catecholaminergic system. Therefore, the neurotransmit- tition between alanine and tyrosine to get transported. ters dopamine and norepinephrine have been implicated The transport of tryptophan in fibroblast cells is in the pathophysiology of ADHD. However, an involve- mediated by multiple transporters that are active at dif- ment of serotonin and the serotonergic system in the ferent substrate concentrations [22]. However, at physio- pathophysiology of ADHD have also regained the logical tryptophan plasma concentrations the transport researchers’ interest during recent years, since there is an is mainly through the LAT1 isoform of system L [22]. interaction between the dopaminergic and serotonergic When using the fibroblast cell model, previous findings neurotransmitter systems. It is suggested that serotonin by our group [23-29] and others [30] have shown a dis- can modulate the activity of dopamine and an alteration turbed membrane transport of tyrosine in a number of in the serotonergic neurotransmission can alter dopa- psychiatric disorders, such as schizophrenia, autism and mine-mediated behaviour [9,10]. Still, the relevance of bipolar disorder, Moreover, in a previous study by our serotonin in ADHD needs to be further explored. group, we found that children with autism had an The rate of synthesis of the neurotransmitters dopamine, increased transport capacity of alanine across cell mem- norepinephrine and serotonin in the central nervous branes [24]. An elevated transport of alanine across the system (CNS) is partly dependent on the brain’s availability BBB might influence the transport of other amino acids of precursor amino acids [11]. The amino acid tyrosine is that are of vital importance for normal brain activity, the precursor for the synthesis of dopamine and norepi- since amino acids using the same transporter compete nephrine, while tryptophan is the precursor for the synth- for transport [13,15]. The aim of this study was to investigate whether chil- esis of serotonin and both amino acids are essential for the dren with ADHD have changes in the transport of tyrosine brain. Amino acids are polar molecules and are therefore actively transported across cell membranes, like the and/or tryptophan, since these amino acids are the precur- endothelial cells that constitute a part of the blood brain sors for the neurotransmitters implicated in the pathophy- barrier (BBB), by different amino acid transport systems siology of ADHD. Moreover, as there is a competition [12-14]. A competition between amino acids using the between tyrosine and alanine we studied the transport of same transporters exists [13,15]. A number of different alanine in children with ADHD. amino acid transport systems, with different amino acid selectivity, have been identified to date, such as system L, Materials and Methods system A, and system ASC [12]. Children with ADHD In order to study amino acid transport properties, in The study included fibroblast cell lines from 14 boys with various psychiatric disorders, fibroblast cells are being ADHD of the combined type (inattention combined with used as a human experimental model. This model is hyperactivity and impulsivity), according to the DSM-IV suggested to be relevant since fibroblasts have a similar criteria [31]. They were between 6-12 years old (mean 10 expression of amino acid transporters as human brain years) at the time of the study. All were patients at the microvascular endothelial cells (hBME) and express sev- Unit of Neurodevelopmental Disorders, Department of eral neuronal specific receptors and enzymes [15-18]. Pediatrics, Skaraborg Hospital, Mariestad which is a pri- Recent studies have also shown that fibroblast cells can mary pediatric referral center for pharmacological treat- be converted into functional neurons [19]. Moreover, ment of children and adolescents with ADHD. The they are easily obtained from both patients and controls, patients had been diagnosed by an experienced team and in comparison to other cell types they can be within the unit with support from standardized ADHD- grown in larger amounts, are stable for many genera- rating scales used by clinicians, teachers and parents as tions and are not affected by prior medication status of well as with a general pediatric and psychological/cogni- the patient [16]. tive work up. First grade relatives with ADHD or disrup- The transport of both tyrosine and tryptophan has tive disorder, in addition to an uneventful pregnancy and recently been characterized in fibroblast cells. Tyrosine perinatal period, were considered as an indication of Johansson et al. Behavioral and Brain Functions 2011, 7:40 Page 3 of 7 http://www.behavioralandbrainfunctions.com/content/7/1/40 heredity forADHD. Thereweresometrueco-morbid- Cell culturing itiesi.e., twoboyshad migraine andtwo otherboyshad Fibroblast cells were cultured in plastic tissue culture asthma and still three others were overweight. Symptoms flasks containing minimal essential medium (MEM) sup- for motor coordination disorder, oppositional defiant dis- plemented with 10% FBS, L-glutamine (2 mM/L), peni- order, conduct disorder, intellectual disability and autism cillin (100 mg/ml), streptomycin (100 mg/ml) and Amino-Max™. Cells were maintained in a humidified spectrum disorder were checked for but not diagnosed at atmosphere of 5% CO in air at 37°C. Before the mea- the time for the study. Both intellectual disability and surement of amino acid transport, cells were harvested autism spectrum disorder were exclusion criterias. All when confluent and seeded in 2 cm -multi-well plates children were pharmacologically treated, twelve with methylphenidate and two with atomoxetine. and grown to confluence for approximately 5 days. Cell th th lines between 4 and 14 passages (number of splitting) Comparison group were used in the experiments. The comparison group consisted of fibroblast cell lines from 13 boys matched for age and without a diagnosis of a Transport assay of amino acids developmental disorder, between 7 and 13 years old (mean Amino acid transport was measured using the cluster tray 10 years). Five of the 13 cell lines were used as controls in method for rapid measurement of amino acid flux in a previous study and were obtained from a Biobank [24]. adherent fibroblast cells [25,32,33]. Fibroblasts grown in Eight skin biopsies were taken in connection with ear- multi-well plates were washed twice with PBS and incu- nose and throat surgery (such as insertion of transmyrin- bated with PBS containing 1% D-glucose for 1 hour at geal ventilation tubes). 37°C, to deplete the endogenous amino acid pools. After removal of the pre-incubation medium, the cells were Collection of biopsies incubated for 60 seconds at 37°C with a constant amount 14 3 14 A2mm skin punch biopsy was taken after anaesthetizing of C(U)- L-tyrosine or H(5)-L-tryptophan or C(U)-L- the mid-forearm under aseptic conditions as described alanine and 12 different concentrations (varying between previously [24]. The tissue was immediately placed in 0.004 and 1.5 mmol/L for tyrosine, 0.005 to 0.5 mmol/L tubes containing complete culture medium and trans- for tryptophan and 0.02 to 6 mmol/L for alanine) of unla- ported to the laboratory. From the biopsy was fibroblast belled amino acids. Amino acid transport was terminated by rapidly washing the cells twice with ice-cold PBS. The cell lines cultured and stored in a Biobank (-196°C) until cells were then lysed in 0.2 ml of 0.5 mol/L sodium hydro- used for the experiments. xide (NaOH) for approximately 30 minutes. The radioac- tivity of the cell lysate was measured by liquid scintillation Materials All growth media, antibiotics and fetal bovine serum counting from a mixture of cell lysate and scintillation (FBS) were obtained from Gibco Invitrogen cell culture cocktail. The total amino acid uptake was correlated to the (Sweden). total amount of protein in each well, determined by the Tissue culture flasks and multi-well plates were from Bradford method [34], using bovine serum albumin as a Costar Europe Ltd, Costar NY. C (U)-L-tyrosine with standard. specific activity 486 mCi/mmol, H(5)-L-tryptophan with specific activity 30 Ci/mmol and C(U)-L- Alanine with Calculations specific activity 110 Ci/mmol were obtained from Larodan The amino acid kinetic parameters V and K were cal- max m Fine Chemicals AB (Malmö, Sweden). D-Glucose was culated from the obtained uptake values, corrected for dif- obtained from Ambresco (Ohio, USA) and phosphate fusion constant (K ), by using the Lineweaver-Burke plot buffered saline (PBS) was from the National Veterinary equation [1/V =(K /V [S] + (1/V )] as described 0 m max max Institute (SVA) (Uppsala, Sweden). All other chemicals previously [25]. V is the initial transport capacity, [S] is and amino acids were purchased from Sigma-Aldrich the substrate concentration, V is the maximal transport max Sweden AB (Sweden). Scintillation cocktail (Optiphase, capacity (nmol/min/mg protein) and K , is the affinity Hisafe 3) and liquid scintillation counter (Winspectral constant (the concentration at half-saturation, μmol/L). 1414) were from PerkinElmer Life Sciences (USA). Scintil- Each experiment was performed in duplicate at the same lation vials were purchased from Sarstedt AB (Sweden). time point for all amino acid transport assays and a mean Micro-well plates used for protein determination were value was taken for kinetic analysis. purchased from Nunc (Roskilde, Denmark) and readings Statistics were done using Multiscan MS from Labsystems (Hel- All kinetic parameters are presented by descriptive sta- sinki, Finland). All amino acid solutions were made in PBS tistics (mean with standard deviations or median with and the pH was maintained between 7.35 and 7.40. Johansson et al. Behavioral and Brain Functions 2011, 7:40 Page 4 of 7 http://www.behavioralandbrainfunctions.com/content/7/1/40 range). Assumptions about parametric methods were transport in the ADHD group was 1.5 (1.1-3.0) nmol/ fulfilled for K of tyrosine, tryptophan and alanine trans- min/mg protein and for the comparison group 2.0 port, but not for V of tyrosine, tryptophan and ala- (1.3-4.7) nmol/min/mg protein. The mean K for trypto- max m nine transport (determined using Shapiro-Wilk W test). phan transport in the ADHD group was 13.6 (4.7) Significance of the difference in K for tyrosine, trypto- μmol/L and for the comparison group 21.1 (8.2) μmol/L. phan and alanine transport between children with ADHD and the comparison group was analysed using the Alanine Student’s unpaired t-test. Significance of the difference in They ADHD group had a significantly higher V for max V for tyrosine, tryptophan and alanine transport alanine transport (p = 0.031) than the controls, but the max between children with ADHD and the comparison group Km for alanine transport did not differ significantly (p = was analysed using the Mann Whitney U test. 0.086). The median V for alanine was 42.8 (36.3-64.9) max For all statistical analyses a significant level of 5% nmol/min/mg protein in the group of children with (two-tailed) was accepted. All statistical analyses were ADHD, while the median V for the comparison max performed using PASW statistics version 18.0 for group was 32.0 (22.7-63.4) nmol/min/mg protein. The Windows. mean K of alanine transport in the ADHD group was The study was approved by the Regional Ethics Com- 125.7 (41.2) μmol/L and for the comparison group mittee in Gothenburg, Sweden (Dnr: 218-07). A written 100.7 (30.1) μmol/L. and signed consent was obtained from the parent/s and their child before performing the studies. Discussion The main finding in the present study was that the Results group with ADHD had a decreased V of tryptophan max Maximal transport capacity (V ) and mean affinity of transport and an elevated V of alanine transport max max binding site (K ) of tyrosine, tryptophan and alanine across the fibroblast cell membranes. A low V m max transport in children with ADHD and in controls are implies that the transport systems have lower capacity presented in Table 1. for amino acid uptake, while an increased V indicates max the opposite. Hence, the children in the ADHD group Tyrosine had a decreased transport of tryptophan and an elevated There were no significant differences between the ADHD transport of alanine through the cell membrane of fibro- group and controls in any of the kinetic parameters blasts. Several mechanisms could contribute to the dif- regarding the tyrosine transport (V ; p = 0.617, K ;p = ferences found between the ADHD group and the max m 0.645). The median V for tyrosine transport was 15.1 controls; 1) altered expression of transporter proteins max (11.5-25.4) nmol/min/mg protein in the ADHD group and and/or mutation/s in the genes coding for the involved 15.5 (7.8-21.5) nmol/min/mg protein in the comparison transporter proteins; 2) general changes in the cell group. The mean K for tyrosine transport was 20.0 (6.3) membrane, such as a disturbed membrane phospholipid μmol/L in the ADHD group and 18.8 (5.5) μmol/L in the composition (MPC) that could be altering the structure comparison group. of the transporter proteins embedded in the membrane, which in turn might change the functionality of the Tryptophan transporters; 3) the altered amino acid transport could The ADHD group had significantly decreased V (p = be caused by some other molecule(s) affecting the trans- max 0.039) and K (p = 0.010) for tryptophan transport in porter(s) indirectly, e.g. some cytokines are known to comparison to controls. The median V for tryptophan influence amino acid uptake [35-39]. max Table 1 Kinetic parameters of tyrosine, tryptophan and alanine transport in fibroblasts from ADHD children Kinetic parameter Amino acid Children with ADHD Controls p-value (n = 14) (n = 13) V Tyrosine 15.1 (11.5-25.4) 15.5 (7.8-21.5) 0.617 max Tryptophan 1.5 (1.1-3.0) 2.0 (1.3-4.7) 0.039* Alanine 42.8 (36.3-64.9) 32.0 (22.7-63.4) 0.031* K Tyrosine 20.0 (6.3) 18.8 (5.5) 0.645 Tryptophan 13.6 (4.7) 21.1 (8.2) 0.010** Alanine 125.7 (41.2) 100.7 (30.1) 0.086 The results are presented as median (range) for V values and as mean (standard deviation) for K values. ADHD, indicates Attention Deficit/Hyperactivity Disorder, max m V , indicates maximal transport capacity (nmol/min/mg protein) and K , indicates affinity of binding sites for a specific amino acid (μmol/l). *Statistically significant max m (p ≤ 0.05) **Statistically significant (p ≤ 0.01). Johansson et al. Behavioral and Brain Functions 2011, 7:40 Page 5 of 7 http://www.behavioralandbrainfunctions.com/content/7/1/40 The ADHD group also had a decreased K for trypto- of ADHD [45]. However, some children with ADHD not phan transport, which corresponds to an increased affinity responding to single treatment of psychostimulants are between the amino acid and the transport protein, indicat- given a combination with selective serotonin reuptake ing that a lower concentration of extracellular tryptophan inhibitors (SSRI) and show beneficial response. is needed to reach maximal transport capacity. This could be a compensatory mechanism to the decreased V . Implications of an altered alanine transport max However, there was a negative correlation between the The physiological relevance of the increased alanine V and K values for tryptophan transport within the transport that was found in the ADHD group has not max m ADHD group (r = -0.70, p = 0.005). been explored. However, at physiological plasma con- The use of fibroblast cells as a human experimental centrations there is a competition between amino acids model to study amino acid transport across the BBB has for transport across the BBB [13-15]. Although alanine gained support by a number of previous studies [15, is not involved in the synthesis of neurotransmitters, an 23-29]. Moreover, the amino acid transport systems (i.e. elevated transport of alanine might influence the trans- system L and A) and their isoforms are expressed in port of other amino acids that are of essential impor- both fibroblasts and at BBB [15,40]. The present find- tance for normal brain activity. Moreover, since alanine ings of altered amino acid transport in fibroblasts from is involved in many complex and important metabolic children with ADHD might therefore also be present at pathways [46,47], our experiments cannot exclude that the BBB. the elevated alanine transport might be related to other metabolic mechanisms of vital importance for normal Implications of an altered tryptophan transport brain activity. An increased transport of alanine has also The decreased transport of tryptophan that was found in been found in children with autism. ADHD and autism the ADHD group in the present study may imply reduced have high co-morbidity [48,49], and the present finding levels of serotonin in the CNS that might result in a dis- might imply a shared amino acid transport disturbance. turbed serotonergic neurotransmission. A dysfunctional serotonergic system might secondarily lead to distur- Implications of an unaltered tyrosine transport bances in the catecholaminergic systems as these neuro- We did not find any significant differences in the transport transmitter systems have strong anatomical and of tyrosine between the ADHD group and the control functional interactions [41]. group. Since tyrosine and tryptophan are considered to be Although the neurotransmitter most clearly implicated transported across the BBB in similar fashion (i.e. through in ADHD is dopamine, there is a considerable amount of the LAT1 isoform of system L) it is difficult to explain literature associating the serotonergic system with impul- why the tyrosine transport was not altered, whilst the tryp- sivity, i.e. a core symptom in the combined type of ADHD tophan transport was. However, this indicates that it is not [42].There arealsoevidences suggestingthatserotonin the overall capacity (expression) of system L that is may be important in the action of amphetamine to reduce affected; rather the isolated decrease in the V for trypto- max impulsive behaviour, potentially via its interactions with phan could be linked to a more general alteration in the dopaminergic system [43]. The dopamine-serotonin plasma membrane function in ADHD. Altered membrane interaction and evidences for an altered dopaminergic and composition has been implicated in other psychiatric dis- serotonergic contribution to ADHD were reviewed com- orders such as schizophrenia and bipolar disorder [50-53]. prehensively by Oades [9]. The author presented studies Moreover, we measured the total transport of respective supporting the role of serotonergic activity in impulse amino acids, i.e. we did not differentiate between the dif- responses and implications as potential target for pharma- ferent transport systems and their isoforms. cotherapy. Moreover, adolescents with ADHD and disrup- tive behaviour were studied by Malmberg et al (2008) with Possible transporters involved in the altered amino acid respect to MAO-A and 5-HTT genes and platelet MAO-B transport found in the ADHD group activity. The importance of further investigations of the In fibroblasts the tryptophan transport is through differ- serotonergic system, in addition to the dopaminergic sys- ent amino acid transport systems at different substrate tem, in individuals with ADHD and disruptive behaviour concentrations [22]. In this study we measured the trans- disorders was emphasized [44]. port of tryptophan at low concentrations (5 μM-500 μM If the transport of tryptophan is decreased through the of tryptophan), i.e. at physiological plasma concentrations BBB, as our results indicate, increasing plasma concen- of tryptophan (approximately 50 μM, physiological trationoftryptophan(e.g. via tryptophan supplementa- plasma levels of tyrosine and alanine are approximately tion) might not result in more serotonin production. 84 and 250 μM respectively). At this concentration, tryp- This could be one reason for the inconsistent results for tophan is being transported through the LAT1 isoform of tryptophan supplementation approaches in the treatment system L (approximately 80%) and through a high affinity Johansson et al. Behavioral and Brain Functions 2011, 7:40 Page 6 of 7 http://www.behavioralandbrainfunctions.com/content/7/1/40 system, which could be a hitherto undefined transporter necessary. Such exploration should include molecular or a variant of a known transport system with different investigations, looking for polymorphism in gene loci, functional properties due to an altered structure or con- further transport studies including girls with ADHD, formation of the transporter protein. For example, system measure the amount of serotonergic receptors in fibro- L is known to alter the transport properties due to differ- blasts and study the effects of different molecules, such ent light-chain subunits [54,55]. The present results as cytokines, on amino acid uptake. could thus imply that it might be the undefined trypto- phan transporter that is malfunctioning in ADHD and Acknowledgements not the LAT1 transporter, since the tyrosine transport was The authors are indebted to professor Mats Bende and research assistant not altered. If this undefined transporter of tryptophan Christel Larsson for important help with the collection of biopsies from the controls. We also thank the pediatric nurse at the Unit of Developmental also exists at the BBB is not known to date. However, tyro- Disorders, Skaraborgs hospital, Carina Höglund, for valuable collaboration. sine is also transported through the ATA2 isoform of sys- The study was supported by grants from the Research and Development tem A, which is the major transporter of alanine. The Center Skaraborg Hospital (FoU center) and the Faculty of Health and Medicine, Örebro University, Sweden. alanine transport was increased in the ADHD group, indi- cated by an increased V but unchanged K ,and this max m Author details transport disturbance could be caused by different Department of Clinical Medicine, School of Health and Medical Sciences, Örebro University, 701 82 Örebro, Sweden. Department of Pediatrics, mechanisms (see above), such as a higher expression of Skaraborg Hospital, Unit of Neurodevelopmental Disorders, 542 24 Mariestad, the ATA2 transporter protein in the fibroblasts from chil- 3 Sweden. Research and Development Centre, Skaraborg Hospital, 54185 dren with ADHD. Hence, if the LAT1 transporter is dis- Skövde, Sweden. The Gillberg Neuoropsychiatry Center, Sahlgrenska Academy, Gothenburg, Sweden. Strömstad Academy, SE-45280 Strömstad, turbed, tyrosine could compulsorily be transported Sweden. through the ATA2 transporter and therefore the V of max tyrosine transport will not be affected in the ADHD group. Authors’ contributions JJ, carried out 70% of primary cultures from skin biopsy specimens, all tryptophan and alanine transport experiments, literature searches, statistical Limitations analysis and wrote the first draft of the manuscript. ML, participated in the There are certain limitations with the present study. The design of the study and in the recruitment of the patients. EF, participated in the design, coordinated the recruitment of the patients and controls and patient group was relatively small, i.e. fourteen children contributed to the manuscript writing. RV, carried out 30% of primary with ADHD. The plasma levels of amino acids were not cultures from skin biopsy specimens, and all tyrosine transport studies. AÅ, determined at the time of the biopsy collection. A further participated in the design of the study. LB, participated in the design of the study. NV, conceived, participated in the design, took the biopsies, limitation is that only boys were included. However, our interpreted the findings and coordinated the whole study. All authors have intention was to include children with a very similar phe- contributed to and approved the final manuscript. notype of ADHD according to gender, age, type of ADHD Competing interests and etiology. To achieve this we selected only boys (with The authors declare that they have no competing interests. the combined type of ADHD) in a rather narrow age span and for almost all boys there were close relatives/family Received: 23 May 2011 Accepted: 24 September 2011 Published: 24 September 2011 members with the same disorder. Moreover, the patients were on different medications at the time of the biopsy References collection, but it seems rather unlikely that this would 1. Biederman J, Faraone SV: Attention-deficit hyperactivity disorder. Lancet affect the results as the cultivated fibroblasts were seeded 2005, 366:237-248. 2. Polanczyk G, de Lima MS, Horta BL, Biederman J, Rohde LA: The worldwide for multiple generations in vitro before being used in the prevalence of ADHD: a systematic review and metaregression analysis. experiments [16]. Am J Psychiatry 2007, 164:942-948. 3. 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Umeki N, Fukasawa Y, Ohtsuki S, Hori S, Watanabe Y, Kohno Y, Terasaki T: 40. Prasad PD, Wang H, Huang W, Kekuda R, Rajan DP, Leibach FH, mRNA expression and amino acid transport characteristics of cultured Ganapathy V: Human LAT1, a subunit of system L amino acid human brain microvascular endothelial cells (hBME). Drug Metab transporter: molecular cloning and transport function. Biochem Biophys Pharmacokinet 2002, 17:367-373. Res Commun 1999, 255:283-288. 19. Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Sudhof TC, Wernig M: 41. Kapur S, Remington G: Serotonin-dopamine interaction and its relevance Direct conversion of fibroblasts to functional neurons by defined factors. to schizophrenia. Am J Psychiatry 1996, 153:466-476. Nature 2010, 463:1035-1041. 42. Oades RD: Role of the serotonin system in ADHD: treatment 20. Choi TB, Pardridge WM: Phenylalanine transport at the human blood- implications. Expert Rev Neurother 2007, 7:1357-1374. brain barrier. 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Ehrlich BE, Diamond JM: Lithium, membranes, and manic-depressive 28. Wiesel FA, Venizelos N, Bjerkenstedt L, Hagenfeldt L: Tyrosine transport in illness. J Membr Biol 1980, 52:187-200. schizophrenia. Schizophr Res 1994, 13:255-258. 52. Lachman HM, Papolos DF: Abnormal signal transduction: a hypothetical 29. Persson ML, Johansson J, Vumma R, Raita J, Bjerkenstedt L, Wiesel FA, model for bipolar affective disorder. Life Sci 1989, 45:1413-1426. Venizelos N: Aberrant amino acid transport in fibroblasts from patients 53. Horrobin DF: The membrane phospholipid hypothesis as a biochemical with bipolar disorder. Neurosci Lett 2009, 457:49-52. basis for the neurodevelopmental concept of schizophrenia. Schizophr 30. Ramchand CN, Peet M, Clark AE, Gliddon AE, Hemmings GP: Decreased Res 1998, 30:193-208. tyrosine transport in fibroblasts from schizophrenics: implications for 54. Seymour RL, Ganapathy V, Mellor AL, Munn DH: A high-affinity, membrane pathology. Prostaglandins Leukot Essent Fatty Acids 1996, tryptophan-selective amino acid transport system in human 55:59-64. macrophages. J Leukoc Biol 2006, 80:1320-1327. 31. American Psychiatric Association, Diagnostics and Statistical Manual of 55. Verrey F: System L: heteromeric exchangers of large, neutral amino acids Mental Disorders (DSM-IV). 4 edition. Washington DC: American Psychiatric involved in directional transport. Pflugers Arch 2003, 445:529-533. Press; 1994. doi:10.1186/1744-9081-7-40 32. Gazzola GC, Dall’Asta V, Franchi-Gazzola R, White MF: The cluster-tray Cite this article as: Johansson et al.: Altered tryptophan and alanine method for rapid measurement of solute fluxes in adherent cultured transport in fibroblasts from boys with attention-deficit/hyperactivity cells. Anal Biochem 1981, 115:368-374. disorder (ADHD): an in vitro study. Behavioral and Brain Functions 2011 7:40. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Behavioral and Brain Functions Springer Journals

Altered tryptophan and alanine transport in fibroblasts from boys with attention-deficit/hyperactivity disorder (ADHD): an in vitro study

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Copyright © 2011 by Johansson et al; licensee BioMed Central Ltd.
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Biomedicine; Neurosciences; Neurology; Behavioral Therapy; Psychiatry
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Abstract

Background: The catecholaminergic and serotonergic neurotransmitter systems are implicated in the pathophysiology of attention-deficit/hyperactivity disorder (ADHD). The amino acid tyrosine is the precursor for synthesis of the catecholamines dopamine and norepinephrine, while tryptophan is the precursor of serotonin. A disturbed transport of tyrosine, as well as other amino acids, has been found in a number of other psychiatric disorders, such as schizophrenia, bipolar disorder and autism, when using the fibroblast cell model. Hence, the aim of this study was to explore whether children with ADHD may have disturbed amino acid transport. Methods: Fibroblast cells were cultured from skin biopsies obtained from 14 boys diagnosed with ADHD and from 13 matching boys without a diagnosis of a developmental disorder. Transport of the amino acids tyrosine, tryptophan and alanine across the cell membrane was measured by the cluster tray method. The kinetic parameters, maximal transport capacity (V ) and affinity constant (K ) were determined. Any difference between max m the two groups was analyzed by Student’s unpaired t-test or the Mann Whitney U test. Results: The ADHD group had significantly decreased V (p = 0.039) and K (increased affinity) (p = 0.010) of max m tryptophan transport in comparison to controls. They also had a significantly higher V of alanine transport (p = max 0.031), but the Km of alanine transport did not differ significantly. There were no significant differences in any of the kinetic parameters regarding tyrosine transport in fibroblasts for the ADHD group. Conclusions: Tryptophan uses the same transport systems in both fibroblasts and at the blood brain barrier (BBB). Hence, a decreased transport capacity of tryptophan implies that less tryptophan is being transported across the BBB in the ADHD group. This could lead to deficient serotonin access in the brain that might cause disturbances in both the serotonergic and the catecholaminergic neurotransmitter systems, since these systems are highly interconnected. The physiological importance of an elevated transport capacity of alanine to the brain is not known to date. Background As for many other neurodevelopmental disorders no Attention-deficit/hyperactivity disorder (ADHD) is a single etiology of ADHD has been provided, but a num- neurodevelopmental disorder with a prevalence in chil- ber of underlying theories exist. The disorder is highly dren of about 5-7% worldwide [1,2]. It is clinically char- heritable, based on family, twin and adoption studies [5], but the genetic architecture of ADHD is suspected to be acterized by a persistent pattern of inattention and/or hyperactivity-impulsivity that affect cognitive, beha- complex. However, a number of candidate genes have vioural, emotional and social functioning and symptoms been identified and among these are several genes asso- may also persist into adulthood [1,3,4]. ciated with the catecholaminergic system [6]. Also, a recent study shows that children with ADHD have an increased rate of large copy number variants (CNVs) * Correspondence: nikolaos.venizelos@oru.se especially at chromosome 16 [7], which further support Department of Clinical Medicine, School of Health and Medical Sciences, ADHD as a genetic disorder. Örebro University, 701 82 Örebro, Sweden Full list of author information is available at the end of the article © 2011 Johansson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Johansson et al. Behavioral and Brain Functions 2011, 7:40 Page 2 of 7 http://www.behavioralandbrainfunctions.com/content/7/1/40 Many neuro-imaging studies have identified abnormal- was found mainly to be transported by system L, with ities of brain structure and function in ADHD and in LAT1 as the main transporter [15], in accordance with particular, a dysfunction of the fronto-subcortical path- the transport of tyrosine through human brain micro- ways in the brain has been implicated [8]. These path- vascular endothelial cells [18,20,21]. Tyrosine is also, to ways control attention and motor behaviour, and it is a less extent, transported through system A, by its iso- known that the catecholamines; dopamine and norepi- form ATA2 [12]. System A is known to transport short- chain amino acids, such as alanine, but in a study by nephrine, are vital for their function [8]. Moreover, sti- Vumma et al in 2008 [15] it was demonstrated that mulant agents, such as methylphenidate, amphetamine approximately 50% of alanine was transported through and atomoxetine, which currently are the most common drugs used for treatment of ADHD, act primarily on the the LAT1 isoform of system L, which denotes a compe- catecholaminergic system. Therefore, the neurotransmit- tition between alanine and tyrosine to get transported. ters dopamine and norepinephrine have been implicated The transport of tryptophan in fibroblast cells is in the pathophysiology of ADHD. However, an involve- mediated by multiple transporters that are active at dif- ment of serotonin and the serotonergic system in the ferent substrate concentrations [22]. However, at physio- pathophysiology of ADHD have also regained the logical tryptophan plasma concentrations the transport researchers’ interest during recent years, since there is an is mainly through the LAT1 isoform of system L [22]. interaction between the dopaminergic and serotonergic When using the fibroblast cell model, previous findings neurotransmitter systems. It is suggested that serotonin by our group [23-29] and others [30] have shown a dis- can modulate the activity of dopamine and an alteration turbed membrane transport of tyrosine in a number of in the serotonergic neurotransmission can alter dopa- psychiatric disorders, such as schizophrenia, autism and mine-mediated behaviour [9,10]. Still, the relevance of bipolar disorder, Moreover, in a previous study by our serotonin in ADHD needs to be further explored. group, we found that children with autism had an The rate of synthesis of the neurotransmitters dopamine, increased transport capacity of alanine across cell mem- norepinephrine and serotonin in the central nervous branes [24]. An elevated transport of alanine across the system (CNS) is partly dependent on the brain’s availability BBB might influence the transport of other amino acids of precursor amino acids [11]. The amino acid tyrosine is that are of vital importance for normal brain activity, the precursor for the synthesis of dopamine and norepi- since amino acids using the same transporter compete nephrine, while tryptophan is the precursor for the synth- for transport [13,15]. The aim of this study was to investigate whether chil- esis of serotonin and both amino acids are essential for the dren with ADHD have changes in the transport of tyrosine brain. Amino acids are polar molecules and are therefore actively transported across cell membranes, like the and/or tryptophan, since these amino acids are the precur- endothelial cells that constitute a part of the blood brain sors for the neurotransmitters implicated in the pathophy- barrier (BBB), by different amino acid transport systems siology of ADHD. Moreover, as there is a competition [12-14]. A competition between amino acids using the between tyrosine and alanine we studied the transport of same transporters exists [13,15]. A number of different alanine in children with ADHD. amino acid transport systems, with different amino acid selectivity, have been identified to date, such as system L, Materials and Methods system A, and system ASC [12]. Children with ADHD In order to study amino acid transport properties, in The study included fibroblast cell lines from 14 boys with various psychiatric disorders, fibroblast cells are being ADHD of the combined type (inattention combined with used as a human experimental model. This model is hyperactivity and impulsivity), according to the DSM-IV suggested to be relevant since fibroblasts have a similar criteria [31]. They were between 6-12 years old (mean 10 expression of amino acid transporters as human brain years) at the time of the study. All were patients at the microvascular endothelial cells (hBME) and express sev- Unit of Neurodevelopmental Disorders, Department of eral neuronal specific receptors and enzymes [15-18]. Pediatrics, Skaraborg Hospital, Mariestad which is a pri- Recent studies have also shown that fibroblast cells can mary pediatric referral center for pharmacological treat- be converted into functional neurons [19]. Moreover, ment of children and adolescents with ADHD. The they are easily obtained from both patients and controls, patients had been diagnosed by an experienced team and in comparison to other cell types they can be within the unit with support from standardized ADHD- grown in larger amounts, are stable for many genera- rating scales used by clinicians, teachers and parents as tions and are not affected by prior medication status of well as with a general pediatric and psychological/cogni- the patient [16]. tive work up. First grade relatives with ADHD or disrup- The transport of both tyrosine and tryptophan has tive disorder, in addition to an uneventful pregnancy and recently been characterized in fibroblast cells. Tyrosine perinatal period, were considered as an indication of Johansson et al. Behavioral and Brain Functions 2011, 7:40 Page 3 of 7 http://www.behavioralandbrainfunctions.com/content/7/1/40 heredity forADHD. Thereweresometrueco-morbid- Cell culturing itiesi.e., twoboyshad migraine andtwo otherboyshad Fibroblast cells were cultured in plastic tissue culture asthma and still three others were overweight. Symptoms flasks containing minimal essential medium (MEM) sup- for motor coordination disorder, oppositional defiant dis- plemented with 10% FBS, L-glutamine (2 mM/L), peni- order, conduct disorder, intellectual disability and autism cillin (100 mg/ml), streptomycin (100 mg/ml) and Amino-Max™. Cells were maintained in a humidified spectrum disorder were checked for but not diagnosed at atmosphere of 5% CO in air at 37°C. Before the mea- the time for the study. Both intellectual disability and surement of amino acid transport, cells were harvested autism spectrum disorder were exclusion criterias. All when confluent and seeded in 2 cm -multi-well plates children were pharmacologically treated, twelve with methylphenidate and two with atomoxetine. and grown to confluence for approximately 5 days. Cell th th lines between 4 and 14 passages (number of splitting) Comparison group were used in the experiments. The comparison group consisted of fibroblast cell lines from 13 boys matched for age and without a diagnosis of a Transport assay of amino acids developmental disorder, between 7 and 13 years old (mean Amino acid transport was measured using the cluster tray 10 years). Five of the 13 cell lines were used as controls in method for rapid measurement of amino acid flux in a previous study and were obtained from a Biobank [24]. adherent fibroblast cells [25,32,33]. Fibroblasts grown in Eight skin biopsies were taken in connection with ear- multi-well plates were washed twice with PBS and incu- nose and throat surgery (such as insertion of transmyrin- bated with PBS containing 1% D-glucose for 1 hour at geal ventilation tubes). 37°C, to deplete the endogenous amino acid pools. After removal of the pre-incubation medium, the cells were Collection of biopsies incubated for 60 seconds at 37°C with a constant amount 14 3 14 A2mm skin punch biopsy was taken after anaesthetizing of C(U)- L-tyrosine or H(5)-L-tryptophan or C(U)-L- the mid-forearm under aseptic conditions as described alanine and 12 different concentrations (varying between previously [24]. The tissue was immediately placed in 0.004 and 1.5 mmol/L for tyrosine, 0.005 to 0.5 mmol/L tubes containing complete culture medium and trans- for tryptophan and 0.02 to 6 mmol/L for alanine) of unla- ported to the laboratory. From the biopsy was fibroblast belled amino acids. Amino acid transport was terminated by rapidly washing the cells twice with ice-cold PBS. The cell lines cultured and stored in a Biobank (-196°C) until cells were then lysed in 0.2 ml of 0.5 mol/L sodium hydro- used for the experiments. xide (NaOH) for approximately 30 minutes. The radioac- tivity of the cell lysate was measured by liquid scintillation Materials All growth media, antibiotics and fetal bovine serum counting from a mixture of cell lysate and scintillation (FBS) were obtained from Gibco Invitrogen cell culture cocktail. The total amino acid uptake was correlated to the (Sweden). total amount of protein in each well, determined by the Tissue culture flasks and multi-well plates were from Bradford method [34], using bovine serum albumin as a Costar Europe Ltd, Costar NY. C (U)-L-tyrosine with standard. specific activity 486 mCi/mmol, H(5)-L-tryptophan with specific activity 30 Ci/mmol and C(U)-L- Alanine with Calculations specific activity 110 Ci/mmol were obtained from Larodan The amino acid kinetic parameters V and K were cal- max m Fine Chemicals AB (Malmö, Sweden). D-Glucose was culated from the obtained uptake values, corrected for dif- obtained from Ambresco (Ohio, USA) and phosphate fusion constant (K ), by using the Lineweaver-Burke plot buffered saline (PBS) was from the National Veterinary equation [1/V =(K /V [S] + (1/V )] as described 0 m max max Institute (SVA) (Uppsala, Sweden). All other chemicals previously [25]. V is the initial transport capacity, [S] is and amino acids were purchased from Sigma-Aldrich the substrate concentration, V is the maximal transport max Sweden AB (Sweden). Scintillation cocktail (Optiphase, capacity (nmol/min/mg protein) and K , is the affinity Hisafe 3) and liquid scintillation counter (Winspectral constant (the concentration at half-saturation, μmol/L). 1414) were from PerkinElmer Life Sciences (USA). Scintil- Each experiment was performed in duplicate at the same lation vials were purchased from Sarstedt AB (Sweden). time point for all amino acid transport assays and a mean Micro-well plates used for protein determination were value was taken for kinetic analysis. purchased from Nunc (Roskilde, Denmark) and readings Statistics were done using Multiscan MS from Labsystems (Hel- All kinetic parameters are presented by descriptive sta- sinki, Finland). All amino acid solutions were made in PBS tistics (mean with standard deviations or median with and the pH was maintained between 7.35 and 7.40. Johansson et al. Behavioral and Brain Functions 2011, 7:40 Page 4 of 7 http://www.behavioralandbrainfunctions.com/content/7/1/40 range). Assumptions about parametric methods were transport in the ADHD group was 1.5 (1.1-3.0) nmol/ fulfilled for K of tyrosine, tryptophan and alanine trans- min/mg protein and for the comparison group 2.0 port, but not for V of tyrosine, tryptophan and ala- (1.3-4.7) nmol/min/mg protein. The mean K for trypto- max m nine transport (determined using Shapiro-Wilk W test). phan transport in the ADHD group was 13.6 (4.7) Significance of the difference in K for tyrosine, trypto- μmol/L and for the comparison group 21.1 (8.2) μmol/L. phan and alanine transport between children with ADHD and the comparison group was analysed using the Alanine Student’s unpaired t-test. Significance of the difference in They ADHD group had a significantly higher V for max V for tyrosine, tryptophan and alanine transport alanine transport (p = 0.031) than the controls, but the max between children with ADHD and the comparison group Km for alanine transport did not differ significantly (p = was analysed using the Mann Whitney U test. 0.086). The median V for alanine was 42.8 (36.3-64.9) max For all statistical analyses a significant level of 5% nmol/min/mg protein in the group of children with (two-tailed) was accepted. All statistical analyses were ADHD, while the median V for the comparison max performed using PASW statistics version 18.0 for group was 32.0 (22.7-63.4) nmol/min/mg protein. The Windows. mean K of alanine transport in the ADHD group was The study was approved by the Regional Ethics Com- 125.7 (41.2) μmol/L and for the comparison group mittee in Gothenburg, Sweden (Dnr: 218-07). A written 100.7 (30.1) μmol/L. and signed consent was obtained from the parent/s and their child before performing the studies. Discussion The main finding in the present study was that the Results group with ADHD had a decreased V of tryptophan max Maximal transport capacity (V ) and mean affinity of transport and an elevated V of alanine transport max max binding site (K ) of tyrosine, tryptophan and alanine across the fibroblast cell membranes. A low V m max transport in children with ADHD and in controls are implies that the transport systems have lower capacity presented in Table 1. for amino acid uptake, while an increased V indicates max the opposite. Hence, the children in the ADHD group Tyrosine had a decreased transport of tryptophan and an elevated There were no significant differences between the ADHD transport of alanine through the cell membrane of fibro- group and controls in any of the kinetic parameters blasts. Several mechanisms could contribute to the dif- regarding the tyrosine transport (V ; p = 0.617, K ;p = ferences found between the ADHD group and the max m 0.645). The median V for tyrosine transport was 15.1 controls; 1) altered expression of transporter proteins max (11.5-25.4) nmol/min/mg protein in the ADHD group and and/or mutation/s in the genes coding for the involved 15.5 (7.8-21.5) nmol/min/mg protein in the comparison transporter proteins; 2) general changes in the cell group. The mean K for tyrosine transport was 20.0 (6.3) membrane, such as a disturbed membrane phospholipid μmol/L in the ADHD group and 18.8 (5.5) μmol/L in the composition (MPC) that could be altering the structure comparison group. of the transporter proteins embedded in the membrane, which in turn might change the functionality of the Tryptophan transporters; 3) the altered amino acid transport could The ADHD group had significantly decreased V (p = be caused by some other molecule(s) affecting the trans- max 0.039) and K (p = 0.010) for tryptophan transport in porter(s) indirectly, e.g. some cytokines are known to comparison to controls. The median V for tryptophan influence amino acid uptake [35-39]. max Table 1 Kinetic parameters of tyrosine, tryptophan and alanine transport in fibroblasts from ADHD children Kinetic parameter Amino acid Children with ADHD Controls p-value (n = 14) (n = 13) V Tyrosine 15.1 (11.5-25.4) 15.5 (7.8-21.5) 0.617 max Tryptophan 1.5 (1.1-3.0) 2.0 (1.3-4.7) 0.039* Alanine 42.8 (36.3-64.9) 32.0 (22.7-63.4) 0.031* K Tyrosine 20.0 (6.3) 18.8 (5.5) 0.645 Tryptophan 13.6 (4.7) 21.1 (8.2) 0.010** Alanine 125.7 (41.2) 100.7 (30.1) 0.086 The results are presented as median (range) for V values and as mean (standard deviation) for K values. ADHD, indicates Attention Deficit/Hyperactivity Disorder, max m V , indicates maximal transport capacity (nmol/min/mg protein) and K , indicates affinity of binding sites for a specific amino acid (μmol/l). *Statistically significant max m (p ≤ 0.05) **Statistically significant (p ≤ 0.01). Johansson et al. Behavioral and Brain Functions 2011, 7:40 Page 5 of 7 http://www.behavioralandbrainfunctions.com/content/7/1/40 The ADHD group also had a decreased K for trypto- of ADHD [45]. However, some children with ADHD not phan transport, which corresponds to an increased affinity responding to single treatment of psychostimulants are between the amino acid and the transport protein, indicat- given a combination with selective serotonin reuptake ing that a lower concentration of extracellular tryptophan inhibitors (SSRI) and show beneficial response. is needed to reach maximal transport capacity. This could be a compensatory mechanism to the decreased V . Implications of an altered alanine transport max However, there was a negative correlation between the The physiological relevance of the increased alanine V and K values for tryptophan transport within the transport that was found in the ADHD group has not max m ADHD group (r = -0.70, p = 0.005). been explored. However, at physiological plasma con- The use of fibroblast cells as a human experimental centrations there is a competition between amino acids model to study amino acid transport across the BBB has for transport across the BBB [13-15]. Although alanine gained support by a number of previous studies [15, is not involved in the synthesis of neurotransmitters, an 23-29]. Moreover, the amino acid transport systems (i.e. elevated transport of alanine might influence the trans- system L and A) and their isoforms are expressed in port of other amino acids that are of essential impor- both fibroblasts and at BBB [15,40]. The present find- tance for normal brain activity. Moreover, since alanine ings of altered amino acid transport in fibroblasts from is involved in many complex and important metabolic children with ADHD might therefore also be present at pathways [46,47], our experiments cannot exclude that the BBB. the elevated alanine transport might be related to other metabolic mechanisms of vital importance for normal Implications of an altered tryptophan transport brain activity. An increased transport of alanine has also The decreased transport of tryptophan that was found in been found in children with autism. ADHD and autism the ADHD group in the present study may imply reduced have high co-morbidity [48,49], and the present finding levels of serotonin in the CNS that might result in a dis- might imply a shared amino acid transport disturbance. turbed serotonergic neurotransmission. A dysfunctional serotonergic system might secondarily lead to distur- Implications of an unaltered tyrosine transport bances in the catecholaminergic systems as these neuro- We did not find any significant differences in the transport transmitter systems have strong anatomical and of tyrosine between the ADHD group and the control functional interactions [41]. group. Since tyrosine and tryptophan are considered to be Although the neurotransmitter most clearly implicated transported across the BBB in similar fashion (i.e. through in ADHD is dopamine, there is a considerable amount of the LAT1 isoform of system L) it is difficult to explain literature associating the serotonergic system with impul- why the tyrosine transport was not altered, whilst the tryp- sivity, i.e. a core symptom in the combined type of ADHD tophan transport was. However, this indicates that it is not [42].There arealsoevidences suggestingthatserotonin the overall capacity (expression) of system L that is may be important in the action of amphetamine to reduce affected; rather the isolated decrease in the V for trypto- max impulsive behaviour, potentially via its interactions with phan could be linked to a more general alteration in the dopaminergic system [43]. The dopamine-serotonin plasma membrane function in ADHD. Altered membrane interaction and evidences for an altered dopaminergic and composition has been implicated in other psychiatric dis- serotonergic contribution to ADHD were reviewed com- orders such as schizophrenia and bipolar disorder [50-53]. prehensively by Oades [9]. The author presented studies Moreover, we measured the total transport of respective supporting the role of serotonergic activity in impulse amino acids, i.e. we did not differentiate between the dif- responses and implications as potential target for pharma- ferent transport systems and their isoforms. cotherapy. Moreover, adolescents with ADHD and disrup- tive behaviour were studied by Malmberg et al (2008) with Possible transporters involved in the altered amino acid respect to MAO-A and 5-HTT genes and platelet MAO-B transport found in the ADHD group activity. The importance of further investigations of the In fibroblasts the tryptophan transport is through differ- serotonergic system, in addition to the dopaminergic sys- ent amino acid transport systems at different substrate tem, in individuals with ADHD and disruptive behaviour concentrations [22]. In this study we measured the trans- disorders was emphasized [44]. port of tryptophan at low concentrations (5 μM-500 μM If the transport of tryptophan is decreased through the of tryptophan), i.e. at physiological plasma concentrations BBB, as our results indicate, increasing plasma concen- of tryptophan (approximately 50 μM, physiological trationoftryptophan(e.g. via tryptophan supplementa- plasma levels of tyrosine and alanine are approximately tion) might not result in more serotonin production. 84 and 250 μM respectively). At this concentration, tryp- This could be one reason for the inconsistent results for tophan is being transported through the LAT1 isoform of tryptophan supplementation approaches in the treatment system L (approximately 80%) and through a high affinity Johansson et al. Behavioral and Brain Functions 2011, 7:40 Page 6 of 7 http://www.behavioralandbrainfunctions.com/content/7/1/40 system, which could be a hitherto undefined transporter necessary. Such exploration should include molecular or a variant of a known transport system with different investigations, looking for polymorphism in gene loci, functional properties due to an altered structure or con- further transport studies including girls with ADHD, formation of the transporter protein. For example, system measure the amount of serotonergic receptors in fibro- L is known to alter the transport properties due to differ- blasts and study the effects of different molecules, such ent light-chain subunits [54,55]. The present results as cytokines, on amino acid uptake. could thus imply that it might be the undefined trypto- phan transporter that is malfunctioning in ADHD and Acknowledgements not the LAT1 transporter, since the tyrosine transport was The authors are indebted to professor Mats Bende and research assistant not altered. If this undefined transporter of tryptophan Christel Larsson for important help with the collection of biopsies from the controls. We also thank the pediatric nurse at the Unit of Developmental also exists at the BBB is not known to date. However, tyro- Disorders, Skaraborgs hospital, Carina Höglund, for valuable collaboration. sine is also transported through the ATA2 isoform of sys- The study was supported by grants from the Research and Development tem A, which is the major transporter of alanine. The Center Skaraborg Hospital (FoU center) and the Faculty of Health and Medicine, Örebro University, Sweden. alanine transport was increased in the ADHD group, indi- cated by an increased V but unchanged K ,and this max m Author details transport disturbance could be caused by different Department of Clinical Medicine, School of Health and Medical Sciences, Örebro University, 701 82 Örebro, Sweden. Department of Pediatrics, mechanisms (see above), such as a higher expression of Skaraborg Hospital, Unit of Neurodevelopmental Disorders, 542 24 Mariestad, the ATA2 transporter protein in the fibroblasts from chil- 3 Sweden. Research and Development Centre, Skaraborg Hospital, 54185 dren with ADHD. Hence, if the LAT1 transporter is dis- Skövde, Sweden. The Gillberg Neuoropsychiatry Center, Sahlgrenska Academy, Gothenburg, Sweden. Strömstad Academy, SE-45280 Strömstad, turbed, tyrosine could compulsorily be transported Sweden. through the ATA2 transporter and therefore the V of max tyrosine transport will not be affected in the ADHD group. Authors’ contributions JJ, carried out 70% of primary cultures from skin biopsy specimens, all tryptophan and alanine transport experiments, literature searches, statistical Limitations analysis and wrote the first draft of the manuscript. ML, participated in the There are certain limitations with the present study. The design of the study and in the recruitment of the patients. EF, participated in the design, coordinated the recruitment of the patients and controls and patient group was relatively small, i.e. fourteen children contributed to the manuscript writing. RV, carried out 30% of primary with ADHD. The plasma levels of amino acids were not cultures from skin biopsy specimens, and all tyrosine transport studies. AÅ, determined at the time of the biopsy collection. A further participated in the design of the study. LB, participated in the design of the study. NV, conceived, participated in the design, took the biopsies, limitation is that only boys were included. However, our interpreted the findings and coordinated the whole study. All authors have intention was to include children with a very similar phe- contributed to and approved the final manuscript. notype of ADHD according to gender, age, type of ADHD Competing interests and etiology. To achieve this we selected only boys (with The authors declare that they have no competing interests. the combined type of ADHD) in a rather narrow age span and for almost all boys there were close relatives/family Received: 23 May 2011 Accepted: 24 September 2011 Published: 24 September 2011 members with the same disorder. Moreover, the patients were on different medications at the time of the biopsy References collection, but it seems rather unlikely that this would 1. Biederman J, Faraone SV: Attention-deficit hyperactivity disorder. Lancet affect the results as the cultivated fibroblasts were seeded 2005, 366:237-248. 2. Polanczyk G, de Lima MS, Horta BL, Biederman J, Rohde LA: The worldwide for multiple generations in vitro before being used in the prevalence of ADHD: a systematic review and metaregression analysis. experiments [16]. Am J Psychiatry 2007, 164:942-948. 3. 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