Objectives: To determine whether intracellular glutathione-mediated antioxidant capacity in resting and activated peripheral blood mononuclear cells (PBMC) differs between children with autism and age-matched control children and whether decreased antioxidant capacity is accompanied by elevated ROS production in primary monocytes and lymphocytes.
Methods: Subjects included 80 children, ages 3-10, who were participants in the autism IMAGE study (Integrated Metabolic And Genomic Endeavor) at Arkansas Children’s Hospital Research Institute. The IMAGE cohort consisted of 40 children with autism, 16 unaffected siblings and 24 unaffected control children. Children with autistic disorder were diagnosed using DSM-IV (299.0), ADOS and/or CARS >30. PBMC were isolated from whole blood using standard density gradient centrifugation. PBMC were left untreated or were stimulated for 4 hr with 0.1 µg/mL LPS or 10 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 µg/mL ionomycin. Monocytes and CD4+ T-cells were then isolated from the stimulated cells using magnetic bead based technology. Glutathione (GSH) was measured using HPLC elution and electrochemical detection. ROS production was determined by flow cytometric measures of median fluorescence intensity of cells pre-loaded with DCFDA, a membrane permeable probe that fluoresces when oxidized by intracellular free radicals.
Results: Resting PBMC from children with autism exhibited a significantly higher concentration of oxidized glutathione (GSSG) compared to age-matched control children. Similarly, resting PBMC and activated monocytes isolated from children with autism exhibited significantly higher percent oxidized glutathione equivalents as well as a significant decrease in the intracellular redox ratio (GSH/GSSG) compared to age-matched control children. Activated CD4+ T-cells isolated from children with autism exhibited a significant decrease in intracellular GSH and GSH/GSSG redox ratio as well as an increase in oxidized glutathione equivalents. Consistent with reduced antioxidant capacity, resting lymphocytes from children with autism produced significantly more intracellular ROS compared to lymphocytes from control children.
Conclusions: Preliminary data indicate that primary immune cells from children with autism have a more oxidized intracellular microenvironment and a decrease in glutathione-mediated antioxidant capacity compared to age-matched unaffected control children.
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