This study builds upon findings from one prior randomized, placebo-controlled trial, which found that the use of sulforaphane led to improvements in behavior and social responsiveness in children and young adults (aged 13–27) with ASD [19]. Our primary goal was to examine changes in metabolites in children with ASD who were taking sulforaphane to determine a possible mechanism of action. We observed that a group of school-age children (mean age 14.8) showed a trend towards improvement in behavior (ABC) and a statistically significant improvement in social responsiveness after 12 weeks of treatment. The magnitude of improvements in the current study (− 9.7 points for the SRS and − 7.1 points for the ABC) were smaller than in the prior study (− 20.4 points for the SRS and − 21.4 points for the ABC), which may be related to the younger age of these participants or other differences in the study populations. One important difference in the study populations is that in the current study, all of the children were attending one specialized school with programs designed for children with ASD. The behavioral interventions in place at the school may have already been producing positive effects (at baseline) and limited the ability to detect further improvement from sulforaphane over the 12-week study (when compared to the prior study, where subjects were not in the same school).
In order to determine which metabolites might mediate clinical improvements, we examined correlations between the change in urinary metabolite levels and change in behavior and social responsiveness over the 12-week study. We defined, a priori, that a correlation with an absolute value of 0.6 or greater was potentially relevant and might indicate a mechanism of action. Of 694 measured urinary metabolites, 77 had correlations with an absolute value of ≥ 0.6. While it is not possible to discuss the implications of all of these correlations, several of these metabolite changes cluster into known pathways (Table 4) that have been reported to be altered in children with ASD. These pathways involve metabolites involved in oxidative stress, amino acid/gut microbiome, neurotransmitters, hormones/stress response, and sphingomyelin metabolism, among others. Sulforaphane may affect these pathways through a variety of mechanisms including Nrf2-mediated induction of phase 2 detoxification enzymes, suppression of cytochrome P450 enzymes, induction of apoptotic pathways, and anti-inflammatory activity that have been described in detail [28].
Many prior studies have found increased oxidative stress in children with ASD, which may be due to increased production or decreased clearance of reactive oxygen species (also known as ROS, or oxygen free-radicals). Children with ASD have been found to have lower levels of the metabolites that process oxygen free radicals (methionine, S-adenosylmethionine, homocysteine, cystathionine, cysteine, and total glutathione), higher levels of metabolites that are involved in the body’s mechanism for reducing oxidative stress (oxidized glutathione, adenosine, and S-adenosylhomocysteine), and markers of protein and DNA oxidative damage [3, 29]. Also, two prior randomized controlled trials—one of the antioxidant NAC, and one of the methyl donor, methyl B12—have found that these treatments improve clinical symptoms in children with ASD [17, 18]. In the methyl B12 supplementation study, clinical improvements were correlated with increases in plasma methionine, decreases in S-adenosyl-homocysteine (SAH), and improvements in the ratio of S-adensylmethionine (SAM) to SAH [18]. Interestingly, in the current study, clinical improvements were correlated with two metabolites known to be involved in redox metabolism. The negative correlations found with γ-glutamylglutamine and methionine sulfone indicate that, as the urinary levels of these metabolites increased, the symptoms scores decreased (improved). This suggests that sulforaphane may mediate beneficial clinical effects through increases in antioxidant capacity, which is one of its well-documented physiological effects [20].
Abnormalities in amino acid metabolism have been reported in children with ASD compared to control children [9, 12, 14, 16], and this may be related to altered processing of amino acids by gut microbiota [30]. We found correlations between clinical improvement and the amino acids tryptophan, tyrosine, and assymetric-dimethylarginine (ADMA, a derivative of the amino acid, arginine). The involvement of altered amino acids in the pathology of ASD is plausible since amino acids are building blocks for many key neurotransmitters and hormones, including catecholamines and serotonin [30]. Six prior studies using urinary metabolomics noted increased urinary tryptophan in children with ASD [7, 9, 12, 14, 16, 30], and tryptophan is a key substrate in the serotonergic metabolic pathway. For tryptophan, tyrosine, and ADMA, the correlations in the current study were all negative, indicating that as the urinary levels of these amino acids increased, symptom scores decreased (improved). We also identified correlations between clinical improvement and changes in a number of other amino acids that are known to be associated with gut microbiota. Six of the eight metabolites in this category had positive correlations, indicating that decreased urinary levels were associated with improved clinical symptoms. It is not yet clear how sulforaphane may affect or improve the amino acid abnormalities (including those associated with the gut microbiome) and lead to clinical improvements.
We also found associations between clinical improvements and changes in five urinary neurotransmitter-related metabolites, including N-methylglutamate, glutamine, hypoxanthine, serotonin, and homovanillate (HMV), which is the normal end product of dopamine degradation and was elevated in the urine of children with ASD in a prior study [14]. For all of the urinary neurotransmitters except N-methylglutamate, the correlations were negative, indicating that increases in the urinary levels of these metabolites were associated with lower scores on the ABC and SRS and therefore improved symptoms. This suggests that either increased production or increased elimination of these metabolites is correlated with beneficial effects. Hypoxanthine, which is part of the purine pathway, was previously found to be elevated in children with ASD [9]. We also found that urinary glutamine was correlated with clinical improvements. Glutamatergic dysfunction has been hypothesized to be involved in the pathogenesis of ASD, with several studies reporting abnormal levels of glutamate in various regions in the brain [13].
We found correlations between clinical improvements and a large number of hormones, some of them stress-related. Prior studies have found higher salivary stress hormones in children with ASD and higher hair cortisol levels, suggesting both acute and chronic elevation in stress hormones [31, 32]. Higher hair cortisol levels were associated with more severe autism symptoms and anxiety [31]. In the current study, both cortisone and cortisol-21-glucuronide had negative correlations with ASD-related behavior, indicating that increased urinary levels were associated with improved symptoms. ASD behavior and social responsiveness were also related to a large number of other hormones, and all of these correlations were negative, again indicating that increased urinary levels of various hormones were associated with improved symptoms. It is not clear if this indicates that increased production or increased excretion is associated with improvement, but it highlights changes in hormonal function in ASD as an area for further study.
A novel finding of the current study is that improvement in behavior was correlated with seven different chemical forms of sphingomyelin. Seven sphingomyelin metabolites were each strongly negatively correlated with behavior, such that increased urinary levels were associated with improved behavior. Sphingomyelin is a sphingolipid found in animal cell membranes, especially in the membranous myelin sheath that surrounds nerve cells and axons. To our knowledge, there have been no prior reports of abnormalities in sphingolipid levels in children with ASD, but there are numerous studies documenting abnormalities in the size, number, and morphology of dendrites in autism, which is related to altered synapse function [33]. Furthermore, sphingomyelin abnormalities have been noted in a number of other central nervous system disorders, including depression, anxiety, Alzheimer’s disease, and amyotrophic lateral sclerosis, suggesting that it may have a central role in normal brain development and function [34,35,36,37]. It is not clear how sulforaphane might alter sphingomyelin metabolism or availability and whether this is related to clinical benefits, but if this association is confirmed, it has important clinical and treatment implications.
The current study has a number of limitations. Importantly, this was a pilot study to investigate whether metabolomics might be a useful tool to suggest pathways that may be involved in the mechanism of action of treatments (in this case, sulforaphane) in ASD. The study was open label and parent raters may have rated more positively knowing that their child was taking the sulforaphane, although the magnitude of benefit is lower than in the one prior randomized controlled trial. The ratings were also limited to the ABC and SRS to minimize respondent burden, and while these are widely used outcome tools in ASD, we did not include other measures of repetitive behavior or adaptive function, which might have shown clinical changes in other important areas. Many factors affect urinary metabolomics, including diet, environment, stress, sleep, age, and other factors. In the current study, the wide age range (7–21), different gender and pubertal state of subjects and the small sample size all limit the strength of the conclusions regarding urinary metabolomic changes. The variation in these environmental effects may have been minimized by having all the children attending the same school 5 days a week. However, the findings of this study should be viewed as hypothesis generating and should be confirmed in future studies with larger sample sizes. Future studies would also benefit from plasma biochemical assessments of antioxidant status pre- and post-treatment since this is likely a key pathway in the mechanism of action of sulforaphane. We found that change in the two outcome measures, ABC and SRS, were associated with mostly different metabolites. We believe this is to be expected since the measures assess different components of human behavior and are likely influenced by different metabolic pathways. Finally, we used a different delivery method of sulforaphane than in a prior randomized controlled trial in autism [19] by providing a precursor, glucoraphanin, along with a conversion enzyme, myrosinase. Although the dosing was designed to produce a similar level of sulforaphane, it is possible that differences in bioavailable sulforaphane levels between the two studies could have led to differences in clinical results.