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Aberrant amino acid transport in fibroblasts from patients with bipolar disorder
Neuroscience Letters 457 (2009) 49–52
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Aberrant amino acid transport in fibroblasts from patients with bipolar disorder M.L. Persson a , J. Johansson b , R. Vumma b , J. Raita a , L. Bjerkenstedt c , F.-A. Wiesel d,1 , N. Venizelos b,∗ a
Stockholm County Council, Center for Dependency Disorder, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden Department of Clinical Medicine, School of Health and Medical Sciences, Örebro University, 701 82 Örebro, Sweden c Psychiatry Section, Dept of Clinical and Experimental Medicine, Linköping University, 581 81 Linköping, Sweden d Department of Neuroscience, Psychiatry, University Hospital, Uppsala University, 751 85 Uppsala, Sweden b
a r t i c l e
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Article history: Received 29 January 2009 Received in revised form 25 March 2009 Accepted 25 March 2009 Keywords: Bipolar disorder Amino acid transport Fibroblasts Tyrosine Tryptophan
a b s t r a c t Aberrant tyrosine transport is a repeated finding in fibroblasts from schizophrenic patients. The transport aberration could lead to disturbances in the dopaminergic and noradrenergic neurotransmitter systems. Tyrosine and tryptophan are the precursors of the neurotransmitters dopamine and serotonin. Disturbed dopaminergic, noradrenergic and serotoninergic systems are implicated as causes of bipolar disorder. Hence, the aim of this study was to explore whether patients with bipolar disorder have an aberrant transport of tyrosine and/or tryptophan. Fibroblast cell lines from patients with bipolar type-1 disorder (n = 10) and healthy controls (n = 10) were included in this study. All patients fulfilled the DSM-IV diagnostic criteria. The transport of amino acids across the cell membranes was measured by the cluster tray method. The kinetic parameters, maximal transport velocity (Vmax ) and affinity constant (Km ) were determined. A significantly lower Vmax for tyrosine (p = 0.027) was found in patients with bipolar type-1 disorder in comparison to healthy controls. No significant differences in Km for tyrosine and in the kinetic parameters of tryptophan between patients with bipolar type-1 disorder and healthy controls were observed. The decreased tyrosine transport (low Vmax ) found in this study may indicate less access of dopamine in the brain, resulting in disturbed dopaminergic and/or noradrenergic neurotransmission, that secondarily could lead to disturbances in other central neurotransmitter systems, such as the serotoninergic system. However, as sample size was small in this study and an age difference between patients and controls existed, the present findings should be considered as pilot data. Further studies with larger sample number are needed to elucidate the transport aberration and the significance of these findings. © 2009 Elsevier Ireland Ltd. All rights reserved.
Bipolar disorder is a chronic psychiatric disorder, clinically characterized by recurrent episodes of mania or hypomania and depression. It has a worldwide prevalence of about 3–5% and it affects women and men almost equally [37]. The disorder interferes with cognition and behaviour and may thus severely impact patients and their social relations [28]. Moreover, patients with bipolar disorder have an increased risk of suicidal ideation, attempts as well as completions [30]. The pathophysiology of the disorder is still not well understood, mainly due to the complexity of brain function [37]. It has been proposed that bipolar disorder may be related to alterations in brain regions involved in processing emotions [5,22] and there are implications for an aberration of neurotransmitter function or regulation in the pathophysiology of bipolar disorder [2]. However, the role of specific neurotransmitters that may contribute to the disorder still
∗ Corresponding author. Tel.: +46 019 30 10 28. E-mail address: [email protected] (N. Venizelos). URL: http://www.oru.se/templates/oruExtNormal38061.aspx (N. Venizelos). 1 Deceased. 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.03.095
needs to be elucidated. For this matter Bjerkenstedt et al. [7] presented a strategy for the simultaneous determination of amino acid and monoamine metabolism, as early as 1985. The amino acids, tyrosine and tryptophan are the precursors of the monoamines dopamine, noradrenaline and serotonin. Repeated studies by our group [8,14,15,31,43,45] and by others [33] have found an aberrant amino acid transport in psychiatric disorders, such as schizophrenia and autism, by using a fibroblast cell model. These transport aberrations may imply that there is a limited access of essential amino acids to the brain, which could lead to disturbances in central neurotransmitter systems [8]. Monoamines that function as neurotransmitters in regulating brain areas are known to have importance in psychiatric disorders. Dopamine, one of the key neurotransmitters in the brain, is a powerful regulator in different aspects of cognitive functions and alterations in dopaminergic activity may lead to cognitive impairment [29]. Quite recently, the kinetics of tyrosine, the precursor of dopamine, has shown to be connected with cognitive functioning [44]. Consistent findings concerning the role of dopamine in the neurobiology of bipolar disorder are that dopaminergic agonists stimulate manic and hypo manic episodes in patients with subjacent bipolar disorder, whereas a
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decreased dopaminergic activity can be associated with depression [2,22]. Abnormal dopaminergic neurotransmission is also implicated in Alzheimer’s disease, attention deficit hyperactivity disorder (ADHD), autism and schizophrenia, these disorders like bipolar disorder show cognitive dysfunctions [29]. Moreover, findings showing a hypo function of the noradrenergic system in depressive states and a hyper function in manic states [2,22], implicate the noradrenergic neurotransmission system in the pathophysiology of bipolar disorder. Serotonin (5 hydroxytryptamine, 5-HT), an other key neurotransmitter in the brain, regulates several physiological and behavioural functions, such as control of impulses, aggressiveness and suicidal tendencies [38]. The involvement of 5-HT in the neurobiology of bipolar disorder is based on observations that patients with bipolar disorder, manic and depressed patients and suicide attempters have lower levels of the serotonin metabolite (5 hydroxyindolacetic acid, 5-HIAA) in the cerebrospinal fluid (CSF) compared to controls [2,3,27,38,40]. Since 5-HIIA is believed to reflect serotonin turnover in the brain [38], the findings of low levels of this metabolite could implicate a reduction in central 5-HT function in both mania and depression. Also, treating depressed patients with selective serotonin re-uptake inhibitors (SSRIs) has shown to be successful [17], which further enhances the importance of this neurotransmitter in psychiatric disorders. Tyrosine belongs to the group of large neutral amino acids (LNAAs). It is transported trough the blood–brain barrier (BBB) and human fibroblasts mainly via the sodium independent system-L and sodium dependent system-A [19,31,42]. System-L consists of four isoforms LAT1 [32], LAT2 [35], LAT3 [4] and LAT4 [9] and these transporters are widely expressed in the body and are present in both BBB and fibroblasts [21]. It was recently shown by our group that LAT1 is the main transporter of tyrosine with high affinity in fibroblast cells [42]. Tyrosine is also transported via ATA2 [21], one of the isoforms of system-A, that is present at the BBB [25] and in fibroblasts [31]. Tryptophan transport has still not been characterized, but being a LNAA, tryptophan is considered to be transported via system-L [19]. Recently, a new transporter was described, which has high affinity and specificity for tryptophan, but the organ expression pattern of this transporter is still not elucidated [36]. The aim of this study was to investigate whether patients with bipolar type-1 disorder have an aberrant transport of tyrosine and/or tryptophan, since these amino acids are the precursors of the important neurotransmitters dopamine and serotonin, which are speculated to have disturbed function or regulation in patients with bipolar disorder. In order to culture fibroblast cell lines a 2 mm2 skin punch biopsy was taken after anaesthetizing the mid-forearm under aseptic conditions as described previously [14]. The tissue was immediately placed in tubes containing complete culture medium and transported to the laboratory. This study included ten fibroblast cell lines from patients with bipolar type-1 disorder, according to the DSM-IV criteria [1]. The group consisted of six men and four women aged 29–77 years (mean 45 years). All were patients at the Karolinska University Hospital, Huddinge, during January and February 2006, and had been assessed and diagnosed by experienced psychiatrists. All of them had been hospitalised several times with both manic and depressive episodes. At the time of interview, eight of patients were euthymic, one was depressed and one was hypo manic. Nine out of ten patients had episodes with psychotic features. The patients were on different medications. Seven patients were on lithium and out of these, two patients were medicated with lithium and lamotrigine, four patients were on lithium and an atypical neuroleptic and one patient was on lithium in combination with an atypical neuroleptic and an antidepressant (a SSRI).
In 50% of the patients, there was found heredity for bipolar as well as schizophrenic disorder. The study was approved by the Ethical Committee at Karolinska University Hospital. An informed consent was obtained from both patients and controls before entering the study. Ten fibroblast cell lines from healthy volunteers were used as controls in this study. The group consisted of two men and eight women (for tyrosine transport assay) aged 21–39 years (mean 30 years) and five men and five women (for tryptophan transport assay) aged 21–41 years (mean 30 years). Cell lines from healthy controls were obtained from a Biobank [15]. All growth media, antibiotics and fetal bovine serum (FBS) were obtained from Gibco Invitrogen cell culture (Sweden). Tissue culture flasks and multi-well plates were from Costar Europe Ltd., Costar NY. 14 C (U)-l-tyrosine with specific activity 391 mCi/mmol was purchased from Moravek Biochemical’s, Inc. (California, USA). [3 H]-l-Tryptophan with specific activity 30 Ci/mmol was obtained from Larodan Fine Chemicals AB (Malmö, Sweden). D-Glucose was obtained from Ambresco (Ohio, USA) and phosphate buffered saline (PBS) was from the National Veterinary Institute (SVA) (Uppsala, Sweden). All other chemicals and amino acids were purchased from Sigma–Aldrich Sweden AB (Sweden). Scintillation cocktail (Optiphase, Hisafe 3) and liquid scintillation counter (Winspectral 1414) were from PerkinElmer Life Sciences (USA). Scintillation vials were purchased from Sarstedt AB (Sweden). Micro-well plates used for protein determination were purchased from Nunc (Roskilde, Denmark) and readings were done using Multiscan MS from Labsystem (Helsinki, Finland). All amino acid solutions were made in PBS and the pH was maintained between 7.35 and 7.40. Fibroblast cells were cultured in plastic tissue culture flasks containing minimal essential medium (MEM) supplemented with 10% FBS, l-glutamine (2 mM/l), penicillin (100 mg/ml), streptomycin (100 mg/ml) and Amino-MaxTM . Cells were maintained in a humidified atmosphere of 5% CO2 in air at 37 ◦ C. Before the measurement of amino acid transport, cells were harvested when confluent and seeded in 2 cm2 -multi-well plates and grown to confluence for approximately 5 days. Cell lines between 4th and 19th passages (number of splitting) were used in the experiments. Amino acid transport was measured using the cluster tray method for rapid measurement of amino acid flux in adherent fibroblast cells [15,16,18]. Fibroblasts grown in multi-well plates were washed twice with PBS and incubated with PBS containing 1% d-glucose for 1 h at 37 ◦ C, to deplete the endogenous amino acid pools. After removal of the pre-incubation medium, the cells were incubated for 60 s at 37 ◦ C with a constant amount of 14 C(U)-labelled tyrosine or 3 H-labelled tryptophan and 12 different concentrations (varying between 0.004 and 1.5 mmol/l for tyrosine and between 0.005 and 2 mmol/l for tryptophan) of unlabelled amino acids. Amino acid transport was terminated by rapidly washing the cells twice with ice-cold PBS. The cells were then lysed in 0.2 ml of 0.5 mmol/l sodium hydroxide (NaOH) for approximately 30 min. The radioactivity of the cell lysate was measured by liquid scintillation counting from a mixture of cell lysate and scintillation cocktail. The total amino acid uptake was correlated to total amount of protein in each well, determined by the Bradford method [10], using Bovine Serum Albumin as a standard. The amino acid kinetic parameters were calculated from the Lineweaver–Burke plot equation [1/V0 = (Km /Vmax [S] + (1/Vmax )], by using computerized software as described in previous study [15]. V0 is the initial transport capacity and [S] is the transport substrate concentration, Vmax is the maximum transport capacity at a saturating concentration of tyrosine or tryptophan (nmol/min × mg protein) and Km , is the affinity constant (the concentration at halfsaturation mol/l). Each experiment was performed in duplicate at the same time point for both tyrosine and tryptophan transport assay.
M.L. Persson et al. / Neuroscience Letters 457 (2009) 49–52 Table 1 Kinetic parameters (Vmax and Km ) of tyrosine and tryptophan transport in cultured fibroblasts from patients with bipolar disorder and controls. Kinetic parameter
Patients with bipolar disorder (n = 10)
Controls (n = 10)
p-Value
Tyrosine, Vmax Tyrosine, Km Tryptophan, Vmax Tryptophan, Km
7.2 (2.53) 14.5 (4.88) 4.5 (1.40) 12.0 (2.46)
9.8 (2.34) 13.5 (3.21) 4.6 (1.58) 11.6 (2.90)
0.027 0.617 0.883 0.787
The results are presented as mean with standard deviation in parenthesis. Vmax , indicates maximal transport capacity (nmol/min × mg protein). Km , indicates affinity of binding sites for a specific amino acid (mol/l).
All amino acid incubations (e.g. tyrosine and tryptophan) were performed in duplicates, and a mean value was taken for kinetic analysis. Assumptions behind parametric methods were fulfilled for Vmax and Km (determined using Shapiro–Wilk W-test), hence all variables are presented by standard descriptive statistics (mean with standard deviations). Significance of the differences in Vmax and Km between patients and controls was analysed using Student’s unpaired t-test assuming equal variance. For all statistical analyses a significant level of 5% percent (two-tailed) was accepted. All statistical analyses were performed using SPSS version 14.0 for Windows. The Vmax of tyrosine transport for patients with bipolar type1 disorder was 7.2 (2.53) nmol/min × mg protein and for controls 9.8 (2.34) nmol/min × mg protein. The Km of tyrosine transport for patients with bipolar type-1 disorder was 14.5 (4.88) mol/l and for controls it was 13.5 (3.21) mol/l (Table 1). For patients with bipolar type-1 disorder, the Vmax of tyrosine transport was significantly lower (p = 0.027) compared to controls, but the Km of tyrosine transport did not differ significantly between the two groups (p = 0.617). For tryptophan transport the Vmax was found to be 4.5 (1.40) nmol/min × mg protein for patients with bipolar type-1 disorder and 4.6 (1.58) nmol/min × mg protein for controls. The Km of tryptophan transport was 12.0 (2.46) mol/l for patients with bipolar type-1 disorder and 11.6 (2.90) mol/l for controls (Table 1). No significant differences were found in Vmax (p = 0.883) and Km (p = 0.787) between patients with bipolar type-1 disorder and controls concerning tryptophan transport. The main finding in this study was that fibroblasts from patients with bipolar type-1 disorder have a decreased Vmax of tyrosine transport, while the Km of tyrosine did not differ between the patients and controls. The low Vmax of tyrosine transport implies that the transport systems have lower capacity for amino acid uptake, which could be due to lower expression of transporting proteins and/or a point mutation in one of the genes coding for the transporters involved. It could also be due to a disturbed membrane phospholipid composition (MPC), indicated in bipolar disorder and schizophrenia [12,13,20,23] that could be altering the structure of transporter proteins, which in turn might change the functionality of the transporters. It is important to note that the brain has no storage of amino acids and therefore the brain is dependent on a stable and balanced influx of tyrosine across the BBB in order to maintain adequate brain functioning. Hence, a satisfactory level of tyrosine in the brain is critical for the maintenance of dopamine and noradrenaline levels. This is of vital importance also for adequate cognitive functions [44]. Thus, a reduced transport of tyrosine, demonstrated in this study by a low Vmax , could indicate that patients with bipolar type-1 disorder have less access of tyrosine in the brain, which may result in central dopamine and noradrenaline imbalance or deficiency. A low Vmax and/or Km have repeatedly been found in patients with schizophrenia [15,18,31,33,45]. As bipolar disorder and schizophrenia share many features, like treatment strategies, onset of illness, life long persistence, increased risk of suicide and
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evidence of shared genetic susceptibility exists, it is speculated whether the two disorders are related [6,24]. The present results are in accordance with previous findings in schizophrenic patients i.e. a decreased tyrosine transport exists in both disorders, but they present with different clinical symptoms. Maybe this implicates a shared amino acid transport aberration in bipolar disorder and schizophrenia. No differences in the tryptophan kinetic parameters (Vmax and Km ) were found in this study, which indicates that the transport of tryptophan is not dysfunctional in patients with bipolar type1 disorder. This is also in accordance with our previous findings in schizophrenia [18]. However, it is suggested that a disturbed dopamine metabolism could cause a dysfunctional dopaminergic neurotransmission, which in turn could lead to disturbances in other neurotransmission systems, like the serotoninergic system [8,34,46]. Hence, the proposed dysfunctional serotoninergic neurotransmission implicated in bipolar disorder could be caused by a disturbed dopamine metabolism/transmission. Treatment with citalopram gives indirect support for this view, since remission of depressive symptomatology occurred during significant elevation of HVA levels and significant reduction of 5-HIAA levels, in the CSF of depressed patients [7]. Also treatment with the recently introduced noradrenergic and partial dopamine re-uptake inhibitor (NDRI) bupropion hydrochloride [39] offers even stronger support for this theory, since this drug “acts” directly on the proposed dopamine deficiency. In this study of patients with bipolar type-1 disorder, an aberrant transport of tyrosine across the fibroblast cell membranes was demonstrated. There might be a corresponding disturbance of tyrosine transport across the BBB in this group of patients with bipolar type-1 disorder, as LAT1, the major transporter of tyrosine [42], is present in both fibroblasts and at human brain micro-capillary endothelial cells [11,41]. The use of fibroblast cells as a human experimental model to study amino acid transport across the BBB in various psychiatric disorders, gained further support in a recent study by Vumma et al. [42]. There are certain limitations with this study. First of all monoamine metabolites in CSF were not determined. At the brain neuron level it is important to consider the balance between dopamine and noradrenaline synthesis, thus one should measure the metabolites of dopamine and noradrenaline in the CSF of patients with bipolar type-1 disorder. The patient group was small, only ten patients with bipolar type-1 disorder was included, which gives a low power of the statistical analyses. On the other hand, the accuracy of results from in vitro studies is high and for that reason fewer patients are needed. The age was differing between the patients with bipolar type-1 disorder and the controls; however there is no statistical correlation between age and the kinetic parameters of both tyrosine and tryptophan. Medication status of the patients with bipolar type-1 disorder was collected at the time of the sample collection (described above) and because of the heterogeneity of the medication utilized and the ignorance of their neurobiological effects it could be rather difficult to draw any conclusions from this study. However, it seems unlikely that prior medication status or the condition of the patient at the time of the biopsy could influence the results, as the cells were growing in vitro for several generations [26]. Rather, the findings observed in the fibroblasts from patients with bipolar type-1 disorder may reflect a hereditary trait that can pass on through many cell-generations. In conclusion, patients with bipolar type-1 disorder have a decreased transport of tyrosine across the membranes of fibroblast cells. Hence, our findings show that there might be disturbances of amino acid transport mechanisms in patients with bipolar type-1 disorder, at least at the membrane level. This could imply that these patients also have a decreased access of tyrosine to or in the
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Aberrant amino acid transport in fibroblasts from patients with bipolar disorder M.L. Persson a , J. Johansson b , R. Vumma b , J. Raita a , L. Bjerkenstedt c , F.-A. Wiesel d,1 , N. Venizelos b,∗ a
Stockholm County Council, Center for Dependency Disorder, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden Department of Clinical Medicine, School of Health and Medical Sciences, Örebro University, 701 82 Örebro, Sweden c Psychiatry Section, Dept of Clinical and Experimental Medicine, Linköping University, 581 81 Linköping, Sweden d Department of Neuroscience, Psychiatry, University Hospital, Uppsala University, 751 85 Uppsala, Sweden b
a r t i c l e
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Article history: Received 29 January 2009 Received in revised form 25 March 2009 Accepted 25 March 2009 Keywords: Bipolar disorder Amino acid transport Fibroblasts Tyrosine Tryptophan
a b s t r a c t Aberrant tyrosine transport is a repeated finding in fibroblasts from schizophrenic patients. The transport aberration could lead to disturbances in the dopaminergic and noradrenergic neurotransmitter systems. Tyrosine and tryptophan are the precursors of the neurotransmitters dopamine and serotonin. Disturbed dopaminergic, noradrenergic and serotoninergic systems are implicated as causes of bipolar disorder. Hence, the aim of this study was to explore whether patients with bipolar disorder have an aberrant transport of tyrosine and/or tryptophan. Fibroblast cell lines from patients with bipolar type-1 disorder (n = 10) and healthy controls (n = 10) were included in this study. All patients fulfilled the DSM-IV diagnostic criteria. The transport of amino acids across the cell membranes was measured by the cluster tray method. The kinetic parameters, maximal transport velocity (Vmax ) and affinity constant (Km ) were determined. A significantly lower Vmax for tyrosine (p = 0.027) was found in patients with bipolar type-1 disorder in comparison to healthy controls. No significant differences in Km for tyrosine and in the kinetic parameters of tryptophan between patients with bipolar type-1 disorder and healthy controls were observed. The decreased tyrosine transport (low Vmax ) found in this study may indicate less access of dopamine in the brain, resulting in disturbed dopaminergic and/or noradrenergic neurotransmission, that secondarily could lead to disturbances in other central neurotransmitter systems, such as the serotoninergic system. However, as sample size was small in this study and an age difference between patients and controls existed, the present findings should be considered as pilot data. Further studies with larger sample number are needed to elucidate the transport aberration and the significance of these findings. © 2009 Elsevier Ireland Ltd. All rights reserved.
Bipolar disorder is a chronic psychiatric disorder, clinically characterized by recurrent episodes of mania or hypomania and depression. It has a worldwide prevalence of about 3–5% and it affects women and men almost equally [37]. The disorder interferes with cognition and behaviour and may thus severely impact patients and their social relations [28]. Moreover, patients with bipolar disorder have an increased risk of suicidal ideation, attempts as well as completions [30]. The pathophysiology of the disorder is still not well understood, mainly due to the complexity of brain function [37]. It has been proposed that bipolar disorder may be related to alterations in brain regions involved in processing emotions [5,22] and there are implications for an aberration of neurotransmitter function or regulation in the pathophysiology of bipolar disorder [2]. However, the role of specific neurotransmitters that may contribute to the disorder still
∗ Corresponding author. Tel.: +46 019 30 10 28. E-mail address: [email protected] (N. Venizelos). URL: http://www.oru.se/templates/oruExtNormal38061.aspx (N. Venizelos). 1 Deceased. 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.03.095
needs to be elucidated. For this matter Bjerkenstedt et al. [7] presented a strategy for the simultaneous determination of amino acid and monoamine metabolism, as early as 1985. The amino acids, tyrosine and tryptophan are the precursors of the monoamines dopamine, noradrenaline and serotonin. Repeated studies by our group [8,14,15,31,43,45] and by others [33] have found an aberrant amino acid transport in psychiatric disorders, such as schizophrenia and autism, by using a fibroblast cell model. These transport aberrations may imply that there is a limited access of essential amino acids to the brain, which could lead to disturbances in central neurotransmitter systems [8]. Monoamines that function as neurotransmitters in regulating brain areas are known to have importance in psychiatric disorders. Dopamine, one of the key neurotransmitters in the brain, is a powerful regulator in different aspects of cognitive functions and alterations in dopaminergic activity may lead to cognitive impairment [29]. Quite recently, the kinetics of tyrosine, the precursor of dopamine, has shown to be connected with cognitive functioning [44]. Consistent findings concerning the role of dopamine in the neurobiology of bipolar disorder are that dopaminergic agonists stimulate manic and hypo manic episodes in patients with subjacent bipolar disorder, whereas a
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decreased dopaminergic activity can be associated with depression [2,22]. Abnormal dopaminergic neurotransmission is also implicated in Alzheimer’s disease, attention deficit hyperactivity disorder (ADHD), autism and schizophrenia, these disorders like bipolar disorder show cognitive dysfunctions [29]. Moreover, findings showing a hypo function of the noradrenergic system in depressive states and a hyper function in manic states [2,22], implicate the noradrenergic neurotransmission system in the pathophysiology of bipolar disorder. Serotonin (5 hydroxytryptamine, 5-HT), an other key neurotransmitter in the brain, regulates several physiological and behavioural functions, such as control of impulses, aggressiveness and suicidal tendencies [38]. The involvement of 5-HT in the neurobiology of bipolar disorder is based on observations that patients with bipolar disorder, manic and depressed patients and suicide attempters have lower levels of the serotonin metabolite (5 hydroxyindolacetic acid, 5-HIAA) in the cerebrospinal fluid (CSF) compared to controls [2,3,27,38,40]. Since 5-HIIA is believed to reflect serotonin turnover in the brain [38], the findings of low levels of this metabolite could implicate a reduction in central 5-HT function in both mania and depression. Also, treating depressed patients with selective serotonin re-uptake inhibitors (SSRIs) has shown to be successful [17], which further enhances the importance of this neurotransmitter in psychiatric disorders. Tyrosine belongs to the group of large neutral amino acids (LNAAs). It is transported trough the blood–brain barrier (BBB) and human fibroblasts mainly via the sodium independent system-L and sodium dependent system-A [19,31,42]. System-L consists of four isoforms LAT1 [32], LAT2 [35], LAT3 [4] and LAT4 [9] and these transporters are widely expressed in the body and are present in both BBB and fibroblasts [21]. It was recently shown by our group that LAT1 is the main transporter of tyrosine with high affinity in fibroblast cells [42]. Tyrosine is also transported via ATA2 [21], one of the isoforms of system-A, that is present at the BBB [25] and in fibroblasts [31]. Tryptophan transport has still not been characterized, but being a LNAA, tryptophan is considered to be transported via system-L [19]. Recently, a new transporter was described, which has high affinity and specificity for tryptophan, but the organ expression pattern of this transporter is still not elucidated [36]. The aim of this study was to investigate whether patients with bipolar type-1 disorder have an aberrant transport of tyrosine and/or tryptophan, since these amino acids are the precursors of the important neurotransmitters dopamine and serotonin, which are speculated to have disturbed function or regulation in patients with bipolar disorder. In order to culture fibroblast cell lines a 2 mm2 skin punch biopsy was taken after anaesthetizing the mid-forearm under aseptic conditions as described previously [14]. The tissue was immediately placed in tubes containing complete culture medium and transported to the laboratory. This study included ten fibroblast cell lines from patients with bipolar type-1 disorder, according to the DSM-IV criteria [1]. The group consisted of six men and four women aged 29–77 years (mean 45 years). All were patients at the Karolinska University Hospital, Huddinge, during January and February 2006, and had been assessed and diagnosed by experienced psychiatrists. All of them had been hospitalised several times with both manic and depressive episodes. At the time of interview, eight of patients were euthymic, one was depressed and one was hypo manic. Nine out of ten patients had episodes with psychotic features. The patients were on different medications. Seven patients were on lithium and out of these, two patients were medicated with lithium and lamotrigine, four patients were on lithium and an atypical neuroleptic and one patient was on lithium in combination with an atypical neuroleptic and an antidepressant (a SSRI).
In 50% of the patients, there was found heredity for bipolar as well as schizophrenic disorder. The study was approved by the Ethical Committee at Karolinska University Hospital. An informed consent was obtained from both patients and controls before entering the study. Ten fibroblast cell lines from healthy volunteers were used as controls in this study. The group consisted of two men and eight women (for tyrosine transport assay) aged 21–39 years (mean 30 years) and five men and five women (for tryptophan transport assay) aged 21–41 years (mean 30 years). Cell lines from healthy controls were obtained from a Biobank [15]. All growth media, antibiotics and fetal bovine serum (FBS) were obtained from Gibco Invitrogen cell culture (Sweden). Tissue culture flasks and multi-well plates were from Costar Europe Ltd., Costar NY. 14 C (U)-l-tyrosine with specific activity 391 mCi/mmol was purchased from Moravek Biochemical’s, Inc. (California, USA). [3 H]-l-Tryptophan with specific activity 30 Ci/mmol was obtained from Larodan Fine Chemicals AB (Malmö, Sweden). D-Glucose was obtained from Ambresco (Ohio, USA) and phosphate buffered saline (PBS) was from the National Veterinary Institute (SVA) (Uppsala, Sweden). All other chemicals and amino acids were purchased from Sigma–Aldrich Sweden AB (Sweden). Scintillation cocktail (Optiphase, Hisafe 3) and liquid scintillation counter (Winspectral 1414) were from PerkinElmer Life Sciences (USA). Scintillation vials were purchased from Sarstedt AB (Sweden). Micro-well plates used for protein determination were purchased from Nunc (Roskilde, Denmark) and readings were done using Multiscan MS from Labsystem (Helsinki, Finland). All amino acid solutions were made in PBS and the pH was maintained between 7.35 and 7.40. Fibroblast cells were cultured in plastic tissue culture flasks containing minimal essential medium (MEM) supplemented with 10% FBS, l-glutamine (2 mM/l), penicillin (100 mg/ml), streptomycin (100 mg/ml) and Amino-MaxTM . Cells were maintained in a humidified atmosphere of 5% CO2 in air at 37 ◦ C. Before the measurement of amino acid transport, cells were harvested when confluent and seeded in 2 cm2 -multi-well plates and grown to confluence for approximately 5 days. Cell lines between 4th and 19th passages (number of splitting) were used in the experiments. Amino acid transport was measured using the cluster tray method for rapid measurement of amino acid flux in adherent fibroblast cells [15,16,18]. Fibroblasts grown in multi-well plates were washed twice with PBS and incubated with PBS containing 1% d-glucose for 1 h at 37 ◦ C, to deplete the endogenous amino acid pools. After removal of the pre-incubation medium, the cells were incubated for 60 s at 37 ◦ C with a constant amount of 14 C(U)-labelled tyrosine or 3 H-labelled tryptophan and 12 different concentrations (varying between 0.004 and 1.5 mmol/l for tyrosine and between 0.005 and 2 mmol/l for tryptophan) of unlabelled amino acids. Amino acid transport was terminated by rapidly washing the cells twice with ice-cold PBS. The cells were then lysed in 0.2 ml of 0.5 mmol/l sodium hydroxide (NaOH) for approximately 30 min. The radioactivity of the cell lysate was measured by liquid scintillation counting from a mixture of cell lysate and scintillation cocktail. The total amino acid uptake was correlated to total amount of protein in each well, determined by the Bradford method [10], using Bovine Serum Albumin as a standard. The amino acid kinetic parameters were calculated from the Lineweaver–Burke plot equation [1/V0 = (Km /Vmax [S] + (1/Vmax )], by using computerized software as described in previous study [15]. V0 is the initial transport capacity and [S] is the transport substrate concentration, Vmax is the maximum transport capacity at a saturating concentration of tyrosine or tryptophan (nmol/min × mg protein) and Km , is the affinity constant (the concentration at halfsaturation mol/l). Each experiment was performed in duplicate at the same time point for both tyrosine and tryptophan transport assay.
M.L. Persson et al. / Neuroscience Letters 457 (2009) 49–52 Table 1 Kinetic parameters (Vmax and Km ) of tyrosine and tryptophan transport in cultured fibroblasts from patients with bipolar disorder and controls. Kinetic parameter
Patients with bipolar disorder (n = 10)
Controls (n = 10)
p-Value
Tyrosine, Vmax Tyrosine, Km Tryptophan, Vmax Tryptophan, Km
7.2 (2.53) 14.5 (4.88) 4.5 (1.40) 12.0 (2.46)
9.8 (2.34) 13.5 (3.21) 4.6 (1.58) 11.6 (2.90)
0.027 0.617 0.883 0.787
The results are presented as mean with standard deviation in parenthesis. Vmax , indicates maximal transport capacity (nmol/min × mg protein). Km , indicates affinity of binding sites for a specific amino acid (mol/l).
All amino acid incubations (e.g. tyrosine and tryptophan) were performed in duplicates, and a mean value was taken for kinetic analysis. Assumptions behind parametric methods were fulfilled for Vmax and Km (determined using Shapiro–Wilk W-test), hence all variables are presented by standard descriptive statistics (mean with standard deviations). Significance of the differences in Vmax and Km between patients and controls was analysed using Student’s unpaired t-test assuming equal variance. For all statistical analyses a significant level of 5% percent (two-tailed) was accepted. All statistical analyses were performed using SPSS version 14.0 for Windows. The Vmax of tyrosine transport for patients with bipolar type1 disorder was 7.2 (2.53) nmol/min × mg protein and for controls 9.8 (2.34) nmol/min × mg protein. The Km of tyrosine transport for patients with bipolar type-1 disorder was 14.5 (4.88) mol/l and for controls it was 13.5 (3.21) mol/l (Table 1). For patients with bipolar type-1 disorder, the Vmax of tyrosine transport was significantly lower (p = 0.027) compared to controls, but the Km of tyrosine transport did not differ significantly between the two groups (p = 0.617). For tryptophan transport the Vmax was found to be 4.5 (1.40) nmol/min × mg protein for patients with bipolar type-1 disorder and 4.6 (1.58) nmol/min × mg protein for controls. The Km of tryptophan transport was 12.0 (2.46) mol/l for patients with bipolar type-1 disorder and 11.6 (2.90) mol/l for controls (Table 1). No significant differences were found in Vmax (p = 0.883) and Km (p = 0.787) between patients with bipolar type-1 disorder and controls concerning tryptophan transport. The main finding in this study was that fibroblasts from patients with bipolar type-1 disorder have a decreased Vmax of tyrosine transport, while the Km of tyrosine did not differ between the patients and controls. The low Vmax of tyrosine transport implies that the transport systems have lower capacity for amino acid uptake, which could be due to lower expression of transporting proteins and/or a point mutation in one of the genes coding for the transporters involved. It could also be due to a disturbed membrane phospholipid composition (MPC), indicated in bipolar disorder and schizophrenia [12,13,20,23] that could be altering the structure of transporter proteins, which in turn might change the functionality of the transporters. It is important to note that the brain has no storage of amino acids and therefore the brain is dependent on a stable and balanced influx of tyrosine across the BBB in order to maintain adequate brain functioning. Hence, a satisfactory level of tyrosine in the brain is critical for the maintenance of dopamine and noradrenaline levels. This is of vital importance also for adequate cognitive functions [44]. Thus, a reduced transport of tyrosine, demonstrated in this study by a low Vmax , could indicate that patients with bipolar type-1 disorder have less access of tyrosine in the brain, which may result in central dopamine and noradrenaline imbalance or deficiency. A low Vmax and/or Km have repeatedly been found in patients with schizophrenia [15,18,31,33,45]. As bipolar disorder and schizophrenia share many features, like treatment strategies, onset of illness, life long persistence, increased risk of suicide and
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evidence of shared genetic susceptibility exists, it is speculated whether the two disorders are related [6,24]. The present results are in accordance with previous findings in schizophrenic patients i.e. a decreased tyrosine transport exists in both disorders, but they present with different clinical symptoms. Maybe this implicates a shared amino acid transport aberration in bipolar disorder and schizophrenia. No differences in the tryptophan kinetic parameters (Vmax and Km ) were found in this study, which indicates that the transport of tryptophan is not dysfunctional in patients with bipolar type1 disorder. This is also in accordance with our previous findings in schizophrenia [18]. However, it is suggested that a disturbed dopamine metabolism could cause a dysfunctional dopaminergic neurotransmission, which in turn could lead to disturbances in other neurotransmission systems, like the serotoninergic system [8,34,46]. Hence, the proposed dysfunctional serotoninergic neurotransmission implicated in bipolar disorder could be caused by a disturbed dopamine metabolism/transmission. Treatment with citalopram gives indirect support for this view, since remission of depressive symptomatology occurred during significant elevation of HVA levels and significant reduction of 5-HIAA levels, in the CSF of depressed patients [7]. Also treatment with the recently introduced noradrenergic and partial dopamine re-uptake inhibitor (NDRI) bupropion hydrochloride [39] offers even stronger support for this theory, since this drug “acts” directly on the proposed dopamine deficiency. In this study of patients with bipolar type-1 disorder, an aberrant transport of tyrosine across the fibroblast cell membranes was demonstrated. There might be a corresponding disturbance of tyrosine transport across the BBB in this group of patients with bipolar type-1 disorder, as LAT1, the major transporter of tyrosine [42], is present in both fibroblasts and at human brain micro-capillary endothelial cells [11,41]. The use of fibroblast cells as a human experimental model to study amino acid transport across the BBB in various psychiatric disorders, gained further support in a recent study by Vumma et al. [42]. There are certain limitations with this study. First of all monoamine metabolites in CSF were not determined. At the brain neuron level it is important to consider the balance between dopamine and noradrenaline synthesis, thus one should measure the metabolites of dopamine and noradrenaline in the CSF of patients with bipolar type-1 disorder. The patient group was small, only ten patients with bipolar type-1 disorder was included, which gives a low power of the statistical analyses. On the other hand, the accuracy of results from in vitro studies is high and for that reason fewer patients are needed. The age was differing between the patients with bipolar type-1 disorder and the controls; however there is no statistical correlation between age and the kinetic parameters of both tyrosine and tryptophan. Medication status of the patients with bipolar type-1 disorder was collected at the time of the sample collection (described above) and because of the heterogeneity of the medication utilized and the ignorance of their neurobiological effects it could be rather difficult to draw any conclusions from this study. However, it seems unlikely that prior medication status or the condition of the patient at the time of the biopsy could influence the results, as the cells were growing in vitro for several generations [26]. Rather, the findings observed in the fibroblasts from patients with bipolar type-1 disorder may reflect a hereditary trait that can pass on through many cell-generations. In conclusion, patients with bipolar type-1 disorder have a decreased transport of tyrosine across the membranes of fibroblast cells. Hence, our findings show that there might be disturbances of amino acid transport mechanisms in patients with bipolar type-1 disorder, at least at the membrane level. This could imply that these patients also have a decreased access of tyrosine to or in the
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