Review Article
Oxidative stress-related biomarkers in autism: Systematic review and meta-analyses

https://doi.org/10.1016/j.freeradbiomed.2012.03.011Get rights and content

Abstract

Autism spectrum disorders (ASDs) are rarely diagnosed in children younger than 2 years, because diagnosis is based entirely on behavioral tests. Oxidative damage may play a central role in this pathogenesis, together with the interconnected transmethylation cycle and transsulfuration pathway. In an attempt to clarify and quantify the relationship between oxidative stress-related blood biomarkers and ASDs, a systematic literature review was carried out. For each identified study, mean biomarker levels were compared in cases and controls providing a point estimate, the mean ratio, for each biomarker. After meta-analysis, the ASD patients showed decreased blood levels of reduced glutathione (27%), glutathione peroxidase (18%), methionine (13%), and cysteine (14%) and increased concentrations of oxidized glutathione (45%) relative to controls, whereas superoxide dismutase, homocysteine, and cystathionine showed no association with ASDs. For the C677T allele in the methylene tetrahydrofolate reductase gene (MTHFR), homozygous mutant subjects (TT) showed a meta-OR of 2.26 (95% CI 1.30–3.91) of being affected by ASD with respect to the homozygous nonmutant (CC). Case-control studies on blood levels of vitamins suggest a lack of association (folic acid and vitamin B12) or rare association (vitamins A, B6, C, D, E). Sparse results were available for other biomarkers (ceruloplasmin, catalase, cysteinylglycine, thiobarbituric acid-reactive substances, nitric oxide) and for polymorphisms in other genes. Existing evidence is heterogeneous and many studies are limited by small sample size and effects. In conclusion, existing evidence suggests a role for glutathione metabolism, the transmethylation cycle, and the transsulfuration pathway, although these findings should be interpreted with caution, and larger, more standardized studies are warranted.

Highlights

► We reviewed studies on biomarkers related to oxidative stress in autism. ► Glutathione (GSH), transmethylation, and transsulfuration pathways were altered. ► Blood GSH, glutathione peroxidase, methionine, and cysteine decreased, whereas blood oxidized glutathione increased. ► No association with blood vitamin levels was found. ► Carriers of the mutant C677T allele in the MTHFR gene had higher risk of autism.

Introduction

Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by varying degrees of dysfunctional communication and social abilities and repetitive and stereotypic behaviors. The spectrum of ASDs encompasses autistic disorder (AD), Asperger syndrome (AS), and pervasive developmental disorder not otherwise specified (PDD-NOS) [1]. ASDs are clinically diagnosed in early childhood, with a male-to-female ratio of 4:1. The prevalence has been increasing over the past 2 decades, and the current estimate is 1/110 [2] in the United States. No effective cure is available for autism, as most behavioral or pharmacological treatments are unable to improve ASD core symptoms [3], [4], [5]. ASDs are rarely diagnosed in children younger than 2 years of age because diagnosis is based on behavioral tests designed for older children. Early recognition of ASD symptoms would help affected children to develop their adaptation skills, allowing a better social integration, and eventually lead to a lower level of handicap. Encouraging results have been reported from studies using most advanced techniques of imaging [6], although the interplay between genetic and environmental factors is still the most likely etiologic mechanism underlying autistic symptoms.

Several attempts have been made to validate biomarkers for screening purposes, but as yet none have been found to unequivocally predict ASDs. Important results are expected from prospective studies such as the Autism Birth Cohort, which combines genetic, proteomic, immunologic, metagenomic, and microbiological endpoints to exploit serial biological samples in a “nested case-control” design [7]. Another prospective study, namely the “1-Year Well-Baby Check-up Approach,” investigates multiple endpoints in ASD patients (early brain overgrowth, cerebellar functions, gene expression, and immune system functionality), to attempt to identify early markers of disease [8].

Several etiologic hypotheses have been proposed so far, including genetic susceptibility, immunologic alterations, and environmental exposures [9]. The increasing prevalence of ASDs has been questioned based on advances in detection procedures, increased public awareness, and the broadening of the diagnostic criteria [10], but most of the increase in incidence seems to be real [11]. The role of environmental factors in the etiology of ASDs is supported by extensive literature [12]. Exposure to heavy metals and xenobiotics is a feature of contemporary life and it may also contribute to neurodegenerative disorders, including Parkinson and Alzheimer diseases [13], [14], indicating that the human brain is an especially sensitive target. Most of these agents directly or indirectly influence cellular redox status and the associated pathways of sulfur metabolism by promoting cellular oxidative stress in vulnerable individuals and initiating adaptive responses that include reduced methylation activity [15], [16]. Methylation has an important role in the synthesis of myelin basic protein, an essential component that confers compactness to myelin. This is a critical step because the correct synthesis and assembling of myelin are fundamental in the development of the central nervous system [17], [18]. In addition, decreased DNA methylation increases expression of genes under the negative influence of methylation, disrupting epigenetic silencing of chromosomal regions linked to ASDs and leading to developmental delay, deficit in attention, and neuronal synchronization, which are typical hallmarks of autism [16], [19].

Oxidative stress modifies the normal intracellular balance between reactive oxygen species (ROS) produced during aerobic metabolism and antioxidant defense mechanisms, which perform the function of free radical inactivation. Both enzymatic and nonenzymatic protective mechanisms exist. Enzymatic systems include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and ceruloplasmin with their bound metallic elements. Genetic polymorphisms of these and other enzymes have been the object of several studies [20], [21].

Nonenzymatic mechanisms include glutathione (GSH) and its precursors (the limiting amino acid cysteine plus cysteinylglycine, cystathionine, and homocysteine in the transsulfuration pathway, and methionine in the transmethylation cycle), antioxidant nutrients (vitamins E, C, A, B6; folate), and the phenolic compounds [22]. Oxidative stress can also trigger or enhance mast-cell activation, which induces the secretion of numerous vasoactive, neurosensitizing, and proinflammatory molecules relevant to ASDs, including bradykinin, histamine, IL-6, nitric oxide, tryptase, vascular endothelial growth factor, and VIP [23]. These molecules disrupt the gut–blood–brain barrier, permitting enterotoxic molecules to enter the brain and induce neuroinflammation [9].

Oxidative stress may play a central role in the pathogenesis of ASDs as a result of the cumulative influence of toxic environmental insults, which can promote neuronal damage in genetically predisposed individuals. The mammalian brain is especially sensitive to oxidative damage, because it accounts for only a few percent of body weight, whereas it is responsible for about 20% of basal O2 consumption. The discrepancy is even more striking in young children, who have much smaller bodies but not proportionately smaller brains. The role of oxidative stress in neurological dysfunction has been reviewed by Halliwell [24], and various animal studies have been carried out to assess the effect of oxidative stress on the central nervous system in animal models [25], [26], [27].

The involvement of an imbalance between oxidant and antioxidant systems in disease pathophysiology has been proposed also for other mental disorders, including schizophrenia and bipolar disorder [28], [29], [30], and neurodegenerative pathologies, e.g., Alzheimer disease [31] There is increasing evidence that autistic patients present excessive ROS production and reduced methylation capacity. Brain tissues from autistic cases showed higher levels of oxidative stress biomarkers than healthy controls in postmortem analysis [32], [33], [34], [35], [36].

Moreover, mitochondrial abnormalities – a potential source of elevated oxidative stress – were reported in autistic case studies and have been recently reevaluated through meta-analysis [37].

It is likely that the action of oxidative stress has extensive and profound consequences in the developing central nervous system.

The association between oxidative damage and autism has been investigated in several studies with various biomarkers, sometimes with conflicting results [38], [39]. Both markers of oxidative stress and markers of systemic antioxidant capacity have been extensively studied, including reduced and oxidized glutathione with their precursors, amino acids, vitamins, thiobarbituric acid-reactive substances, and oxidoreductases with their genetic polymorphisms.

To clarify and quantify the relationship between oxidative stress-related biomarkers and ASDs in humans, we carried out a systematic review and meta-analysis of the published literature in this area of research.

Section snippets

Bibliographic search

Identification of the studies was carried out through an extensive literature search using the PubMed database (National Library of Medicine, National Institutes of Health, Bethesda, MD, USA; http://www.ncbi.nih.gov/PubMed) mainly based on specific keywords (mesh terms [mesh]), without any language restriction, and was updated to May 6, 2011.

The first search strategy included the terms Autistic Disorder[mesh] AND (Antioxidants[mesh] OR Oxidoreductases[mesh] OR Vitamins[Pharmacological Action]

Study characteristics

Twenty-nine articles reporting data on antioxidant biomarkers levels in blood of autistic patients in comparison to healthy controls fulfilled selection criteria and are illustrated in detail in Table 1.

Most identified papers were published after 2002. Twelve studies were conducted in the United States, 8 were from Europe, 4 were from Asia, 3 were from Africa, and 1 was from South America. The study size was always rather small, with the number of cases ranging from 16 [71] to 80 [60] and the

Discussion

This study addresses an important area in autism research. A role for oxidative stress in the etiology of this disease has been hypothesized for decades and several biomarkers have been the focus of clinical studies, by comparing levels in the blood of ASD patients to those of healthy controls. The reviewed data from the scientific community and the results of these meta-analyses point to a role for the transmethylation and transsulfuration pathways and glutathione metabolism, although evidence

Acknowledgments

We thank Jill James (Little Rock, AR, USA) and Pierandrea Muglia (Ballerup, Denmark), for helpful discussion and valuable suggestions; Marcello Ceppi (Genoa, Italy), for his help with meta-analysis techniques; and Lucio Da Ros (Verona, Italy), for making this work possible.

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    These authors contributed equally to this work.

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