1. Introduction
Autism spectrum disorder (ASD) is a neurodevelopmental disorder defined by the Diagnostic and Statistical Manual of Mental Disorders, fifth edition (DSM-5) as observable behavioral deficits in social communication and social interaction across multiple contexts. Oftentimes, patients with ASD present with difficulties relating to others in the form of repetitive patterns of behaviors and limited interests. Although ASD can present at any age, the disorder usually manifests early in childhood and can be reliably diagnosed by age 2 [
1]. DSM-5 (
Appendix A) replaced and folded the DSM-IV subtypes of Pervasive Developmental Disorder (PDD), Autistic Disorder, Asperger’s Disorder, Childhood Disintegrative Disorder under a single umbrella term of ASD. According to the most recent data from the CDC’s Autism and Developmental Disabilities Monitoring (ADDM) network published in December 2021, an average of 1 in every 44 (2.3%) 8-year-old children were estimated to have ASD in 2018 [
2]. Additionally, ASD is 4.2 times as prevalent among boys (3.7%) compared to girls (0.9%) [
2].
The cause of ASD is currently unknown and involves a mix of genetics and environmental factors. Based on the current literature, ASD involves many complex biological processes that manifest as physiologic and metabolic abnormalities. Namely, ASD has been reported to be in association with impaired neurotransmission, immune dysregulation in the brain, mitochondrial dysfunction, and increased oxidative stress [
3,
4,
5].
The neurodevelopmental nature of ASD is partially explained by the pathophysiologic imbalance of neurochemical signaling in the central nervous system. ASD has been observed to be associated with changes to neurotransmission involving gamma aminobutyric acid (GABA), glutamate, serotonin, dopamine, acetylcholine, N-acetyl aspartate, and endogenous opioids, amongst others [
6]. Changes to the production, degradation, and response pathways of these neurotransmitters may affect cellular functions including differentiation, migration, and apoptosis.
There is growing evidence that supports a role for immune dysregulation in the brain in ASD. Neurobiological studies on children with ASD have demonstrated an excess in neuronal synapses in the cerebral cortex. An impairment in synaptic pruning is observed to lead to social and behavioral dysregulation, as is seen in ASD. On an immunologic level, brain microglia are crucial in the synaptic refinement process [
7]. Specifically, studies have shown that the loss of mTOR-dependent autophagy pathways impairs synaptic pruning which further underscores immune dysregulation as a contributor to the development of ASD [
8,
9].
Synaptic transmission is an energy intensive process that requires large amounts of ATP produced by mitochondria. The synthesis of proteins and dendrites for synaptic plasticity is also another energy intensive process. As such, functional mitochondria are crucial to neurodevelopment. An early study in 1985 cited lactic acidosis in a subset of children with ASD, which further paved the way to explore the association between mitochondrial dysfunction in ASD [
10]. Current studies on mitochondrial dysfunction hint at enzymatic dysregulation in the tricarboxylic acid cycle and the electron transport chain [
3,
4]. Mitochondria also play other roles in synaptic regulation including calcium buffering, the regulation of neurotransmitter release, and reduction in oxidative stress [
11]. Mitochondrial dysfunction is also noted to be associated with other ASD-like neurodevelopmental disorders including Down syndrome, DiGeorge syndrome, Rett syndrome, and tuberous sclerosis complex [
11,
12,
13,
14].
Lastly, an imbalance in oxidation–reduction (redox) reactions in cells favor pro-oxidant agents, such as free radicals, which promote oxidative stress. More specifically, the generation of ROS outweighs the body’s ability to remove them. In low concentrations, reactive oxygen species act as signaling molecules that induce apoptosis and dysregulates cell proliferation via changes to gene expression [
15]. On a molecular level, ROS can destroy polyunsaturated fatty acids that make up the cell membrane. Additionally, ROS can oxidize amino acids leading to DNA strand breakage and DNA protein crosslinking resulting in mutations. ROS damage is a positive feedback system whereby more ROS are released downstream, resulting in further cellular damage. The resulting pro-oxidative state and cytotoxicity causes brain inflammation, the disruption of the blood–brain barrier, edema, and presents as the phenotypic symptoms of ASD [
16,
17].
In order to protect itself, the body utilizes an antioxidant defense mechanism that includes both enzymatic and non-enzymatic ways to remove ROS. Enzymatic defense includes the biological action of superoxide dismutase, catalase, peroxiredoxins, glutathione peroxidase, and several enzymes in the ascorbate-glutathione pathway [
18]. Non-enzymatic defense includes vitamins (vitamins C and E), β-carotene, uric acid, and glutathione, among others [
19]. An emerging topic of research is the restoring of the redox balance between bodily oxidative systems and anti-oxidative responses. The literature focuses on the hypothesis that reducing oxidative burden improves symptoms and social functioning in patients with ASD.
One of the most studied antioxidants is glutathione due to its ubiquitous nature and its role as the most abundant non-protein thiol antioxidant in mammalian tissue. Glutathione is a naturally occurring cysteine-glutamate-glycine tripeptide that is synthesized primarily in the liver with significant antioxidative action. In addition to its key role in redox signaling, crucial in the detoxification of xenobiotics, glutathione is an important factor in regulating cell proliferation, apoptosis, immune function, and fibrogenesis [
20,
21]. Decreased levels of glutathione are also associated with other psychiatric conditions including bipolar disorder, major depressive disorder, and schizophrenia [
22]. Glutathione exists primarily in its thiol reduced form (GSH) accounting for >98% of total glutathione versus its disulfide-oxidized form (GSSG) [
20]. The antioxidant function of glutathione is mediated by glutathione peroxidase, which reduces ROS such as hydrogen peroxide and lipid peroxide, resulting in GSSG. GSH is regenerated via GSSG reductase and NADPH for further antioxidative action (
Figure 1). The synthesis of glutathione is primarily limited by the rate-limiting agent, cysteine. Cysteine can be derived in several ways including diet, protein breakdown and recycling in the liver, the transsulfuration of methionine (
Figure 2) [
20]. Another important molecule is N-acetylcysteine (NAC), which is an acetylated cysteine residue that can serve as a glutamatergic modulator, a free radical scavenger, and as a cysteine donor to maintain GSH status [
23].
NAC has been a target of interest primarily for its antioxidative role in a variety of disorders including neural cell survival, cell signaling, neurodegenerative diseases, multiple sclerosis, traumatic brain injuries, and other psychotic disorders [
24,
25]. Its antioxidant effects can be extrapolated to play a role in the redox balance in ASD. There have been recent placebo-controlled pilot studies that have looked at NAC in ASD patients directed toward the treatment of irritability, aggression, self-injurious behavior and tantrums [
26,
27]. Harden et al. found a significant reduction in irritability symptoms that were measured by the Aberrant Behavior Checklist in a placebo-controlled pilot trial of NAC in children with ASD [
28]. Another 12-week, randomized, double-blind, placebo-controlled trial of oral NAC in children with ASD found NAC to be safe. It was not noted to have a significant impact on core social impairment seen in ASD, but the authors did indicate that larger scaled trials are needed in order to predict a good treatment response [
26]. A meta-analysis by Lee et al. found that NAC supplementation alleviated hyperactivity and irritability in ASD, based on the Aberrant Behavior Checklist. More importantly, the study demonstrated the safety and tolerability of NAC supplementation in children with ASD [
29].
Further research into anti-oxidative agents for ASD begs the question of whether direct supplementation with glutathione is equally or more efficacious than NAC supplementation due to bypassing the rate-limiting transsulfuration step (cysteine) in glutathione synthesis. Despite its proposed benefits, oral glutathione supplementation failed to show changes to the observed biomarkers of oxidative stress and glutathione status, due to the enzymatic degradation of supplemental glutathione in the intestine [
30]. The issue of poor bioavailability was recently circumvented with the development and introduction of a novel glutathione that aids in gastrointestinal uptake [
31]. One such formulation, termed Opitac
TM glutathione, is derived from the fermentation of Torula yeast. When administered at 50 mg/kg body weight, the level of Opitac
TM glutathione in the protein-bound fraction of plasma significantly increased 60–120 min after supplementation and the level of glutathione and related compounds (γGlu-Cys and Cys-Gly) were sustained for at least 2 h [
32]. Other studies on the oral administration of glutathione also show significantly elevated body stores of glutathione in addition to improved markers of immune function [
31,
33]. This open-label pilot case series examines the use of Opitac
TM glutathione in the symptomatic treatment of ASD in children.
4. Discussion
Our pilot investigation examined the utility and tolerability of oral supplementation with glutathione as a treatment for patients with ASD. A similar study by Kern et al. looked at glutathione supplementation for the treatment of ASD. Kern et al. demonstrated the safety profile of oral glutathione supplementation and significant increases in plasma reduced glutathione using glutathione dosages significantly lower (ranging from 50–200 mg/30 lbs or 3.67–14.7 mg/kg) than those used in this pilot study; however, they did not demonstrate a statistically significant difference in the increased whole blood glutathione. As previously mentioned in the study design, patients with cystic fibrosis require ~65 mg/kg of glutathione for adequate absorption. Our study utilizes a more conservative approach of ~32.5 mg/kg of glutathione, which is still well above the range used by Kern et al. Even at higher glutathione doses, our study once again demonstrates a good safety profile for glutathione use.
The study by Kern et al. also showed statistically significant increases in plasma transsulfuration metabolites including sulfate, cysteine, and taurine following glutathione supplementation, but they were unable to delineate whether the increased metabolites were truly due to glutathione’s effect on the transsulfuration pathway or whether the increases in transsfulfuration metabolites were due to the increased breakdown of glutathione [
35]. Our case series redemonstrates an increase in transsulfuration metabolites with oral glutathione supplementation, but the changes are not statistically significant. Supplementation with oral glutathione may improve oxidative markers but does not necessarily translate to the observed clinical improvement of subjects with ASD.
Glutathione was generally well-tolerated except in the case of one subject, who experienced a significant increase in irritability and ultimately discontinued their participation in the study. Stomach upset was reported in four of the six subjects in the study as a side effect with oral glutathione treatment. Stomach upset and GI side effects may not necessarily be attributed to oral glutathione alone. With the exception of irritability, all other reported side effects with oral glutathione (
Table 6) did not persist and did not serve as a limitation to continuing treatment. Studies have shown that the prevalence of patients with ASD and associated gastrointestinal dysfunction ranges from 9–91% [
36,
37]. Children with ASD and anxiety seem to have greater risk for lower GI issues, suggesting an exaggerated stress response. Additionally, it is suggested that a subset of children with ASD have automatic nervous system dysfunction leading to greater GI instability [
37]. The GI symptoms of abdominal pain, gas, diarrhea, and constipation are associated with a greater risk for worsened ASD symptoms including withdrawal, irritability, and hyperactivity [
38]. Recent studies have demonstrated that the dysbiosis of gut microbiota can lead to distinct autistic phenotypes ranging from mild irritability to extreme behavioral concerns in ASD [
37,
39]. Certain bacteria and viruses can alter gut microbiome by producing ROS and triggering inflammatory pathways. Higher levels of nitrites among other biomarkers such as short-chain fatty acids, lipopolysaccharides, beta-cresol, and bacterial toxins in children with ASD supports the theory that ASD is a brain–gut–microbiome disorder [
39,
40]. There is a major research focus on protecting the gut microbiome as a treatment for ASD.
The baseline oxidative stress analysis was crucial in understanding the redox imbalance in the patients with ASD. In the case of the subject that dropped out of the study, the pre-treatment laboratory work shows a baseline depletion of the glutathione reserve, high levels of superoxide dismutase despite normal glutathione peroxidase, and elevated lipid peroxides. Another subject shows baseline low levels of antioxidant capacity, significantly elevated glutathione peroxidase, and significantly elevated superoxide dismutase. Both subjects had high oxidative burdens prior to treatment and did not seem to experience any clinical or redox benefit with oral glutathione supplementation. A possible explanation for this phenomenon is that these subjects are at maximum physiologic compensation with a high baseline oxidative burden. The further addition of glutathione is unable to surpass the physiologic cap on the redox balance. The results may indicate that subjects with high baseline oxidative burden may be poor responders to oral glutathione supplementation. All other subjects showed improved total antioxidant capacity, increased sulfate levels, and significant increases in glutathione peroxidase and superoxide dismutase. Another observation is that oxidative burden does not necessarily translate to clinical severity, as indicated via the CGI scores.
The clinical progress in this study was monitored via ABC. There were decreases in the post-treatment mean scores across all ABC domains as compared to the pre-treatment scores, but the mean differences were not statistically significant. However, it is important to note that despite the lack of statistical significance, there was a mild improvement in the severity of ASD symptoms in 66.7% of the patients, according to the CGI-I. This translates to an observable clinical significance and improvement in the quality of life of some patients with ASD.
As referenced, an imbalance in redox reactions is only one of the many factors that contribute to ASD. Impaired neurotransmission, immune dysregulation in the brain, mitochondrial dysfunction, and a mix of environmental factors may also contribute to treatment response. A recent study by Huang et al. demonstrates the critical role of glutathione in astrocytes to help maintain blood–brain barrier stability by suppressing endothelial cell tight junction phosphorylation and delocalization [
41]. The maintenance of blood–brain barrier homeostasis prevents the intrusion of ROS, neurotoxic debris, and inflammatory cytokines from disrupting the brain parenchyma.
Given the association between ASD and gastrointestinal abnormalities, food picking, and food aversion, many individuals with ASD have concurrent deficiencies in macro and micronutrients. The most well-studied agents are vitamin A, vitamin B
1 (thiamine), vitamin B
6 (pyridoxine), and vitamin B
12 (cobalamin). Vitamin B
1 plays a role in multiple systems including the regulation of apoptosis in response to oxidative stress [
19]. Vitamin B
1 deficiency is associated with delayed language development in childhood [
42]. Vitamin A plays a role in multiple systems including the regulation of apoptosis, neurogenesis, immune health, and serotonin systems [
19]. Supplementation with vitamin A has been shown to have a positive effect on social memory, communication, and coordination [
43]. Vitamin B
6 is an important coenzyme required for the general metabolic maintenance of cells including the degradation of amino acids and the synthesis of many neurotransmitters including dopamine, GABA, serotonin, noradrenaline, and histamine, among others [
19]. Supplementation with concurrent vitamin B
6 with Mg
2+ (cofactor) has shown improvements in social interaction and restrictive behaviors [
44]. Vitamin B
12 is an important cofactor in the methionine cycle, which ultimately generates cysteine to be consumed in the transsulfuration pathway to produce reduced GSH. Simply put, vitamin B
12 plays a crucial role in maintaining the redox balance in the body by generating antioxidant species. Supplementation with subcutaneous vitamin B
12 injections has been shown to improve sleep, gastrointestinal symptoms, hyperactivity, and non-verbal intellectual quotient [
45]. Other nutrients such as vitamin B
7, vitamin D, zinc, omega fatty acids, sulforaphane, and chelating agents have also been research targets for the symptomatic management of ASD; no single treatment has been shown to be effective as a monotherapy [
45,
46,
47]. Oral glutathione may work synergistically with other nutritional supplementation to generate a greater effect size in treating ASD.
Another key component in the baseline redox balance involves genetics. Glutathione S-transferases (GST) are a family of enzymes that aid in the conjugation of reduced GSH to xenobiotics for detoxification and also helps reduce endogenous oxidative species. All four major classes of GST have been shown to exhibit genetic polymorphisms reducing their antioxidative ability and are associated with psychiatric pathologies such as ASD [
48,
49]. Common GST polymorphisms include GSTA1, GSTM1, GSTT1, and GSTP1. Some protective polymorphisms include the GSTA1*CC genotype, which has predicted lower non-verbal communication; the GSTP1*allele genotype, which is associated with better cognitive functioning in ASD; and the GSTM1-active genotype, which has predicted higher adaptive functioning [
50]. Furthermore, the GSTM1-active genotype contributes to the overall antioxidant capacity and is moderated by external factors such as maternal smoking during pregnancy [
50]. The null phenotypes and moderated phenotypes lack the protective benefit against ASD and other psychiatric illnesses. It is important to keep in mind that ASD is a complex developmental disorder. Understanding the interaction between genetic predisposition and environmental risks may help mitigate ASD impairment to improve cognitive functioning and behaviors.
The method of delivery is an important consideration in this study. Opitac
TM glutathione was used as a way to increase bioavailability, with better central nervous system (CNS) penetration. There is a growing body of research that takes this idea one step further to explore the tolerability and efficacy of intranasal glutathione. Intranasal administration bypasses the blood–brain barrier, which filters out 98–100% of lipophilic molecules. Although intranasal glutathione has not been studied for ASD, treatment with intranasal glutathione has been studied for Parkinson’s disease and shows an excellent penetration rate and increased levels of GSH in brain tissue [
51]. Newer advances in bioengineering utilize nasal permeability enhancers, gelling agents, and nanoparticle formulations that optimize drug delivery into brain tissue [
52]. Other considerations for parenteral glutathione administration for bioavailability include the intravenous route. There is no current data on the use of IV glutathione in the treatment of ASD, but IV glutathione has been used for Parkinson’s disease and as a skin lightening therapy in the literature [
53,
54,
55]. Current studies on IV glutathione have failed to evaluate the long-term safety profile of its use [
55]. Additionally, it may be of limited clinical benefit in the ASD population based on behavioral limitations to adhere with continuous treatment. Numerous studies have explored the use of NAC in the context of ASD, and this represents the second study focusing on the application of glutathione. To gain a more comprehensive understanding of the redox system’s involvement in ASD, it is essential that future research endeavors include comparative studies between NAC and glutathione in the context of this disorder. Such investigations will shed light on the relative efficacy and mechanism of redox and its role in ASD. Utilizing biomarkers and newly acquired genetic profiles for autism, along with insights into the redox systems, can pave the way for future studies to embrace precision medicine and personalized interventions in heterogeneous groups and phenotypes.