Blood and brain glutamate levels in children with autistic disorder

Research in Autism Spectrum Disorders 7 (2013) 541–548

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Blood and brain glutamate levels in children with autistic disorder Tamer H. Hassan a, Hadeel M. Abdelrahman a, Nelly R. Abdel Fattah b, Nagda M. El-Masry b, Haitham M. Hashim b,*, Khaled M. El-Gerby c, Nermin R. Abdel Fattah d a

Department of Pediatrics, Faculty of Medicine, Zagazig University, Zagazig, Egypt Department of Psychiatry, Faculty of Medicine, Zagazig University, Zagazig, Egypt c Department of Radiodiagnosis, Faculty of Medicine, Zagazig University, Zagazig, Egypt d Department of Medical Biochemistry, Faculty of Medicine, Zagazig University, Zagazig, Egypt b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 7 September 2012 Received in revised form 14 December 2012 Accepted 19 December 2012

Despite of the great efforts that move forward to clarify the pathophysiologic mechanisms in autism, the cause of this disorder, however, remains largely unknown. There is an increasing body of literature concerning neurochemical contributions to the pathophysiology of autism. We aimed to determine blood and brain levels of glutamate in children with autistic disorder and to correlate between them. The study included 10 children with autism and 10 age- and sex-matched healthy controls. Blood glutamate levels were measured using high performance liquid chromatography technique. Brain glutamate levels were measured using proton magnetic resonance spectroscopy. The mean blood and brain glutamate levels were significantly higher in patients than controls (p < 0.001). There was highly significant positive correlation between blood glutamate level and brain glutamate levels in the four tested brain regions (p < 0.001). Glutamate plays an important role in the pathogenesis of autism. Further larger studies are required to support our findings. ß 2012 Elsevier Ltd. All rights reserved.

Keywords: Blood glutamate Brain glutamate Autism

1. Introduction Autism spectrum disorder (ASD) is characterized by social deficits, impaired verbal, and nonverbal communication as well as the presence of restricted and stereotyped behaviors or circumscribed interests. These signs all begin before a child is three years old (American Psychiatric Association, 2000 [APA]). ASD affects information processing in the brain by altering how nerve cells and their synapses connect and organize; how this occurs is not well understood (Levy, Mandell, & Schultz, 2009). Biochemical and pharmacological studies have unveiled dysfunction of various neurotransmitter systems as causing autism. Major neurotransmitters that are implicated in autism include serotonin, dopamine, glutamate, GABA, etc. (Burgess, Sweeten, McMahon, & Fujinam, 2006; Lam, Aman, & Arnold, 2006; Rolf, Grotemeyer, Haarman, & Kehren, 1993). Glutamate is the most prominent excitatory neurotransmitter. It is ubiquitous throughout the central nervous system where it modulates synaptic plasticity, vital to memory, learning and regulation, and modulates gene expression. Overstimulation of glutamate receptors leads to excitotoxicity, creating oxidative stress, mitochondrial damage and ultimately may play a role in neurodegeneration (Debanne, Daoudal, Sourdet, & Russier, 2003).

* Corresponding author. Tel.: +20 1000089614. E-mail address: [email protected] (H.M. Hashim). 1750-9467/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rasd.2012.12.005

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T.H. Hassan et al. / Research in Autism Spectrum Disorders 7 (2013) 541–548

Glutamate is deemed to play a role in the pathophysiology of some neuropsychiatric disorders (Sheldon & Robinson, 2007). Recently, Shimmura et al. (2011) suggested that hyperglutamatergia in the brain is involved in the pathophysiology of autism. Shimmura et al. (2011) measured plasma levels of 25 amino acids in male children with high-functioning autism (HFA) and normally developed healthy male controls. They found that, compared with the normal control group, the HFA group had significantly higher levels of plasma glutamate and lower levels of plasma glutamine. No significant group difference was found in the remaining 23 amino acids. Similarly, Shinohe et al. (2006) in a study on 18 adult patients with autism found that serum levels of glutamate in patients with autism were significantly higher than those of normal controls. Neuroimaging studies have contributed substantially to the understanding of the neurobiology of autism (Williams & Minshew, 2007). Imaging studies have revealed abnormalities in individuals with autistic disorder, which suggest that abnormal brain growth in many major brain structures such as cerebellum, cerebral cortex, amygdala, hippocampus, corpus collosum, basal ganglia, and brain stem may contribute to behavioral abnormalities in autism (Acosta & Pearl, 2004; Courchesne et al., 2001). Potential irregularities in brain neurochemistry can be investigated using proton magnetic resonance spectroscopic imaging (1H MRSI), which can be used to detect various low-molecular-weight metabolites in vivo including the excitatory neurotransmitter glutamate (DeVito et al., 2007). Neuroimaging assays of regional levels of glutamate may help evaluate glutamatergic theories of ASD and inform potential therapies targeting glutamate in specific brain structures (Bejjani et al., 2012). On the basis of these considerations, we designed this study to determine blood and brain levels of glutamate in children with autistic disorder and to correlate between both. Simply we had to clarify that whether there is positive correlation or not and whether it is limited to one or more area or with all brain areas. 2. Methods 2.1. Participants This case–control study was done in Psychiatry, Pediatrics, Radiodiagnosis, and Medical Biochemistry Departments of Zagazig University, Zagazig, Egypt, in the year 2012. It included 10 children with ASD (4 female; age (mean  SD): 11.4  2.7 years, range: 6–14 years) and 10 age- and sex-matched healthy control subjects (5 female; age (mean  SD): 11.3  2.7 years, range: 6.4–13.7 years). Children with ASD recruited from the Pediatric and Psychiatric Outpatients Clinics, Faculty of Medicine, Zagazig University, during their follow up visits. ASD patients fulfilled the criteria for a diagnosis of autism according to the Diagnostic and Statistical Manual of Mental Disorders-IV criteria (APA, 2000). Control children were recruited from the Pediatric Outpatients Clinic, Children’s Hospital, Zagazig University. They were the siblings of children attending this clinic because of a minor illness likes common cold, tonsillitis, acute bronchitis, gastroentritis, etc. None of the control participants recruited was related to the children with autism. All 20 subjects were right handed. 2.2. Procedure 2.2.1. Clinical assessment Clinical evaluation of autistic children was based on clinical history taking, clinical examination, and neuropsychiatric evaluation, during which the diagnosis of autism was confirmed according to the Diagnostic and Statistical Manual of Mental Disorders-IV criteria for research (DSM-IV) (APA, 2000). Patients with associated neurological diseases, immune and metabolic disorders were excluded from the study. All control children underwent a comprehensive assessment of their medical history to exclude those with neurological, psychiatric, or other medical disorders. 2.2.2. Laboratory assessment Blood and brain levels of glutamate were measured in all children. 2.2.2.1. Measurement of blood glutamate level. Blood collection procedure. Plasma samples of the participants in both ASD and control groups were all collected between 11:00 a.m. and 12:00 p.m. before lunch to minimize potential effects of food intake. Blood was taken into 7-ml blood collection tubes containing EDTA-2Na, and samples were immediately centrifuged at 1000  g for 15 min to obtain platelet-poor plasma. The plasma was decanted, aliquoted to avoid multiple freeze-thaw cycles and stored at 80 8C until analysis. Glutamic acid measurement. The plasma samples obtained were homogenized in 1.5 volumes of 5% 5-sulfosalicylic acid (final concentration: 2.0%). The homogenates were centrifuged immediately at 12,000 rpm 4 8C for 10 min to remove precipitated protein. The supernatants were collected and used for glutamic acid measurement using an automatic HPLC system (L-8500A; Hitachi High-Technologies Corporation, Tokyo, Japan). Briefly, amino acids, separated by cation-exchange chromatography, were detected spectrophotometrically after postcolumn reaction with ninhydrin reagent (Shimmura et al., 2011).

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2.2.2.2. Measurement of brain glutamate level. MR imaging of the brain was performed for all children on Philips Achieva class II MRI 1.5-T scanner (Philips Medical Systems, Best, the Netherlands) using T1-weighted (T1W) sagittal spin-echo [TR = 500 ms; TE = 15 ms, field of view (FOV) = 240 mm, matrix = 256  256, slice thickness = 5 mm], axial T2-weighted transverse fast spin-echo [TR = 3300 ms; TE = 100 ms, flip angle = 308, FOV = 240 mm, matrix = 320  320, slice thickness = 5 mm], and axial fluid attenuation inversion recovery (FLAIR) [TR = 8000 ms, TE = 110 ms, T1 = 2400 ms, inversion time = 2500 ms, FOV = 220 mm, matrix = 256  256, slice thickness = 5 mm] sequences. In addition, proton magnetic resonance imaging was performed. T2-weighted images were used for positioning the volumes of interest (VOI). The magnet was shimmed on the water signal of the VOI to a line width of about 8 Hz before the 1H MRS image acquisition to achieve magnetic field homogenization. During the four obtained acquisitions, the radiologist placed the voxels (2 mm  2 mm  2 mm) at bilateral anterior cingulate, left striatum, left cerebellar hemisphere, and left frontal lobe. The single-voxel acquisition used a spin-echo sequence [TR/TE = 1500/30 ms, averages = 3, flip angle = 908] with point-resolved spectroscopy (PRESS) technique. The voxels were positioned excluding contamination of signal from the skull and subcutaneous fat. Preparation scans were used and water saturation applied. MR imaging post processing. The 1H MRS data sets were processed with spectroscopic imaging software on a workstation (Philips Medical Systems, Best, the Netherlands). The post processing procedure consisted of the following steps: zero filling, filtering (Hanning), 2D fast Fourier transformation, frequency shift correction, baseline correction, and phase correction with constant phase angle. Glutamate resonance in the spectra was curve-fitted, and peak areas were obtained from all voxels. 2.3. Ethics The study was undertaken in accordance with the ethical standards and with the Helsinki Declaration of 1964, as revised in 2000 and was approved by our local ethical committee. An informed written consent was obtained from the parents of each participant. The study was explained in simple language to the children and verbal assent was obtained from higher functioning children who were capable of understanding the study process. 2.4. Statistical analysis Data were checked, entered and analyzed using SPSS version 11 (SPSS Inc., Chicago, IL, USA). Data were expressed as mean  SD for quantitative variables, number and percentage for qualitative ones. Paired t-test was used to compare blood and brain glutamate levels between patients and controls. Correlation coefficient was used to correlate blood to brain glutamate levels. A p-value < 0.05 was considered to be statistically significant. 3. Results 3.1. Blood glutamate level in patients and controls The mean blood glutamate level was significantly higher in patients than controls (37.0  9.17 and 20.30  3.65 respectively, p value < 0.001) (Table 1). 3.2. Brain glutamate level in patients and controls The mean brain glutamate level was significantly higher in ASD than controls for the four tested brain regions; bilateral anterior cingulated (8.31  0.97 and 6.55  0.95 respectively, p value < 0.001), left striatum (8.41  1.02 and 6.54  0.91 respectively, p value < 0.001), left cerebellar hemisphere (8.40  0.97 and 6.42  0.75 respectively, p value < 0.001), and left frontal lobe (7.94  1.14 and 6.33  0.79 respectively, p value < 0.001) (Table 1, Figs. 1 and 2). Axial T2-weighted images in left striatum (Fig. 1) showing an increased glutamate level in children with ASD compared to control group. Sagittal T2-weighted images in anterior cingulate (Fig. 2) also, showing an increased glutamate level in children with ASD compared to control group.

Table 1 Blood and brain glutamate levels in patients and controls. Parameter

Blood glutamate level (mM) Glutamate level in bilateral anterior cingulate (institutional unit) Glutamate level in left striatum (institutional unit) Glutamate level in left cerebellar hemisphere (institutional unit) Glutamate level in left frontal lobe (institutional unit)

Patients (n = 10)

Controls (n = 10)

Mean  SD

Mean  SD

37.0  9.17 8.31  0.97 8.41  1.02 8.40  0.97 7.94  1.14

20.30  3.65 6.55  0.95 6.54  0.91 6.42  0.75 6.33  0.79

t value

p value

5.35 4.10 4.32 5.12 3.69

<0.001 <0.001 <0.001 <0.001 <0.001

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Fig. 1. Brain glutamate level in left striatum in patients and controls. Axial T2-weighted images depicting volume of interest in left striatum (A) with its corresponding spectra from control (B) and autistic patient (C), showing an increased glutamate level at 2.2 ppm (arrows) in the later.

3.3. Correlation between blood and brain glutamate levels in patients There was highly significant positive correlation between blood glutamate level and brain glutamate levels in the four tested brain regions; bilateral anterior cingulated (r = 0.94, p value < 0.001), left striatum (r = 0.93, p value < 0.001), left cerebellar hemisphere (r = 0.98, p value < 0.001), and left frontal lobe (r = 0.97, p value < 0.001) (Fig. 3). 4. Discussion It has recently been hypothesized that hyperglutamatergia in the brain is involved in the pathophysiology of ASD (Blaylock & Strunecka, 2009; Fatemi, 2008). In this study we determined blood and brain levels of glutamate in children with autistic disorder and the correlation between both. We found that the mean blood glutamate level was significantly higher in ASD patients than controls. Our results are in line with those of Shimmura et al. (2011), and Shinohe et al. (2006). In addition, elevated glutamate levels have been reported in blood serum, plasma, and platelets in ASD (Aldred, Moore, Fitzgerald, & Waring, 2003; Moreno et al., 1992; Moreno-Fuenmayor, Borjas, Arrieta, Valera, & Socorro-Candanoza, 1996; Rolf et al., 1993). We found that, the mean brain glutamate level was significantly higher in ASD patients than controls in the four tested brain regions which are bilateral anterior cingulate, left striatum, left cerebellar hemisphere, and left frontal lobe. Our result was in line with an earlier study using 1H MRS, O’Neill, Levitt, McCracken, Toga, and Alger (2003) examined eight autistic children and nine matched healthy controls and found that, compared to controls, autistics exhibited 27.9% higher glutamate level in midline anterior cingulated. But there was 27.2% lower glutamate level in left amygdala. Also, Page et al. (2006) found an increased excitatory glutamate + glutamine (Glx) levels in the amygdala–hippocampal complex of persons with autism using 1H MRS measures at 1.5 T. In addition, Brown, Youngpeter, Singel, Hepburn, and Rojas (2011) found higher left side glutamate concentration in the hippocampus in the autistic group versus the controls, with no significant differences on the right side. Fatemi et al. (2002) have shown that levels of GAD 65 kDa and GAD 67 kDa proteins, both of which are involved in converting glutamate to GABA, are reduced in the brains of individuals with autism, resulting in increased levels of glutamate in the brain substrate. On the other hand, our result was inconsistent with those

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Fig. 2. Brain glutamate level in anterior cingulate in patients and controls. Sagittal T2-weighted images depicting volume of interest in anterior cingulate (A) with its corresponding spectra from control (B) and autistic patient (C), showing an increased glutamate level at 2.2 ppm (arrows) in the later.

of DeVito et al. (2007) who reported wide spread Glx reduction in frontal and occipital gray matter and in cerebellum in children with ASD. In addition, Bernardi et al. (2011) in an adult sample not taking psychotropic medications, compared to healthy control group, the ASD group showed significantly lower Glx concentration in right anterior cingulate cortex. But both studies did not determine if the changes in Glx are due to glutamine, glutamate, or both. However, Friedman et al. (2006), and Hardan et al. (2008) did not find significant differences in Glx levels in the thalamus or elsewhere in the white and gray matter by 1.5 T 1H MRS comparing children and adolescents with ASD to healthy controls. Prior MRS studies reporting glutamate or Glx in ASD have yielded mixed results. This may be due to differences in subject samples and MRS methods. We measured glutamate levels in four brain regions which are bilateral anterior cingulate, left striatum, left cerebellar hemisphere, and left frontal lobe according to previous studies points to involvement of these regions in ASD. As cerebellum is involved in several processes that include cognitive, affective and sensory functions in addition to motor tasks. Attention related cerebellar function is usually reduced in autistic individuals (Allen & Courchesne, 2003). Another study relates anterior cingulate dysfunction to deficits in joint attention and social orienting in ASD (Mundy, 2003), and different parts of the striatum may participate in different types of memories (Packard & Knowlton, 2002). In addition, the frontal lobe is central to many functions that are associated with autism, such as language and executive functions such as working memory, inhibition, planning, organizing, set-shifting, and cognitive flexibility (DeVito et al., 2007). Hyperglutamatergic hypothesis of autism was supported by preliminary genetic studies reported an association between ASD and alleles encoding for kainite and metabotropic glutamate receptors (Jamain et al., 2002; Serajee, Zhong, Nabi, & Huq, 2003; Shuang et al., 2004), the mitochondrial aspartate/glutamate carrier (Ramoz et al., 2004), the enzyme glutamate decarboxylase (Buttenschøn et al., 2009), and astrocytic glutamate transporter proteins (Purcell, Jeon, Zimmerman, Blue, & Pevsner, 2001). Our results of elevated glutamate levels in brain are compatible with evidence suggesting the relationship between a dysregulation of glutamine/glutamate metabolism and increased levels of gliosis in the brains of individuals with autism (Shimmura et al., 2011). The increase in gliosis, is characterized by enhanced activation of astrocytes and microglia(Laurence

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12

Brain glutamate(left cerebellar hemisphere)

Brain glutamate (bilateral anterior cingulate)

546

10 8 6 4 2

r = 0.94, P<0.001

0 0

20

40

12 10 8 6 4 2

r = 0.98, P<0.001

0

60

0

12 10 8 6 4 2

r = 0.93, P<0.001

0 0

20

40

Blood glutamate

20

40

60

Blood glutamate

Brain glutamate(left frontal lobe)

Brain glutamate( left striatum)

Blood glutamate

60

12 10 8 6 4 2

r = 0.97, P<0.001

0 0

20

40

60

Blood glutamate

Fig. 3. Correlation between blood and brain glutamate levels in patients. There was highly significant positive correlation between blood glutamate level and brain glutamate levels in the four tested brain regions.

& Fatemi, 2005; Vargas, Nascimbene, Krishnan, Zimmerman, & Pardo, 2005). Ortinski et al. (2010) have reported that activated astrocytes downregulate the expression of glutamine synthetase, whereby glutamate is converted into glutamine, which in turn results in reduced glutamine coupled with elevated glutamate. In addition, glutaminase, another enzyme which convert glutamine into glutamate, has been shown to be upregulated in activated microglia (Pais, Figueiredo, Peixoto, Braz, & Chatterjee, 2008). Thus, the process of gliosis activate astrocytes and/or microglia, which may disturb the regulation of certain types of enzymes and thereby alter the metabolism of glutamate/glutamine (Shimmura et al., 2011). We found highly significant positive correlation between blood glutamate level and brain glutamate levels in the four tested brain regions. We are the first to report this positive correlation in ASD patients. Thus, the peripheral glutamate level can be postulated to reflect the glutamate level in the brain per se. In agreement with our study, Alfredsson, Wiesel, and Tylec (1988), McGale, Pye, Stonier, Hutchinson, and Aber (1977) found that the level of glutamate in the blood is positively correlated with the cerebrospinal fluid (CSF) level of glutamate in humans. Previous researches reported that glutamate is not to readily cross the blood–brain barrier (BBB) (Sheldon & Robinson, 2007). However, our results can be explained by the role of glutamate transporters (excitatory amino acid transporters; EAATs) which exist exclusively in the abluminal membranes of BBB and shift glutamate from the extracellular fluids to the endothelial cells through an active efflux pump mechanism where glutamate is free to diffuse into blood on facilitative carriers (Hosoya, Sugawara, Asaba, & Terasaki, 1999). In our study we tried to prove this positive correlation in different brain areas. This would be very helpful because we can rely on blood glutamate to reflect brain glutamate as measurement of blood glutamate is much easier than measurement of brain glutamate. Some limitations of this study include small subject number and we did not correlate between glutamate levels in plasma and brain with the severity of symptoms. Also individuals with autism have some intellectual impairment so controlling for the Intelligence Quotient (IQ) or excluding those individuals with autism who haveintellectual disabilities should be taken in consederation in further studies

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5. Conclusion We conclude that glutamate plays an important role in the pathogenesis of autism. These results provide new insights into the pathophysiology of autism, which may be particularly helpful for the development of novel diagnostic and therapeutic strategies to eliminate excess toxic glutamate. Further studies with a larger sample are required to support our findings.

References Acosta, M. T., & Pearl, P. L. (2004). Imaging data in autism: From structure to malfunction. Seminars in Pediatric Neurology, 11(3), 205–213. Aldred, S., Moore, K. M., Fitzgerald, M., & Waring, R. H. (2003). Plasma amino acid levels in children with autism and their families. Journal of Autism and Developmental Disorders, 33(1), 93–97. Alfredsson, G., Wiesel, F. A., & Tylec, A. (1988). Relationships between glutamate and monoamine metabolites in cerebrospinal fluid and serum in healthy volunteers. Biological Psychiatry, 23(7), 689–697. Allen, G., & Courchesne, E. (2003). Differential effects of developmental cerebellar abnormality on cognitive and motor functions in the cerebellum: An fMRI study of autism. American Journal of Psychiatry, 160(2), 262–273. American Psychiatric Association. (2000). Diagnostic and Statistical Manual of Mental Disorders. 4th, text revision (DSM-IV-TR) ed., ISBN 0-89042-025-4. Diagnostic criteria for 299.00 Autistic Disorder. Bejjani, A., O’Neill, J., Kim, J. A., Frew, A. J., Yee, V. W., Ly, R., et al. (2012). Elevated glutamatergic compounds in pregenual anterior cingulate in pediatric autism spectrum disorder demonstrated by 1H MRS and 1H MRSI. Public Library of Science ONE, 7(7), e38786. Bernardi, S., Anagnostou, E., Shen, J., Kolevzon, A., Buxbaum, J. D., Hollander, E., et al. (2011). In vivo 1H-magnetic resonance spectroscopy study of the attentional networks in autism. Brain Research, 1380(22), 198–205. Blaylock, R. L., & Strunecka, A. (2009). Immune-glutamatergic dysfunction as a central mechanism of the autism spectrum disorders. Current Medicinal Chemistry, 16(2), 157–170. Brown, M. S., Youngpeter, K., Singel, D., Hepburn, S., & Rojas, D. C. (2011). A 1H-MRS study of the auditory cortex in persons with autism spectrum disorder (ASD). Proceedings of the International Society for Magnetic Resonance in Medicine, 19, 2528. Burgess, N. K., Sweeten, T. L., McMahon, W. M., & Fujinam, R. S. (2006). Hyperserotoninemia and altered immunity in autism. Journal of Autism and Developmental Disorders, 36(5), 697–704. Buttenschøn, H. N., Lauritsen, M. B., El Daoud, A., Hollegaard, M., Jorgensen, M., Tvedegaard, K., et al. (2009). A population-based association study of glutamate decarboxylase as a candidate gene for autism. Journal of Neural Transmission, 116(3), 381–388. Courchesne, E., Karns, C. M., Davis, H. R., Ziccardi, R., Carper, R. A., Tigue, Z. D., et al. (2001). Unusual brain growth patterns in early life in patients with autistic disorder: An MRI study. Neurology, 57(2), 245–254. Debanne, D., Daoudal, G., Sourdet, V., & Russier, M. (2003). Brain plasticity and ion channels. Journal of Physiology-Paris, 97(4–6), 403–414. DeVito, T. J., Drost, D. J., Neufeld, R. W., Rajakumar, N., Pavlosky, W., Williamson, P., et al. (2007). Evidence for cortical dysfunction in autism: A proton magnetic resonance spectroscopic imaging study. Biological Psychiatry, 61(4), 465–473. Fatemi, S. H. (2008). The hyperglutamatergic hypothesis of autism. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 32(3), 911–913. Fatemi, S. H., Halt, A. R., Stary, J. M., Kanodia, R., Schulz, S. C., & Realmuto, G. R. (2002). Glutamic acid decarboxylase 65 and 67 kDa proteins are reduced in autistic parietal and cerebellar cortices. Biological Psychiatry, 52(8), 805–810. Friedman, S. D., Shaw, D. W., Artru, A. A., Dawson, G., Petropoulos, H., & Dager, S. R. (2006). Gray and white matter brain chemistry in young children with autism. Archives of General Psychiatry, 63, 786–794. Hardan, A. Y., Minshew, N. J., Melhem, N. M., Srihari, S., Jo, B., Bansal, R., et al. (2008). An MRI and proton spectroscopy study of the thalamus in children with autism. Psychiatry Research, 163(2), 97–105. Hosoya, K., Sugawara, M., Asaba, H., & Terasaki, T. (1999). Blood-brain barrier produces significant efflux of L-aspartic acid but not D-aspartic acid: In vivo evidence using the brain efflux index method. Journal of Neurochemistry, 73(3), 1206–1211. Jamain, S., Betancur, C., Quach, H., Philippe, A., Fellous, M., Giros, B., et al. (2002). Paris Autism Research International Sibpair S. Linkage and association of the glutamate receptor 6 gene with autism. Molecular Psychiatry, 7(3), 302–310. Lam, K. S., Aman, M. G., & Arnold, L. E. (2006). Neurochemical correlates of autistic disorder: A review of the literature. Research in Developmental Disabilities, 27(3), 254–289. Laurence, J. A., & Fatemi, S. H. (2005). Glial fibrillary acidic protein is elevated in superior frontal, parietal and cerebellar cortices of autistic subjects. Cerebellum, 4(3), 206–210. Levy, S. E., Mandell, D. S., & Schultz, R. T. (2009). Autism. Lancet, 374(9701), 1627–1638. McGale, E. H., Pye, I. F., Stonier, C., Hutchinson, E. C., & Aber, G. M. (1977). Studies of the inter-relationship between cerebrospinal fluid and plasma amino acid concentrations in normal individuals. Journal of Neurochemistry, 29(2), 291–297. Moreno, H., Borjas, L., Arrieta, A., Sae, L., Prassad, A., Este´vez, J., et al. (1992). Heterogeneidad clı´nica del sı´ndrome autista: Un estudio en sesenta familias. Investigacio´n Clinica, 33(1), 13–31. Moreno-Fuenmayor, H., Borjas, L., Arrieta, A., Valera, V., & Socorro-Candanoza, L. (1996). Plasma excitatory amino acids in autism. Investigation Clinical Journal, 37(2), 113–128. Mundy, P. (2003). Annotation: The neural basis of social impairments in autism: The role of the dorsal medial-frontal cortex and anterior cingulate system. Journal of Child Psychology and Psychiatry, 44(6), 793–809. O’Neill, J., Levitt, J., McCracken, J., Toga, A., & Alger, J. (2003). Anterior cingulate and amygdalar 1H MRS abnormalities in childhood autism. Proceedings of the International Society for Magnetic Resonance in Medicine, 11, 338. Ortinski, P. I., Dong, J., Mungenast, A., Yue, C., Takano, H., Watson, D., et al. (2010). Selective induction of astrocytic gliosis generates deficits in neuronal inhibition. Nature Neuroscience, 13(5), 584–591. Packard, M. G., & Knowlton, B. J. (2002). Learning and memory functions of the basal ganglia. Annual Review of Neuroscience, 25, 563–593. Page, L. A., Daly, E., Schmitz, N., Simmons, A., Toal, F., Deeley, Q., et al. (2006). In vivo 1H-magnetic resonance spectroscopy study of amygdala-hippocampal and parietal regions in autism. American Journal of Psychiatry, 163(12), 2189–2192. Pais, T. F., Figueiredo, C., Peixoto, R., Braz, M. H., & Chatterjee, S. (2008). Necrotic neurons enhance microglial neurotoxicity through induction of glutaminase by a MyD88-dependent pathway. Journal of Neuroinflammation, 5, 43. Purcell, A. E., Jeon, O. H., Zimmerman, A. W., Blue, M. E., & Pevsner, J. (2001). Postmortem brain abnormalities of the glutamate neurotransmitter system in autism. Neurology, 57(9), 1618–1628. Ramoz, N., Reichert, J. G., Smith, C. J., Silverman, J. M., Bespalova, I. N., Davis, K. L., et al. (2004). Linkage and association of the mitochondrial aspartate/glutamate carrier SLC25A12 gene with autism. American Journal of Psychiatry, 161(4), 662–669. Rolf, L. H., Grotemeyer, K. H., Haarman, F. Y., & Kehren, H. (1993). Serotonin and amino acid content in platelets of autistic children. Acta Psychiatrica Scandinavica, 87(5), 312–316. Serajee, F. J., Zhong, H., Nabi, R., & Huq, A. H. (2003). The metabotropic glutamate receptor 8 gene at 7q31: Partial duplication and possible association with autism. Journal of Medical Genetics, 40(4), e42.

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T.H. Hassan et al. / Research in Autism Spectrum Disorders 7 (2013) 541–548

Sheldon, A. L., & Robinson, M. B. (2007). The role of glutamate transporters in neurodegenerative diseases and potential opportunities for intervention. Neurochemistry International, 51(6–7), 333–355. Shimmura, C., Suda, S., Tsuchiya, K. J., Hashimoto, K., Ohno, K., Matsuzaki, H., et al. (2011). Alteration of plasma glutamate and glutamine levels in children with high-functioning autism. Public Library of Science ONE, 6(10), e25340. Shinohe, A., Hashimoto, K., Nakamura, K., Tsujii, M., Iwata, Y., Tsuchiya, K. J., et al. (2006). Increased serum levels of glutamate in adult patients with autism. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 30(8), 1472–1477. Shuang, M., Liu, J., Jia, M. X., Yang, J. Z., Wu, S. P., Gong, X. H., et al. (2004). Family-based association study between autism and glutamate receptor 6 gene in Chinese Han trios. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics, 131B(1), 48–50. Vargas, D. L., Nascimbene, C., Krishnan, C., Zimmerman, A. W., & Pardo, C. A. (2005). Neuroglial activation and neuroinflammation in the brain of patients with autism. Annals of Neurology, 57(1), 67–81. Williams, D. L., & Minshew, N. J. (2007). Understanding autism and related disorders: What has imaging taught us? Neuroimaging Clinics of North America, 17(4), 495–509.