Oxidative Stress and Immune Cytokines in Plasma of Young Children with Autism Spectrum Disorder and Recent Language and/or Social Regression: A Prospective Case-Control Study
Background: Regression occurs in 22%-41% of children with Autism Spectrum Disorder (ASD) at the average age of 20-23 months. At present, no clear contributors to regression have been identified. Increased levels of several immune cytokines have been reported in the subgroup of regressive autism, but no studies have examined the role of oxidative stress in this process.
Objectives: To compare biomarkers of oxidative stress and immune cytokines in children with ASD, with and without regression.
Methods: Three sites in the Autism Speaks Autism Treatment Network: Toronto, ON; Baltimore, MD; and Little Rock, AS, recruited children, aged 18-42 months with ASD, with regression (ASD-R) (n=25), and without regression (ASD-NR) (n=24). The two groups were compared with regards to oxidative stress markers (free reduced glutathione, oxidized glutathione, homocysteine, cysteine, 3-nitrotyrosine and 3-chlorotyrosine) and immune cytokines (interleukin 1, macrophage chemoattractant protein 1, macrophage inflammatory protein 1A, interferon gamma, TNF alpha, and CD 40L). Regression was defined as having established a skill for at least one month, followed by a significant loss of that skill (more than 90% for language, and 75% for social) for more than 1 month. Regression was operationalized using a modified Regression Validation Interview and inter rater reliability for the assessment of “definite” or “no” regression was 89%. The ASD-R group completed a standardized medical investigation including screening for metabolic and genetic disorders. Children met criteria for an ASD according to the Autism Diagnostic Observation Schedule and the DSM-IV.
Results: The mean ages of the ASD-R and ASD-NR groups were similar, 2.5 and 2.6 years respectively. The mean age of regression in the ASD-R group was 1.9 ± 0.4 years and the duration from when regression was definitively noted to plasma analysis was 8.0 ± 4.5 months.
The ASD-R group had a significantly lower mean free reduced glutathione level (1.71 ± 0.13) compared to the ASD-NR group (1.86 ± 0.20) (p=0.006).
There was also an increase in a marker for protein oxidative damage, 3-nitrotyrosine (p=0.04) in the ASD-R (74.19 ± 17.14) compared to the ASD-NR (64.78 ± 12.30) group.
There were no differences in other oxidative stress markers or immune cytokines between groups.
Conclusions: In this pilot study, ASD and recent regression in young children was associated with decreased free glutathione and increased 3-nitrotyrosine, supporting evidence of decreased antioxidant potential and increased oxidative damage in plasma, compared to young children with ASD and no regression.
There were no group differences in other biomarkers of oxidative stress. Lower free glutathione levels may be a risk factor for regression in a subgroup of children with ASD and regression.
This study was limited by a small sample size and the analysis of oxidative stress biomarkers and cytokine levels was limited to plasma. Future studies should examine oxidative stress biomarkers in the plasma and cerebrospinal fluid in young children with ASD and recent regression to assess for possible differences in oxidative stress and damage in the central nervous system.
Further Readings of Interest
Glutathione has multiple functions:
- It is the major endogenous antioxidant produced by the cells, participating directly in the neutralization of free radicals and reactive oxygen compounds, as well as maintaining exogenous antioxidants such as vitamins C and E in their reduced (active) forms.
- Regulation of the nitric oxide cycle, which is critical for life but can be problematic if unregulated
- It is used in metabolic and biochemical reactions such as DNA synthesis and repair, protein synthesis, prostaglandin synthesis, amino acid transport, and enzyme activation. Thus, every system in the body can be affected by the state of the glutathione system, especially the immune system, the nervous system, the gastrointestinal system and the lungs.
- It has a vital function in iron metabolism. Yeast cells depleted of or containing toxic levels of GSH show an intense iron starvation-like response and impairment of the activity of extra-mitochondrial ISC enzymes, followed by death.
Nitrotyrosine is a product of tyrosine nitration mediated by reactive nitrogen species such as peroxynitrite anion and nitrogen dioxide. Nitrotyrosine is identified as an indicator or marker of cell damage, inflammation as well as NO (nitric oxide) production. Nitrotyrosine is ifs formed in the presence of the active metabolite NO. Generally in many disease states, oxidative stress increases the production of superoxide (O2–) and NO forming ONOO– (peroxy nitrite, which could lead to the formation of pro-xidant peroxynitrite (ONOO–)  The production of ONOO– is capable of oxidizing several lipoproteins and of nitrating tyrosine residues in many proteins. It is difficult to determine the production of ONOO– so, usually nitrotyrosine in proteins are the detectable marker for indirectly detecting ONOO–. It is detected in large number of pathological conditions and is considered a marker of NO-dependent, reactive nitrogen species-induced nitrative stress. Nitrotyrosine is detected in biological fluids such as plasma, lung aspirants-BALF (Broncho alveolar lining fluid and urine. Increased level of nitrotyrosine is detected in rheumatoid arthritis  septic shock and coeliac disease. In all these studies nitrotyrosine was undetected in healthy subjects. Nitrotyrosine is also found in numerous other disease-affected tissues, such as the cornea in keratoconus. Peroxynitrite and/or nitrative stress may participate in the pathogenesis of diabetes