Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders characterized by social and language deficits, stereotypic behavior, and abnormalities in motor functions. The particular set of behavioral impairments expressed in any given individual is variable across the spectrum. These behavioral abnormalities are consistent with our current understanding of the neuropathology of ASD which suggests abnormalities in the amygdala, temporal and frontal cortexes, hippocampus, and cerebellum. However, regions unrelated to these behavioral deficits appear largely intact. Both genetic predisposition and environmental toxins and toxicants have been implicated in the etiology of autism; the impact of these environmental triggers is associated with increases in oxidative stress, and is further exacerbated when combined with genetic susceptibility. We have previously reported increased levels of 3-nitrotyrosine (3-NT), a marker of oxidative stress, in ASD cerebella. We have also shown that this increase was associated with an elevation in neurotrophin-3 (NT-3) levels. The objectives of the current study were to determine whether the increase in oxidative stress in ASD is brain region-specific, to identify the specific brain regions affected by oxidative stress, and to compare brain region-specific NT-3 expression between ASD and control cases. The levels of 3-NT and NT-3 were measured with specific ELISAs in individual brain regions of two autistic and age- and postmortem interval (PMI)--matched control donors. In the control brain, the levels of 3-NT were uniformly low in all brain regions examined ranging from 1.6 to 12.0 pmol/g. On the other hand, there was a great variation in 3-NT levels between individual brain regions of the autistic brains ranging from 1.7 to 281.2 pmol/g. The particular brain regions with the increased 3-NT and the magnitude of the increase were both different in the two autistic cases. In the older autistic case, the brain regions with highest levels of 3-NT included the orbitofrontal cortex (214.5 pmol/g), Wernicke's area (171.7 pmol/g), cerebellar vermis (81.2 pmol/g), cerebellar hemisphere (37.2 pmol/g), and pons (13.6 pmol/g); these brain areas are associated with the speech processing, sensory and motor coordination, emotional and social behavior, and memory. Brain regions that showed 3-NT increase in both autistic cases included the cerebellar hemispheres and putamen. Consistent with our earlier report, we found an increase in NT-3 levels in the cerebellar hemisphere in both autistic cases. We also detected an increase in NT-3 level in the dorsolateral prefrontal cortex (BA46) in the older autistic case and in the Wernicke's area and cingulate gyrus in the younger case. These preliminary results reveal, for the first time, brain region-specific changes in oxidative stress marker 3-NT and neurotrophin-3 levels in ASD.

It has been suggested that oxidative stress and/or mercury compounds play an important role in the pathophysiology of autism. This study compared for the first time the cerebellar levels of the oxidative stress marker 3-nitrotyrosine (3-NT), mercury (Hg) and the antioxidant selenium (Se) levels between control and autistic subjects. Tissue homogenates were prepared in the presence of protease inhibitors from the frozen cerebellar tissue of control (n=10; mean age, 15.5 years; mean PMI, 15.5 hours) and autistic (n=9; mean age 12.1 years; mean PMI, 19.3 hours) subjects. The concentration of cerebellar 3-NT, determined by ELISA, in controls ranged from 13.69 to 49.04 pmol g 1 of tissue; the concentration of 3-NT in autistic cases ranged from 3.91 to 333.03 pmol g 1 of tissue. Mean cerebellar 3-NT was elevated in autism by 68.9% and the increase was statistically significant (p=0.045). Cerebellar Hg, measured by atomic absorption spectrometry ranged from 0.9 to 35 pmol g 1 tissue in controls (n=10) and from 3.2 to 80.7 pmol g 1 tissue in autistic cases (n=9); the 68.2% increase in cerebellar Hg was not statistically significant. However, there was a positive correlation between cerebellar 3-NT and Hg levels (r=0.7961, p=0.0001). A small decrease in cerebellar Se levels in autism, measured by atomic absorption spectroscopy, was not statistically significant but was accompanied by a 42.9% reduction in the molar ratio of Se to Hg in the autistic cerebellum. While preliminary, the results of the present study add elevated oxidative stress markers in brain to the growing body of data reflecting greater oxidative stress in autism.

Abstract:Oxidative damage has been documented in the peripheral tissues of autism patients. In this study, we sought evidence of oxidative injury in autistic brain. Carboxyethyl pyrrole (CEP) and iso [4]levuglandin (iso[4]LG)E 2 -protein adducts, that are uniquely generated through peroxidation of docosahexaenoate and arachidonate-containing lipids respectively, and heme oxygenase-1 were detected immunocytochemically in cortical brain tissues and by ELISA in blood plasma. Significant immunoreactivity toward all three of these markers of oxidative damage in the white matter and often extending well into the grey matter of axons was found in every case of autism examined. This striking threadlike pattern appears to be a hallmark of the autistic brain as it was not seen in any control brain, young or aged, used as controls for the oxidative assays. Western blot and immunoprecipitation analysis confirmed neurofilament heavy chain to be a major target of CEP-modification. In contrast, in plasma from 27 autism spectrum disorder patients and 11 age-matched healthy controls we found similar levels of plasma CEP (124.5 ± 57.9 versus 110.4 ± 30.3 pmol/mL), iso [4]LGE 2 protein adducts (16.7 ± 5.8 versus 13.4 ± 3.4 nmol/mL), anti-CEP (1.2 ± 0.7 versus 1.2 ± 0.3) and anti-iso [4]LGE 2 autoantibody titre (1.3 ± 1.6 versus 1.0 ± 0.9), and no differences between the ratio of NO 2 Tyr/Tyr (7.81 E-06 ± 3.29 E-06 versus 7.87 E-06 ± 1.62 E-06). These findings provide the first direct evidence of increased oxidative stress in the autistic brain. It seems likely that oxidative injury of proteins in the brain would be associated with neurological abnormalities and provide a cellular basis at the root of autism spectrum disorders.

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder whose etiology is poorly understood. Recent studies have shown that autistic children may be experiencing increased inflammation and oxidative stress. Altered immune regulation may be one contributing factor to inflammation and oxidative stress in autistic children. Sulfur amino acid (SAA) metabolism plays a critical role in regulating blood leukocyte functions and oxidative stress. However, it is not known whether autism impacts SAA metabolism in peripheral immune cells. To address this question, a novel liquid chromatography linked tandem mass spectrometric (LC/MS/MS) method was used to determine the levels of SAA metabolites in peripheral blood mononuclear cells obtained from 11 healthy controls and 31 autistic children. Improved detection sensitivity and selectivity of the LC/MS/MS method allowed accurate quantification using small samples. Results show that leukocytes from autistic children contained significantly lower concentrations of S-adenosylmethionine (-35%; p = 0.01), and elevated levels of intracellular homocysteine content (+80%; p=0.003). Additionally, the levels of intracellular total cysteine and glutathione (GSH) were reduced by 39% (p=0.004) and 25% (p=0.01), respectively. These autism-associated changes were leukocyte specific in that no significant alterations in SAA metabolite concentrations were detected in the plasma samples. Our results provide novel evidence for altered metabolism in immune cells; furthermore, this data suggest the involvement of inflammation in autism. Dietary differences between controls and patients, however, remain a potential confounder.

Background: Autism is a neurologic disorder characterized by impaired communication and social interaction. Results of previous studies showed biochemical evidence for abnormal platelet reactivity and altered blood flow in children with autism. Objective: To evaluate the vascular phenotype in children with autism. Design and Main Outcome Measures: Urinary levels of isoprostane F 2␣-VI, a marker of lipid peroxidation; 2,3-dinor-thromboxane B 2 , which reflects platelet activation; and 6-keto-prostaglandin F 1␣ , a marker of endothelium activation, were measured by means of gas chromatography-mass spectrometry in subjects with autism and healthy control subjects. Setting and Subjects: Children with a clinical diagnosis of autism attending the Pfeiffer Treatment Center. Results: Compared with controls, children with autism had significantly higher urinary levels of isoprostane F 2␣-VI, 2,3-dinor-thromboxane B 2 , and 6-ketoprostaglandin F 1␣. Lipid peroxidation levels directly correlated with both vascular biomarker ratios. Conclusion: Besides enhanced oxidative stress, platelet and vascular endothelium activation also could contribute to the development and clinical manifestations of autism.