*Review* **Cerebral Folate Deficiency, Folate Receptor Alpha Autoantibodies and Leucovorin (Folinic Acid) Treatment in Autism Spectrum Disorders: A Systematic Review and Meta-Analysis**

**Daniel A. Rossignol 1,\* and Richard E. Frye 2,3**


**Citation:** Rossignol, D.A.; Frye, R.E. Cerebral Folate Deficiency, Folate Receptor Alpha Autoantibodies and Leucovorin (Folinic Acid) Treatment in Autism Spectrum Disorders: A Systematic Review and Meta-Analysis. *J. Pers. Med.* **2021**, *11*, 1141. https://doi.org/10.3390/ jpm11111141

Academic Editor: Guido Krenning

Received: 10 October 2021 Accepted: 1 November 2021 Published: 3 November 2021 Corrected: 29 April 2022

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Abstract:** The cerebral folate receptor alpha (FRα) transports 5-methyltetrahydrofolate (5-MTHF) into the brain; low 5-MTHF in the brain causes cerebral folate deficiency (CFD). CFD has been associated with autism spectrum disorders (ASD) and is treated with *d,l*-leucovorin (folinic acid). One cause of CFD is an autoantibody that interferes with the function of the FRα. FRα autoantibodies (FRAAs) have been reported in ASD. A systematic review was performed to identify studies reporting FRAAs in association with ASD, or the use of *d,l*-leucovorin in the treatment of ASD. A meta-analysis examined the prevalence of FRAAs in ASD. The pooled prevalence of ASD in individuals with CFD was 44%, while the pooled prevalence of CFD in ASD was 38% (with a significant variation across studies due to heterogeneity). The etiology of CFD in ASD was attributed to FRAAs in 83% of the cases (with consistency across studies) and mitochondrial dysfunction in 43%. A significant inverse correlation was found between higher FRAA serum titers and lower 5-MTHF CSF concentrations in two studies. The prevalence of FRAA in ASD was 71% without significant variation across studies. Children with ASD were 19.03-fold more likely to be positive for a FRAA compared to typically developing children without an ASD sibling. For individuals with ASD and CFD, meta-analysis also found improvements with *d,l*-leucovorin in overall ASD symptoms (67%), irritability (58%), ataxia (88%), pyramidal signs (76%), movement disorders (47%), and epilepsy (75%). Twenty-one studies (including four placebo-controlled and three prospective, controlled) treated individuals with ASD using *d,l*-leucovorin. *d,l*-Leucovorin was found to significantly improve communication with medium-to-large effect sizes and have a positive effect on core ASD symptoms and associated behaviors (attention and stereotypy) in individual studies with large effect sizes. Significant adverse effects across studies were generally mild but the most common were aggression (9.5%), excitement or agitation (11.7%), headache (4.9%), insomnia (8.5%), and increased tantrums (6.2%). Taken together, *d,l*-leucovorin is associated with improvements in core and associated symptoms of ASD and appears safe and generally well-tolerated, with the strongest evidence coming from the blinded, placebocontrolled studies. Further studies would be helpful to confirm and expand on these findings.

**Keywords:** autism spectrum disorder; cerebral folate deficiency; folate receptor alpha autoantibodies; folinic acid; leucovorin

### **1. Introduction**

Autism spectrum disorder (ASD) is a behaviorally defined disorder that affects approximately 2% of children in the United States [1]. A number of medical comorbidities have been reported in individuals with ASD, with some studies reporting an average of 4–5 comorbidities [2], including allergic rhinitis [3], irritable bowel syndrome [3], attention

deficit hyperactivity disorder [4], ophthalmological conditions [4], sleep problems [5], immune problems [5], mitochondrial dysfunction [6], gastrointestinal abnormalities [7] and epilepsy [8]. Recently, cerebral folate deficiency (CFD) has been reported in a number of studies in individuals with ASD and its treatment, *d,l*-leucovorin calcium (also known as folinic acid) has undergone investigation as a treatment for ASD [9].

The importance of folate for the development of the central nervous system (CNS) was first discovered in animal models of dietary folate deficiency where findings such as hydrocephalus [10], decreased myelin cerebroside [11] and impaired synthesis of neuronal RNA [12] were reported. In humans, studies of serum folate deficiency reported neurological findings including ataxia, nystagmus, areflexia, and confusion [13] as well as organic brain syndrome and damage to the pyramidal tracts [14]. In 1974, low levels of cerebrospinal fluid (CSF) folate were reported in patients with epilepsy that improved with tetrahydrofolate but not folic acid [15]. A study in 1976 reported 2 children with low CSF folate who developed intracranial calcifications [16]. In 1979, cerebral atrophy was reported in a 48-year-old woman with low serum and CSF folate [17]. In 1983, a case of a 23 year old woman with Kearns-Sayre syndrome and CNS deterioration was found to have decreased serum and CSF folate while taking phenytoin and improved with folic acid treatment [18].

In 2002, Ramaekers reported a novel neurometabolic syndrome in five children with low CSF 5-MTHF [19] who manifested severe neurodevelopmental symptoms including irritability, seizures, lower extremity pyramidal signs, spastic paraplegia, psychomotor retardation, dyskinesias, cerebellar ataxia, acquired microcephaly and developmental regression which occurred as young as 4 months of age. Unlike previous studies of folate deficiency associated with CNS abnormalities, this new neurometabolic disorder demonstrated below normal concentration of 5-methyltetrahydrofolate (5-MTHF), one of the active metabolites of folate, in the CSF, but normal systemic folate levels. This condition improved with folinic acid (*d,l*-leucovorin) treatment. In these patients, no genetic abnormalities were identified. Ramaekers et al., later described this condition as "idiopathic CFD" [20].

CFD occurs due to the impaired transport of folates across the blood-brain barrier. CFD is usually caused by dysfunction of the folate receptor-alpha (FRα) [21]. FRα is a receptor which has a high affinity for 5-MTHF and is found on the basolateral endothelial surface of the choroid plexus. FRα transports folates across the blood-brain barrier through adenosine triphosphate (ATP) dependent receptor-mediated endocytosis. Dysfunction of the FRα can occur through several mechanisms. In only rare cases, genetic mutations in the gene encoding for FRα (FOLR1) is a cause of CFD [22,23]. Two other mechanisms are more prevalent in causing FRα dysfunction. First, in the seminal paper describing CFD, two autoantibodies to the FRα (blocking and binding autoantibodies) were described that interfere with the function of the FRα [21]. Second, mitochondrial disease is associated with CFD since folate transportation through the FRα is dependent on ATP [24–27].

Another folate transporter, the reduced folate carrier (RFC/SLC19A1), lies on both the basolateral and apical surface of the choroid plexus and has a lower affinity for folates. *d,l*-leucovorin is a reduced (active) form of folate which can enter the CNS through the RFC and has been reported to normalize 5-MTHF levels in the CSF in individuals with CFD [21]. In some cases, clinical response to *d,l*-leucovorin is dramatic, especially if this treatment is started early in life [19,28] but sometimes improvements can be marked even in adults [29]. *d,l*-leucovorin consists of two diastereomers designated as *d*-leucovorin and *l*-leucovorin. *l*-Leucovorin (5-formyl-(6S)-tetrahydrofolate) is the biologically active isomer. It is rapidly metabolized (via 5,10-methenyltetrahydrofolate then 5,10-methylenetetrahydrofolate) to 5-methyl-(6S)-tetrahydrofolate (L-methyl-folate or 5-MTHF), which, in turn, can be metabolized via other pathways back to tetrahydrofolate and 5,10-methenyltetrahydrofolate. 5,10 methylenetetrahydrofolate is converted to 5-MTHF by an irreversible enzyme-catalyzed reduction using the cofactors FADH<sup>2</sup> and NADPH. The major mechanism of transportation of folates into the brain occurs through the FRα. FRα binds to folate at lower serum

concentrations than the RFC; the latter functions at relatively high concentrations of serum folate and is the predominant method of folate transport in the intestine. Genetic defects in RFC can lead to a rare disorder in intestine folate absorption and genetic abnormalities in FRα may lead to neural tube defects [19].

*d,l*-leucovorin was first approved in the United States in the 1950s and has been used continuously since then to reduce toxicities associated with folate pathway antagonists such as methotrexate (which is typically used in the treatment of osteosarcoma, other cancers and autoimmune diseases). By replenishing intracellular pools of reduced folates, *d,l*-leucovorin can counteract the toxic effects of folate pathway antagonists such as methotrexate which act by inhibiting dihydrofolate reductase (DHFR). The *d*-isomer of leucovorin is not metabolically active and is not metabolized *in vivo* to any significant degree; therefore, only the *l*-isomer can contribute to the direct replenishment of the pools of active folate cofactors. One of the main advantages reported for *d,l*-leucovorin over folic acid (pteroylmonoglutamic acid) is that folic acid is oxidized and must be reduced by DHFR to a biologically active folate in order to become active. High doses of folic acid may also block the FRα which can potentially exacerbate CFD [30]. Many individuals, including those with ASD, have polymorphisms in the DHFR gene which makes the function of this enzyme less efficient [31]. Currently, the most prescribed form of leucovorin in the United States is *d,l*-leucovorin calcium. *d,l*-Leucovorin is only form of folate that has been used to treat CFD except for one case report using 5-MTHF [32].

Since its original description, the phenotype of CFD has expanded. A number of studies have reported ASD in a subset of children with CFD [20,21,33–36]. This is not particularly surprising as two mechanisms for FRα dysfunction, FRα autoantibodies (FRAAs) and mitochondrial dysfunction, are common in ASD. Indeed, studies have reported a high prevalence of FRAAs in individuals with ASD ranging from 58% to 76% [37–45]. Mitochondrial dysfunction is a common medical comorbidity in ASD, with studies reporting 30–50% of individuals with ASD possessing biomarkers of mitochondrial dysfunction [6,46] and up to 80% having abnormal electron transport chain (ETC) activity in immune cells [47,48]. Treatment in some children with CFD and ASD with oral *d,l*-leucovorin has led to clinical improvements ranging from partial improvements in communication, social interaction, attention and stereotypies [21,33,35,36] to complete recovery of both neurological and ASD symptoms [21,34] in up to 21% of treated patients [44]. Of note, *d,l*-leucovorin has been reported to improve symptoms in Down syndrome [49], Rett syndrome [50–52] and schizophrenia [53].

This paper systematically reviews the studies examining an association between CFD and ASD, then examines the prevalence of the major cause of CFD, namely FRAAs, in ASD, typically developing (TD) siblings of individuals with ASD and their parents and TD non-related controls, followed by reviewing the evidence for treatment with *d,l*-leucovorin, the primary treatment for CFD, in individuals with ASD and any associated adverse effects (AEs).

#### **2. Materials and Methods**

#### *2.1. Search Process*

A prospective protocol for this review was developed a priori and the search terms and selection criteria were chosen to capture all pertinent publications. The search included individuals with autistic disorder, Asperger syndrome, pervasive developmental disordernot otherwise specified (PDD-NOS) and ASD. A computer-aided search of PUBMED, Google Scholar, CINAHL, EmBase, Scopus, and ERIC databases from inception through September 2021 was conducted to identify pertinent publications using the search terms "autism", "autistic", "Asperger", "pervasive", "ASD", and "PDD" in all combinations with "folinic acid", "leucovorin", "folate", "folic", "methyl-folate", "5MTHF", "levofolinic", "folinate", and "formyltetrahydrofolate." The references cited in identified publications were also searched to locate additional studies.

#### *2.2. Study Selection and Assessment 2.2. Study Selection and Assessment*

*J. Pers. Med.* **2021**, *11*, x FOR PEER REVIEW 4 of 24

lications were also searched to locate additional studies.

This systematic review and meta-analysis followed PRISMA guidelines [33], the PRISMA Checklist is found in Supplementary Table S1 and the PRISMA Flowchart for *d,l*-leucovorin treatment in ASD is displayed as Figure 1. Studies were included in this systematic review if they: (a) involved individuals with ASD, and (b) either reported on the use of leucovorin in at least one individual with ASD and/or described FRAAs in at least one individual with ASD. Articles that did not present new or unique data (such as review articles or letters to the editor), and animal studies were excluded. Studies on Rett syndrome and Childhood Disintegrative Disorder were also excluded. One reviewer (DR) screened titles and abstracts of all potentially relevant studies for identification purposes. Both reviewers then examined each identified study in-depth and assessed factors such as the risk of bias. As per standardized guidelines [54], selection, performance detection, attrition, and reporting biases were considered. This systematic review and meta-analysis followed PRISMA guidelines [33], the PRISMA Checklist is found in Supplementary Table S1 and the PRISMA Flowchart for *d,l*leucovorin treatment in ASD is displayed as Figure 1. Studies were included in this systematic review if they: (a) involved individuals with ASD, and (b) either reported on the use of leucovorin in at least one individual with ASD and/or described FRAAs in at least one individual with ASD. Articles that did not present new or unique data (such as review articles or letters to the editor), and animal studies were excluded. Studies on Rett syndrome and Childhood Disintegrative Disorder were also excluded. One reviewer (DR) screened titles and abstracts of all potentially relevant studies for identification purposes. Both reviewers then examined each identified study in-depth and assessed factors such as the risk of bias. As per standardized guidelines [54], selection, performance detection, attrition, and reporting biases were considered.

terms "autism", "autistic", "Asperger", "pervasive", "ASD", and "PDD" in all combinations with "folinic acid", "leucovorin", "folate", "folic", "methyl-folate", "5MTHF", "levofolinic", "folinate", and "formyltetrahydrofolate." The references cited in identified pub-

**Figure 1.** PRISMA 2020 flow diagram for this systematic review. **Figure 1.** PRISMA 2020 flow diagram for this systematic review.

As a result of the in-depth review, several studies were excluded from further consideration. One study [55] reported on a child with "autistic personal characteristics" who had CFD, but it was unclear whether this child was diagnosed with ASD. Two studies reported treatment of children with ASD using **folic acid** [56,57]. Finally, one study [43] used an unvalidated, non-clinical FRAA assay in which the functional significance of the autoantibody is unknown. As a result of the in-depth review, several studies were excluded from further consideration. One study [55] reported on a child with "autistic personal characteristics" who had CFD, but it was unclear whether this child was diagnosed with ASD. Two studies reported treatment of children with ASD using **folic acid** [56,57]. Finally, one study [43] used an unvalidated, non-clinical FRAA assay in which the functional significance of the autoantibody is unknown.

#### *2.3. Meta-Analysis*

MetaXL Version 5.2 (EpiGear International Pty Ltd., Sunrise Beach, QLD, Australia) was used with Microsoft Excel Version 16.0.12827.20200 (Redmond, WA, USA) to perform the meta-analysis. Random-effects models, which assume variability in effects from both sampling error and study level differences [58,59], were used to calculate pooled prevalence and odds ratios. The Luis Furuya-Kanamori (LFK) Index derived from Doi plots was reviewed for significant asymmetries (>±2) in the prevalence distribution when there were three or more studies [60,61]. Cochran's Q was calculated to determine heterogeneity of

effects across studies and, when significant, the I<sup>2</sup> statistic (Heterogeneity Index) was calculated to determine the percentage of variation across studies that is due to heterogeneity rather than chance [62,63]. Funnel plots were also reviewed.

Mean FRAA titers were pooled across studies using standard methodology [64]. To compare FRAA titers across groups, pooled Cohen's d' (a measure of effect size) was calculated from the standardized mean difference of outcome measures using the inverse variance heterogeneity model, since it has been shown to resolve issues with underestimation of the statistical error and spuriously overconfident estimates with the random effects model when analyzing continuous outcome measures [65].

The outcome measures used across treatment studies were different in most cases, making a formal meta-analysis of any particular outcome not possible. Thus, the effect size, as measured by Cohen's d', was calculated where possible so the strengths of effects could be compared across studies. For controlled studies, the effect size represented the difference between the treatment and the control groups, whereas for uncontrolled studies the effect size was calculated only for the treatment group. Only a subset of studies contained the information needed to calculate the effect size. For example, for the calculation of effect size, the change in the outcome needed to be reported across the treatment period; reported mean values of the outcome before and after treatment were insufficient to calculate an effect size. Effect sizes were considered small if Cohen's d' was 0.2; medium for Cohen's d' was 0.5; and large if Cohen's d' was 0.8 [66].

#### **3. Results**

This section will first discuss folate pathway abnormalities related to ASD, followed by treatment of folate pathway abnormalities.

### *3.1. Central Folate Pathway Abnormalities and the Folate Receptor Alpha Autoantibody*

#### 3.1.1. ASD Prevalence in CFD

Five case-series [20,21,33,35,67] described 79 children with CFD in which ASD was assessed (Supplementary Table S2) resulting in a prevalence of 44% (21%, 70%) of ASD in CFD (Table 1). Removing the one study with a very high prevalence rate because of asymmetry [33] lowered the pooled prevalence rate to 32% (19%, 45%).

#### 3.1.2. Cerebral Folate Deficiency in Autism Spectrum Disorder

Two case-series [34,68], two case-reports [36,69] and four prospective cohortstudies [37,45,70,71] described 172 individuals with idiopathic ASD who had CSF measurements (See Supplementary Table S3). The pooled prevalence of CFD in ASD was 38% (11%, 71%) with a significant variation across studies due to heterogeneity driven by three studies with very high prevalence rates [34,36,68] and four studies with very low prevalence rates [37,45,70,71]. Two studies with high prevalence rates reported severe patients; one study was an older case series specifically examining low-functioning ASD with neurological deficits [34]; and one was a case report of a child with mental retardation and seizures [36]. Two case series examined patients with mitochondrial disorders with very different prevalence; the series which reported a new type of non-traditional mitochondrial disorder reported a high prevalence (100%) of CFD [68], while the series which reported classical mitochondrial disorders reported a low (5%) prevalence of CFD [71]. The overall pooled prevalence was 43% (0%, 100%) with significant heterogeneity. One study found no correlation between CSF levels of 5-MTHF and measures of autism symptomatology as measured by the Autism Diagnostic Observation Schedule (ADOS) calibrated severity score, adaptive behavior as measured by the Vineland Adaptive Behavior Scale (VABS), and cognitive functioning as measured by the Mullen Scales of Early Learning [70].

**Table 1.** Meta-analysis results for the prevalence of cerebral folate deficiency and folate receptor alpha autoantibodies. Pooled prevalence with 95% confidence interval, Cochran's Q (Q), Heterogeneity Index (I<sup>2</sup> ), Luis Furuya-Kanamori (LFK) Index and number of studies involved (N). Statistics are estimated by a random-effects model. \* *p* < 0.05; \*\* *p* < 0.01; <sup>T</sup> Significant Asymmetry.


3.1.3. Prevalence of Autoantibodies to the Folate Receptor Alpha in ASD

Nine studies examined the prevalence of FRAAs in ASD [37–42,44,45,72]. Two sets of studies [37,39] and [40,41,72] reported on the same cohort of patients. Additionally, one study reported the prevalence in two subsets of patients; those treated with *d,l*-leucovorin (*n* = 82) and those untreated (*n* = 84) [44]. This resulted in six unique studies that examined the prevalence of FRAAs in children with ASD (See Supplementary Table S4).

The correlation between FRAAs and patient characteristics have been outlined in several studies. Two studies [35,37] reported a significant inverse correlation between higher blocking FRAA serum titers and lower 5-MTHF CSF concentrations. One study found that blocking FRAA decreased with age [37] with another study reporting increased blocking FRAA over a 2-year period with continued use of cow's milk [35]. One study suggested that children with ASD were significantly different in physiological and developmental

characteristics depending on whether they had the blocking or binding FRAA; the binding FRAA was associated with higher serum B12 concentration, while the blocking FRAA was associated with better redox metabolism, inflammation markers, communication on the VABS, stereotyped behavior on the Aberrant Behavioral Checklist (ABC) and mannerisms on the Social Responsiveness Scale (SRS) [40]. In another study, FRAA positive children were more likely to have a medical diagnosis of hypothyroidism [37]. Two studies found a positive correlation between the blocking FRAA titers and thyroid stimulating hormone (TSH) concentrations [39,41]. One study examined this relationship in detail, finding that thyroid hormone was rarely outside the normal range, suggesting that the relationship between TSH and thyroid hormone levels were altered in some children with ASD. Interestingly, this study also found that FRAAs bind to prenatal thyroid tissue in early gestation (prior to 18 weeks) suggesting that FRAAs during gestation could affect the programing of the hypothalamic-pituitar*y*-axis regulation of thyroid hormone [37].

Five studies [37,38,40,42,45] from three sets of investigators found a blocking FRAA prevalence of 46% (27%, 64%) in ASD with a significant heterogeneity but no asymmetry, indicating variation in the underlying ASD samples across studies. Four studies [37,40,42,45] from two sets of investigators found a binding FRAA prevalence of 49% (43%, 55%) without significant variation across studies. Five studies [37,40,42,44,45] from three sets of investigators found an overall FRAA prevalence of 71% (64%, 77%) without significant variation across studies. From the three studies reporting both FRAA titers [37,40,42] and one study only measuring blocking FRAAs [38] in ASD, pooled mean blocking FRAA titers was 0.85 pmol of IgG antibody per ml of serum (95% CI: 0.59, 1.11) and binding FRAA was 0.42 pmol of IgG antibody per ml of serum (95% CI: 0.35, 0.49).

One study that looked at the blocking FRAA over a five-week period found that in four patients with predominately negative titers, if tested over several weeks, they may have low positive (~0.3–0.4 pmol of IgG antibody per ml of serum) at some point, while others with the most high titers can have low or negative titers at some time points [45].

Four studies [37,38,42,44] from three sets of investigators examined blocking and binding FRAA prevalence in parents and TD siblings of children with ASD. Prevalence of the blocking, binding and either FRAA was 30% (19%, 44%), 23% (0%, 61%) and 45% (27%, 60%) in parents of children with ASD, respectively. All studies demonstrated significant heterogeneity without asymmetry suggesting variation in the underlying ASD samples across studies. Interestingly, TD siblings of children with ASD appear to have a similar prevalence as the children with ASD themselves with a pooled prevalence of 38% (19%, 58%), 40% (9%, 77%) and 61% (28%, 97%) for blocking, binding and either FRAA, respectively, without significant variation across studies.

The prevalence of FRAAs in TD children without ASD siblings was assessed in two studies [42,45] with a pooled prevalence of 4% (1%, 10%), 10% (10%, 48%) and 15% (0%, 46%) for blocking, binding and either FRAA, respectively; this is much lower than the FRAA prevalence in children with ASD or their siblings. However, there was significant variability in the binding FRAA across these two studies, demonstrating the need for larger cohorts of non-sibling control samples.

One study [38] examined the prevalence of the blocking FRAA in developmentally delayed children without ASD and found a pooled prevalence of 5% (0%, 14%); this is much lower than the FRAAs prevalence in children with ASD or their TD siblings.

3.1.4. Comparison of Prevalence of Autoantibodies to the Folate Receptor Alpha in ASD to Other Groups

A meta-analysis was used to calculate the odds ratio of having FRAAs in children with ASD compared to various groups. Five studies [37,38,42,44,45] reported FRAAs in both children with ASD and their parents. The odds of being positive for the blocking or either FRAA, but not binding alone, was significantly increased in children with ASD as compared to their parents (Table 2). The odds of being positive for the FRAA was not different between children with ASD and their TD siblings. However, children with ASD demonstrated a significantly increased odds of being positive for the blocking, binding and either FRAA as compared to TD children without an ASD sibling, and as compared to developmentally delayed children without ASD for the blocking FRAA.

**Table 2.** Meta-analysis of Odds Ratios with 95% confidence interval for differences between the prevalence of Folate Receptor Alpha Autoantibodies in children with ASD to Various Comparison Groups. Odd ratios that are significant are bolded and italicized. Also listed are Cochran's Q (Q), Heterogeneity Index (I2), Luis Furuya-Kanamori (LFK) Index and number of studies involved (N). Statistics are estimated by a random-effects model. <sup>Γ</sup> *<sup>p</sup>* < 0.05, \*\* *<sup>p</sup>* <sup>≤</sup> 0.001.


Two studies [37,42] compared the serum concentrations of FRAAs in children with ASD compared to parents and/or TD siblings while one study compared FRAA in children with ASD to TD children without ASD siblings [42]. Meta-analysis found that the mean blocking FRAA titer in ASD was significantly higher than parents (d' = 0.26 (0.116, 0.36), *p* < 0.0001) and siblings (d' = 0.29 (0.15, 0.43), *p* < 0.0001) with small-to-medium effect sizes and significantly higher than controls with a very large effect size (d' = 2.93 (1.85, 4.01), *p* < 0.0001). However, the mean binding FRAA titer in ASD was significantly higher than in parents (d' = 0.14 (0.06, 0.22), *p* < 0.001) but not siblings (d' = 0.06 (−0.04, 0.17), *p* = n.s.) with small effect sizes and significantly higher than controls but with a small effect size (d' = 0.16 (0.07, 0.25), *p* < 0.001).

Finally, in one study that measured blocking FRAAs in children with CFD with and without ASD there was no significant difference between groups (Mean (SD) 1.17 (0.84) pmol/mL and 1.78 (1.99) pmol/mL, respectively, t(23) = 0.91, *p* = 0.37) [35].

#### *3.2. Treatment of ASD with d,l-Leucovorin*

As seen in Figure 1, 20 studies were identified which studied *d,l-*leucovorin treatment in individuals with ASD including four placebo-controlled studies [72–75], three prospective, controlled studies [37,44,76], nine prospective studies without a control group [20,21,33–35,77–80] (two studies examined the same cohort of patients [78,79]), and four case reports/series [36,67–69].

A review of these studies on treatment of children with ASD with *d,l*-leucovorin appears to fall into three categories. Firstly, children with ASD and concomitant CFD were studied. Secondly, *d,l*-leucovorin was studied in isolation for treating idiopathic ASD. Thirdly, some studies used *d,l*-leucovorin in combination with other nutritional supplements to treat ASD symptoms. Each of these approaches to using *d,l*-leucovorin is outlined in separate sections below.

#### 3.2.1. Treatment with *d*,*l*-Leucovorin in ASD and Comorbid CFD

Nine unique case-series/reports describe children with ASD and comorbid CFD treated with *d,l*-leucovorin (See Supplementary Tables S2 and S3) [20,21,33–36,67–69]. Two studies, one [36] case report and one case series [33], reported on the same child. A metaanalysis was conducted to determine the prevalence of improvement in symptoms as a result of *d,l*-leucovorin treatment in children with CFD with and without ASD (See Table 3; Supplementary Table S5). Six studies [21,33–35,67,69] reported a response rate of 67% for improvement in ASD symptoms with *d,l*-leucovorin treatment. Response to *d,l*-leucovorin in irritability was studied in children with ASD in three studies [21,34,35] and in children without ASD in three studies [21,33,35]. For those with ASD, irritability improved in 58% while in those without ASD irritability improved in 47% with a wide variation among studies, because one study demonstrated a high response rate of 88% [21] while the other two studies demonstrated much lower response rates of 22% [35] and 0% [33].

**Table 3.** Meta-analysis results for the prevalence response to *d,l*-leucovorin treatment in children with CFD with and without autism spectrum disorder (ASD). Pooled prevalence with 95% confidence interval, Cochran's Q (Q), Heterogeneity Index (I2), Luis Furuya-Kanamori (LFK) Index and number of studies involved (N). Statistics are estimated by a random-effects model. \* *p* < 0.01; <sup>T</sup> Significant Asymmetry.


Five studies examined ataxia in ASD [21,33–35,67] while two studies examined ataxia in children without ASD [21,35] with both groups of children demonstrating a high rate of response of ataxia to *d,l*-leucovorin treatment (88% and 72%, respectively). Pyramidal signs were reported in four studies for those with ASD [21,34,35,67] and in two studies for those without ASD [21,35]. Response was relatively high for those with ASD (76%) while relatively low for those without ASD (33%), although there was wide variation in response rates across studies in both groups.

The response of dyskinesias and other movement disorders to *d,l*-leucovorin treatment was examined in ASD in four studies [21,33–35] and in children without ASD in three studies [21,33,35]. Movement disorders improved with *d,l*-leucovorin in 47% of those with ASD while the response rate was much lower for those without ASD (18%).

Six studies examined epilepsy response for those with ASD [21,33–35,67,69] while four studies examined response to epilepsy for those without ASD [21,33,35,67]. Epilepsy improved in 75% of children with ASD, but the response rate was somewhat lower (54%) and much more variable for those without ASD since there was a large variation across studies, perhaps driven by the overall small number of cases.

Treatment with *d,l-*leucovorin was reported to improve CSF 5-MTHF concentrations in several studies. One year of 0.5–1 mg/kg/day *d,l-*leucovorin treatment normalized 5-MTHF CSF concentrations in 90% of children in one case-series [20]; 0.5–1 mg/kg/day *d,l*leucovorin improved CSF 5-MTHF concentrations in seven children in another study [35]; *d,l-*leucovorin 1–3 mg/kg/day over a 12-month period led to improvements in CSF 5-MTHF concentrations in 21 patients in a third study [34]; *d,l*-leucovorin, 0.5–9.0 mg/kg/day orally, followed by *d,l*-leucovorin, 6 mg/kg Q6 h for 1d IV monthly for 6 months normalized CSF 5-MTHF in a fourth study [69]. Additional treatments which resulted in clinical improvements included the removal of cow's milk in one study [35]. One study reported a trend towards more robust improvements in younger children as compared to older children [34].

#### 3.2.2. Treatment with *d*,*l*-Leucovorin in General ASD: Leucovorin Only

Supplementary Table S6 lists the five studies [37,72,74,80,81] performed by three different sets of investigators which have examined the use of *d,l-*leucovorin in children with idiopathic ASD without additional treatments in order to determine if *d,l-*leucovorin administered by itself is a useful treatment for ASD. Table 4 outlines the effect sizes of some of the key outcome measures used in these studies.

**Table 4.** Outcome measures represented in effect size in key studies which have used *d,l*-leucovorin. Cohen's d' was calculated for studies which provided enough information to make such calculations. For controlled studies, the effect size represented the difference between the treatment and the control group, whereas for uncontrolled studies, the effect size was calculated only for the treatment. Effect sizes were considered small if Cohen's d' was 0.2; medium for Cohen's d' of 0.5, and large if Cohen's d' was 0.8. Effects in bold are statistically significant.


In a medium-sized (*n* = 44) open-label, prospective, controlled study which used a wait-list control group that did not receive any new interventions, children with ASD who were known to be positive for a FRAA were treated with 2 mg/kg/day (max 50 mg per day) of *d,l-*leucovorin over a mean period of 4 months [37]. Using the Parent Rated Autism Symptomatic Change Scale, significant improvements were reported in verbal communication, expressive and receptive language, attention and stereotypy with mostly large effect sizes (See Table 4). Interestingly, improvement in verbal communication and expression language demonstrated greater improvement as age increased in children who were negative for the binding FRAA but demonstrated lesser improvement as age increased for children who were positive for the binding FRAA.

One study reported nonsignificant improvements with *d,l*-leucovorin in 12 patients with ASD using 2 mg/kg/day (max 50 mg/day) of *d,l*-leucovorin over 12 weeks. Nonsignificant improvements were observed on the ABC (2.4-point improvement) and SRS (7.8-point improvement) while a 0.8-point nonsignificant worsening on the PedsQL was also observed. Urinary metabolites showed changes during the study including a 24.8-fold increase in 5MTHF concentrations; a 10.1-fold increase in 1-stearoyl-2-arachidonoyl-GPC; a 9.3-fold increase in 1-stearoyl-2-oleoylGPC; a 9.2-fold increase in alpha-tocopherol; and a 7.8-fold increase in 1-stearoyl-2linoleoyl-GPC (7.8); however, the authors noted that the study lacked to power to determine if changes in urinary metabolites predicted treatment response [80].

Two placebo-controlled studies of *d,l*-leucovorin used *d,l*-leucovorin in children with idiopathic ASD without additional treatments. In a medium-sized (*n* = 48) double-blind, placebo controlled (DBPC) study of 48 children with ASD without known CFD, 23 children received *d,l*-leucovorin calcium (2 mg/kg/day; maximum 50 mg/day) and 25 received a placebo [72]. Significant improvements were seen in the primary outcome measure of verbal communication with an overall medium-to-large effect size and a larger effect size for those positive for at least one FRAA (Table 4). The primary outcome measure exceeded the minimal clinically important difference defined as a change of five standardized points on the language assessments over 3 months. Improvements were also observed in the secondary outcome measures of the VABS daily living skills, ABC irritability, social withdrawal, stereotypy, hyperactivity and inappropriate speech; and in the Autism Symptom Questionnaire (ASQ) stereotypic behavior and total score. ABC social withdrawal, stereotypy, and inappropriate speech and ASQ stereotypic behavior and total score exceeded the predefined minimal clinically important difference. The number needed to treat (NNT) for improvements in verbal communication was 2.4 in all treated children, and 1.8 in children who were positive for at least one FRAA [72].

The second placebo-controlled study was a smaller (*n* = 19) single-blind, placebocontrolled study of 19 children with ASD; 9 children received *d,l-*leucovorin (5 mg twice daily; 0.29–0.63 mg/kg/day) for 12 weeks and 10 children received a placebo [74]. Significant improvements were found in ADOS total score and social interaction subscale with large to very large effect sizes and in the communication subscale with a medium effect size (See Table 4). These changes were significant in the treated group but not in the placebo-control group. The SRS, completed by the parents, showed nonsignificant improvement with a very small effect size.

In a retrospective national survey of 1286 participants with ASD or their parents/ caregivers, a number of nutritional supplements were rated for changes in behaviors and AEs. Higher dose folinic acid (more than 5 mg/day orally) improved cognition in 33%, attention in 29%, and language/communication in 24%. A moderate dose of folinic acid (below 5 mg/day orally) improved language/communication (20%). The overall adverse effect rating was minimal [81].

These series of studies provide evidence that *d,l-*leucovorin is helpful for a wide variety of core and associated ASD symptoms. The three sets of investigators used very different doses of *d,l-*leucovorin. Three studies [37,72,80] used a relatively high dose (2 mg/kg/day; maximum 50 mg/day) while one study [74] used a much lower dose (5 mg twice daily; 0.29–0.63 mg/kg/day) but both dosing parameters were associated with significant clinical improvements.

#### 3.2.3. Treatment with *d,l*-Leucovorin in General ASD: Combined with Other Supplements

Six studies (spanning seven reports) from four sets of investigators used *d,l-*leucovorin in combination with nutritional supplements or other medications to treat ASD (Supplementary Table S7). The most recent study added *d,l*-leucovorin or placebo to risperidone in a DBPC study [75]. One set of investigators examined a multivitaminmineral complex (MVMC) in two controlled studies [73,76] (one study was controlled with a placebo group and one utilized untreated children as controls), while another set of investigators examined *d,l-*leucovorin along with other indicated treatments using a prospective clinical protocol with a control group of ASD children receiving only standard behavioral and educational therapy without medical interventions [44]. Finally, one set of investigators examined *d,l-*leucovorin along with other treatments in an open-label fashion without comparison groups [77–79]. All of these studies were performed on children with ASD without known CFD.

In a medium-size (*n* = 55) DBPC study, children received either *d,l-*leucovorin (2 mg/kg/day up to 50 mg daily) or placebo in two divided daily doses along with risperidone for 10 weeks [75]. Risperidone was started at 0.5 mg and increased by 0.5 mg weekly up to 1 mg for children <20 kg and 2 mg for children ≥20 kg. The ABC inappropriate speech was the primary outcome measure with the remainder of the ABC subscales as the secondary outcome measures. All ABC subscales improved more in the leucovorin group as compared to the placebo group with statistical significance in all subscales except for social withdrawal. The authors used two different measures of effect size Cohen d' and η <sup>2</sup> which provided different estimates of the effect sizes with η 2 showing medium effect sizes and Cohen d' demonstrating extremely large effect sizes. Due to this discrepancy, these results are not listed in Table 4 because of their ambiguity.

In a large (*n* = 141) DBPC study, children with ASD were treated with either a MVMC which contained 550 µg of *d,l-*leucovorin (*n* = 72) or a placebo (*n* = 69) for 3 months. The Parental Global Impressions-Revised (PGI-R) demonstrated improvements in communication and behavior (See Table 4). In addition to the *d,l-*leucovorin, the active treatment contained vitamins A, C, D3, E, K, B1-B6, B12, folic acid, biotin, choline, inositol, mixed carotenoids, mixed tocopherols, CoEnzyme Q10, N-acetylcysteine, calcium, chromium, copper, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, sulfur and zinc [73].

In another medium-sized (*n* = 67) prospective, open-label, controlled study of 67 individuals with ASD, a MVMC containing 600 µg folate mixture (*d,l-*leucovorin, folic acid and 5-MTHF combined) per day was given to 37 individuals with ASD for 12 months, while 30 individuals were untreated [76]. Improvements were found in several of the Parental Global Impressions-2 (PGI-2) measures (Table 4) as well as in the Childhood Autism Rating Scale 2 (CARS2) score, Short Sensory Profile, SRS, ABC, Autism Treatment Evaluation Checklist (ATEC) and VABS. Besides the folate mixture, the treatment contained vitamins A, C, D3, E, K, B1-B6, B12, biotin, choline, inositol, mixed tocopherols, CoEnzyme Q10, N-acetylcysteine, Acetyl-L-Carnitine, calcium, chromium, vanadium, boron, lithium, magnesium, manganese, molybdenum, potassium, selenium, sulfur and zinc.

In a large (*n* = 166) prospective, case-control, open-label study, 82 children with ASD (ages 1–15.9 years) were treated with *d,l-*leucovorin 0.5–2 mg/kg/day (maximum 50 mg/day) for 2 years and were compared to 84 untreated children with ASD (ages 1–16.8 years) who were matched for age, gender, CARS score and FRα autoantibody status [44]. The untreated control group of 84 ASD children received only standard behavioral and educational therapy without additional medical interventions. Blood tests were performed to identify nutritional deficiencies and abnormal oxidative stress biomarkers which were treated with other vitamins and minerals (such as vitamins A, C, D, E, zinc, selenium, manganese, and CoEnzyme Q10, when indicated by testing) in the treated children. *d,l-*

Leucovorin was given to the children who had positive FRAAs (62 of 82 children with ASD had FRAAs, 75.6%). The other 20 children without positive FRAAs did not receive *d,l-*leucovorin. This study reported improvements in mean CARS scores from severe ASD (mean (SD): 41.34 (6.47)) to mild or moderate ASD (mean (SD): 34.35 (6.25)) in all age cohorts of the treated group (See Table 4). In the untreated group of 84 children, there was no significant change in the mean CARS score over the study period. The authors reported "complete recovery" in 17 of 82 children (21%).

Two of the open-label, prospective studies from one set of investigators examined the use of *d,l-*leucovorin in a cohort of individuals with ASD without a control group. In the first study which included eight children with ASD, 800 µg of *d,l-*leucovorin and 1000 mg of betaine was given twice a day for 4 months; significant improvements (*p* ≤ 0.05) were found in the concentrations of methionine, S-adenosylmethionine (SAM), homocysteine, cystathionine, cysteine, total glutathione (tGSH) concentrations, SAM:S-adenosylhomocysteine (SAH) and tGSH:GSSG; clinical improvements in speech and cognition were noted by the attending physician but were not formally quantified [77]. James et al., 2009 [78] studied 40 children with ASD and administered 400 µg of *d,l-*leucovorin twice a day and 75 µg/kg methyl-cobalamin injected subcutaneously twice a week. This treatment led to significant increases in cysteine, cysteinyl-glycine, and glutathione concentrations (all *p* < 0.001); significant improvements were observed in all subscales of the VABS [79].

#### *3.3. Adverse Effects Reported with d,l-Leucovorin Treatment in ASD*

Overall, the placebo-controlled studies support the minimal AEs associated with leucovorin treatment. No significant difference in AE frequency as compared to placebo was reported in the single-blind, placebo-controlled study [74], in two DBPC studies [72,75], or in the last DPBC study for patients who followed the protocol [73].

To investigate the consistency of reported AEs, a meta-analysis was performed on reported AEs for patients on leucovorin treatment, separately for studies examining only leucovorin and for those studies which combined leucovorin with other supplements or treatments (Table 5). For studies which only treated with leucovorin, consistently reported AEs included excitement or agitation (11.7%), aggression (9.5%), insomnia (8.5%), increased tantrums (6.2%), headache (4.9%) and gastroesophageal reflux (2.8%). For studies which used leucovorin in combination with other agents, AEs that were consistently reported included worsening behavior (8.5%) and aggression (1.3%).Interestingly, Frye et al., 2020 [9] examined the reported targeted AE of agitation and excitability every 3 weeks during their previous 12-week study of 2018 [72]. This AE was reported at almost the exact same frequency in the treatment and placebo group until the 9th week of treatment when it precipitously dropped in frequency in the treatment, but not the placebo, group, demonstrating the improvement of this reported AE with longer exposure to the medication.

**Table 5.** Meta-analysis of Adverse Effects Associated with Leucovorin in Children with ASD. Bold and italics indicate significant effects across studies.



**Table 5.** *Cont.*

#### **4. Discussion**

This systemic review found CFD is associated with ASD. One cause of CFD is FRAAs which are a common finding in children with ASD. *d,l*-Leucovorin is a proven treatment for CFD that has been studied in ASD and can normalize 5-MTHF concentrations in the CSF.

The meta-analysis found a pooled prevalence of ASD in CFD of 44%. The pooled prevalence of CFD in ASD was 38% with the etiology attributed to FRAAs in 83% of the cases. The pooled prevalence of blocking, binding and either FRAA in idiopathic ASD was 46%, 49% and 71%, respectively. Children with ASD were more likely than their parents to have blocking or at least one FRAA but were not more likely than typically developing (TD) siblings to have FRAAs. For those with ASD, blocking FRAA titers were significantly higher than their parents or TD siblings, while binding FRAA titers were significantly higher than parents but not TD siblings. Children with ASD were more likely to have positive FRAAs as compared to non-related TD children or children with developmental delay without ASD. Children with ASD demonstrated significantly higher blocking and binding FRAA titers than normal controls with the effect size for blocking FRAAs being very large. FRAAs, particularly blocking FRAAs, are highly prevalent in children with ASD, and may serve as a biomarker for treatment.

This systemic review identified 20 studies which described treating individuals with ASD using *d,l*-leucovorin with a dose typically ranging from 0.5 to 2.5 mg/kg/day. For children with ASD and CFD, *d,l*-leucovorin was particularly effective (>75% response rate) for treating ataxia, pyramidal signs and epilepsy, although it also improved ASD symptoms, irritability and movement disorders in eight case-series. In three controlled studies, *d,l*-leucovorin alone was found to consistently improve communication with medium-to-large effect sizes, but also was shown to have a positive effect on core ASD symptoms and associated behaviors (attention and stereotypy) in individual studies with large effect sizes. In five controlled and uncontrolled studies, *d,l*-leucovorin in combination with vitamin and/or mineral supplements was found to significantly improve core ASD symptoms, communication, behavior and associated symptoms with medium-to-large effect sizes. This systemic review found *d,l*-leucovorin is associated with improvements in core and associated symptoms of ASD with the strongest evidence coming from the blinded, placebo-controlled studies. Most studies reported mild to no AEs, and AEs in the placebo-controlled studies were similar in treated and untreated individuals.

#### *4.1. Dosing of d,l-Leucovorin in ASD*

Most studies used 0.5 to 2.5 mg/kg/day of oral *d,l*-leucovorin but one study reported using up to 9 mg/kg/day in a child with ASD and then added 24 mg/kg/day IV (divided into 4 doses) for one day every month for 6 months with a decrease in severity and seizures along with improved eye contact [69]. Therefore, higher doses of *d,l*-leucovorin appear necessary in order to achieve a higher brain folate level and clinical improvements.

#### *4.2. Time Period Needed for Maximal d,l-Leucovorin Treatment Effects*

One case report of a child with CFD and mitochondrial disease (this child did not have ASD) reported improvements with *d,l*-leucovorin over a 3 year period [26]. Other studies in ASD reported significant improvements over periods of 1 year [34] to 2 years [35,44]. However, other much shorter studies also demonstrated significant improvement in ASD symptoms [37,72,74]. Therefore, although some individuals might show a relatively quick response to *d,l*-leucovorin, it may take 1–2 years to observe maximal clinical improvements.

#### *4.3. The Effect of d,l-Leucovorin on the Core Symptoms of ASD*

Some of the improvements with *d,l*-leucovorin in the reviewed studies were in core ASD symptoms (communication and repetitive and stereotyped behavior). To date, there are no FDA approved medications available to treat the core symptoms of ASD and the only two currently approved medications for ASD (aripiprazole and risperidone) are only approved for treating irritability associated with ASD, which is not considered a core symptom of ASD. In addition, aripiprazole and risperidone have been shown in repeated studies to potentially cause long-term metabolic and neurological adverse effects [82]. Therefore, *d,l*-leucovorin is especially promising since many of the reviewed studies found improvements in core ASD symptomology. *d,l*-Leucovorin also has a much better safety profile and less AEs compared to aripiprazole and risperidone.

#### *4.4. Seizures and Treatment with d,l-Leucovorin*

Six studies reported reductions in seizures in children with ASD and CFD using *d,l*leucovorin [20,33,34,67,69] even in patients with difficult-to-control seizures [20,33]. One child had a breakthrough seizure with discontinuation of *d,l*-leucovorin for 2 weeks [67]. It is possible that epilepsy in these children could be caused by CFD or FRAAs [83]. This is potentially an important finding as many treatments for epilepsy in children treat the seizure condition but not the potential underlying cause or contributing factor. Of note, an animal model reported that certain antiepileptic medications might disrupt folate transportation into the CNS [84]. More studies would be helpful in determining if CFD or FRAAs are an underlying cause of seizures in children with ASD and if treatment with *d,l*-leucovorin is a useful medication for mitigating seizures in these children.

#### *4.5. Treatment of d,l-Leucovorin in Patients with Mitochondrial Dysfunction and ASD*

Mitochondrial dysfunction is a common comorbidity in ASD, with studies reporting 30–50% of individuals with ASD possessing biomarkers of mitochondrial dysfunction [6,46] and up to 80% having abnormal electron transport chain activity in immune cells [47,48]. Several studies have linked CFD to mitochondrial disease in individuals without ASD [24–27] and mitochondrial dysfunction in children with ASD [68]. In children with ASD and CFD, meta-analysis revealed a prevalence of mitochondrial dysfunction of 43% as a potential etiology of CFD. Thus, even in the absence of FRAAs, mitochondrial disease and dysfunction should also be considered as a potential cause of CFD in individuals with ASD.

Treatment of mitochondrial dysfunction with mitochondrial-related cofactors and vitamins, including carnitine [85,86], ubiquinol [87] and a "mitochondrial cocktail" containing carnitine, CoEnzyme Q10 and Alpha-Lipoic Acid [88] has been reported to improve some symptoms of ASD. Folate has also been reported to increase ETC Complex I activity in children with ASD and mitochondrial disease and positively modulate the coupling of ETC Complex I and IV and ETC Complex I and Citrate Synthase [89]. *d,l*-Leucovorin rapidly accumulates in mitochondria [90] and is the preferred form of folate in treating mitochondrial dysfunction [91]. *d,l*-Leucovorin has been reported to improve mitochondrial related symptoms and laboratory findings in some patients with mitochondrial disease in doses

ranging from 1–8 mg/kg/day [24,26,27,92] including in one child with ASD [68]. Since mitochondrial dysfunction is relatively common in individuals with ASD [6,46–48,93] and *d,l*-leucovorin appears to help patients with mitochondrial dysfunction [24,26,27,92], one mechanism by which *d,l*-leucovorin might help improve ASD symptoms is by improvements in mitochondrial function.

#### *4.6. Safety of d,l-Leucovorin in ASD*

*d,l*-Leucovorin was first approved in the United States in the 1950s and has been used continuously since then to reduce toxicities associated with folate pathway antagonists. Therefore, it has a strong and long track record of safety. Most of the reviewed studies reported mild to no AEs with *d,l*-leucovorin. Some studies reported mild behavioral problems, diarrhea/constipation, and aggressive behaviors. In the placebo-controlled studies, AEs were similar in treated and untreated individuals. Two studies used *d,l*leucovorin for up to 2 years [35,44] without significant AEs. Two other studies used *d,l*leucovorin for one year without significant AEs [20,34]. Therefore, the use of *d,l*-leucovorin appears to be safe for at least 2 years of use in most individuals with ASD.

#### *4.7. Screening for FRα Autoantibodies in ASD*

The meta-analysis reported a pooled prevalence of a positive FRAA in children with ASD and concomitant CFD [21,34,35,68] of 83% (69%, 94%) with consistency across studies. Two studies [35,37] reported a significant correlation between higher blocking FRAA concentration and lower CSF levels of 5-MTHF. One study reported that children with ASD who had higher titers of FRAAs had less robust improvements [44]. Only two studies reported one child each with a mutation in the FOLR1 gene which could account for CFD [67,69]. These findings suggest that FRAAs are the major cause of CFD in children with ASD. One set of authors suggested screening young children and infants who have developmental delay and ASD features for FRAAs and starting treatment as soon as possible with *d,l*-leucovorin, especially since younger children generally show more robust improvements [34]. Some patients have intermittently positive FRAA levels and may need to be tested several times if they are negative [45]. Another approach which has been suggested by some authors is an empiric trial of *d,l*-leucovorin in children with ASD without performing a lumbar puncture to confirm CFD, especially given the excellent safety profile of *d,l*-leucovorin [94]. Additionally, one study reported improvements with *d,l*-leucovorin in children with ASD who did not have known CFD and did not possess positive FRAAs [72], further suggesting that empiric treatment in individuals with ASD with *d,l*-leucovorin is a reasonable approach.

Evidence has linked the FRAAs with 5-MTHF concentrations in the CSF in ASD and demonstrated that they can predict response to *d,l*-leucovorin treatment, suggesting that FRAAs are involved in the disruption of folate metabolism in patients with ASD. Additionally, animal models have validated their pathophysiological mechanism [95,96]. However, the meta-analysis suggested a high rate of FRAAs not only in children with ASD but also in their parents and TD siblings, but not in unrelated TD controls or children with developmental delay without ASD. This suggests that FRAA may be one of several mechanisms involved in the disruption of folate metabolism that can contribute to CNS folate disruption. Other factors may be involved. For example, if a child with ASD has FRAAs and other medical comorbidities such as mitochondrial dysfunction, this might explain why a sibling (without these medical comorbidities) can have a positive FRAA but not develop ASD. Polymorphisms in folate genes are also overrepresented in children with ASD and their mothers, suggesting that FRAAs are not alone is disrupting folate metabolism. It is possible that combinations of several mechanisms involved in disrupting folate metabolism many be needed to lead to enough disruption in neurodevelopment to lead to ASD. Timing may also be an important factor as FRAAs and CFD have been related to other psychiatric disorders when they occur outside of childhood such as in schizophrenia [53] and depression [97]. Of note, none of the reviewed studies examined

a potential correlation between ASD severity and FRAA concentrations. In the future, studies that examine this would be useful.

#### *4.8. Adjunctive Treatments Studied for FRAA Positive Patients*

Cow's milk appears to regulate FRAA titers; 6 months of a milk-free diet resulted in a significant decrease in FRAA titers with re-exposure to cow's milk increasing this titer, with the titer often rising above the original titer level before initially discontinuing cow's milk [35]. Concomitant with the decrease in the FRAA titers, patients with CFD who went on a cow's milk free diet demonstrated improvements in ataxia, improved seizure control, and improved ASD symptoms. This is believed to occur because milk contains the FRα protein which may react immunologically in the gut or cause an increase in cross-reactive FRAAs in the blood. Several types of diets (such as a casein-free diet) which have been reported to show some effectiveness in ASD are milk-free diets [98,99] although this has not been found in some studies [100]. Thus, it is possible that dietary treatments not uncommonly used to treat children with ASD may have a therapeutic effect by lowering the concentration of FRAAs in the blood. Unfortunately, large studies have not examined the potential benefit of adding a milk-free diet to *d,l*-leucovorin treatment but it is probably prudent to recommend a milk-free diet when FRAAs are present. Interestingly, in the group of patients with CFD who continued to drink bovine milk, the FRAA concentrations continued to rise over a two-year period [35]. In this study, the use of goat milk caused less elevation in the FRAA concentration and thus might be a better alternative to bovine milk [35]. It is important to recognize that this effect may not generalize to other forms of dairy that do not have intact milk proteins. Indeed, dairy is an important source of calcium, which is a critical nutrient in childhood for bone health.

#### *4.9. Treatments That Support Folate Transport into the Brain*

Presumably, when the FRα is partially blocked, the RFC may be the main alternative transportation mechanism of folates into the brain. In a human cerebral microvascular endothelial cell model, 1,25-dihydroxyvitamin D<sup>3</sup> was shown to up-regulation RFC mRNA and protein expression through activation of the vitamin D receptor [101]. In the same cell model, nuclear respiratory factor 1 and peroxisome proliferator-activated receptor-γ coactivator-1α signaling were found to modulate RFC expression and transport activity with this pathway upregulated by treatment with pyrroloquinoline quinone (PQQ) resulting in increased RFC expression and folate transport activity [102]. Furthermore, 1,25 dihydroxyvitamin D<sup>3</sup> was found to rescue CFD in a knockout FOLR1 mouse model [103].

Interestingly, meta-analysis has shown that vitamin D3 800IU to 2,000IU supplementation improves core ASD symptoms as measured by the Social Responsiveness Scale or CARS in three studies in children with ASD who were not vitamin D deficient [104] and PQQ has been shown to improve social behavior in the bilateral whisker trimming for 10 days after birth in a mouse model which is characterized by its abnormal social behavior [105]. Thus, although no studies have been conducted in ASD or CFD to determine whether these supplements may improve function in children with ASD who have FRAAs or CFD, such supplements may have some utility in these children. Future studies will need to address this possibility.

#### *4.10. Adjunctive Treatments to Support Folate Metabolism*

Folate is essential for several important biochemical pathways, particularly the function of methylation metabolism and the production of purines and pyrimidines. Several studies used *d,l*-leucovorin in combination with other cofactors which could support its metabolism, including methyl-cobalamin, betaine (trimethyl-glycine) and other important cofactors. Many cofactors are essential for optimal functioning of enzymes in the folate cycle; for example, methionine synthase requires cobalamin, and methylenetetrahydrofolate reductase (MTHFR) requires nicotinamide adenine dinucleotide (NAD) which can be derived from niacin. Other factors like betaine support methylation metabolism. No

study has compared the specific cofactors that could best be used in conjunction with *d,l*leucovorin, but it is likely that other cofactors may be useful to optimize folate metabolism. One intriguing possibility is that, along with a CNS folate deficiency, individuals with ASD may also have a CNS cobalamin deficiency [106], so the addition of cobalamin could be critical in some children with ASD.

#### *4.11. Therapeutic Effect of d,l-Leucovorin on Neurotransmitters*

Most important for neurological outcomes for those with a CNS folate deficiency is the connection between folate and the production of neurotransmitters. Being the precursor to purines, folate is essential to produce guanosine-5'-triphosphate which is the precursor of tetrahydrobiopterin (BH4) [107]. BH<sup>4</sup> is an important cofactor for the production of the monoamine neurotransmitters serotonin, dopamine, norepinephrine and epinephrine which are essential for behavioral regulation, mood, social function and cognition. Interestingly, like folate, a central deficiency of BH<sup>4</sup> is associated with ASD [108]; children with ASD respond to BH<sup>4</sup> supplementation [109]; and there is evidence that BH<sup>4</sup> may be transported into the brain through the FRα [110]. The connection between folate and BH<sup>4</sup> can explain how *d,l*-leucovorin can normalize CSF concentrations of serotonin and dopamine in CFD patients [28].

#### *4.12. Implications of FRAAs during Pregnancy*

Rodent models report that exposure of dams to FRAA's during gestation can lead to stereotypies and anxiety in offspring [95]. Further studies showed that *d,l*-leucovorin and/or dexamethasone treatment of dams exposed to FRAAs prevented cognitive, communication and learning problems in the offspring [96]. Human studies have reported an association between FRAAs and subfertility [111], neural tube defects [112], and preterm births [113]. One study found that FRAAs bind to prenatal thyroid tissue, potentially affecting its development and potentially altering hypothalamic-pituitar*y*-axis regulation of thyroid hormones in the offspring [37]. In one case report, a pregnant women with a history of multiple complications in previous pregnancies and who was positive for FRAAs was able to conceive and have a normal pregnancy and delivery with the use of *d,l*-leucovorin, a milk free diet, and a low dose of prednisone given during pregnancy [114].

#### *4.13. Limitation of Published Studies*

Many of the reviewed studies had important limitations. First, there is only one medium-sized [72] and one small-sized [74] blinded, placebo controlled, single center studies that examined treatment only with *d,l*-leucovorin. Thus, clearly larger, multisite trials, which are ongoing [9], are necessary to confirm previous findings. Since many of the studies used *d,l*-leucovorin in combination with other treatments [73,76–79], other treatments may have added to the effects of *d,l*-leucovorin. In addition, studies examining CFD are rather small and do not always use standardized outcome measures [20,21,33–36,67,68].

Interestingly, in the blinded controlled studies, standardized measures of language and social functioning which were obtained by objective and blinded examiners tended to have large effect sizes, whereas parent rated measures tended to have very modest effect sizes. This reflects one of the difficulties in research in ASD where the placebo effect can be large, especially as rated by parents [115]. Such large placebo effects have washed out the effect of the treatment in many studies, resulting in many failed clinical trials. Thus, one of the strengths in the currently conducted blinded trials is the use of standard objective measures of function in blinded observers in addition to parent reported measures. This is especially important when studying more mildly affected children with ASD as the placebo effect appears to be inversely proportional to the severity of the ASD symptoms, so studies with less severe children would be expected to have a larger placebo effect [116]. This effect can explain the larger effect size in parent reported outcomes in the studies which used a non-treatment comparison group [37,76] when compared to the trials which were placebo controlled [72–74].

#### **5. Conclusions**

This systematic review and meta-analysis found *d,l*-leucovorin is associated with improvements in core and associated symptoms of ASD with the strongest evidence coming from the blinded, placebo-controlled studies. FRAAs, particularly blocking FRAAs, are highly prevalent in children with ASD, and may serve as a biomarker for treatment. The high prevalence of FRAAs in families with children with ASD suggests unknown heritability mechanisms that involve additional genetic or environmental factors which contribute to the expression of ASD in those with FRAAs. *d,l*-Leucovorin is an evidencebased treatment for ASD which has significant promise and appears safe and well-tolerated. Further studies would be helpful to confirm and expand on these findings.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/jpm11111141/s1, Table S1: PRISMA Checklist, Table S2: Studies on Children with CFD where ASD was found to be a characteristic, Table S3: Studies on Children with ASD where CSF was measured for possible CFD, Table S4: Studies of FRα autoantibodies in ASD, by year published, Table S5: Response to *d,l*-leucovorin in children with CFD with and without ASD (Total cases with symptoms/total Responses), Table S6: Studies examining only *d,l*-leucovorin in ASD, by year published, Table S7: Studies examining *d,l*-leucovorin along with other supplements or treatments in ASD, by year published.

**Author Contributions:** Conceptualization, methodology, formal analysis, writing—original draft preparation, review and editing, were performed by both D.A.R. and R.E.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** The review did not receive any financial or grant support from any sources.

**Institutional Review Board Statement:** Not applicable. This review is not human research.

**Informed Consent Statement:** Not applicable. This review is not human research.

**Data Availability Statement:** All data are presented within the article.

**Conflicts of Interest:** Frye is funded by the National Institutes of Child Health and Human Development Grant R01HD088528, Department of Defense Grant AR180134 and Autism Speaks Grant 11407 to study the therapeutic effects of leucovorin in autism spectrum disorder. Frye is an uncompensated scientific advisory board member to Iliad Neurosciences Inc. (Plymouth Meeting, PA, USA) which is the commercial laboratory which performs the folate receptor alpha autoantibody tests.

#### **References**


### *Review* **The Effectiveness of Cobalamin (B12) Treatment for Autism Spectrum Disorder: A Systematic Review and Meta-Analysis**

**Daniel A. Rossignol 1,\* and Richard E. Frye <sup>2</sup>**


**Abstract:** Autism spectrum disorder (ASD) is a common neurodevelopmental disorder affecting 2% of children in the United States. Biochemical abnormalities associated with ASD include impaired methylation and sulphation capacities along with low glutathione (GSH) redox capacity. Potential treatments for these abnormalities include cobalamin (B12). This systematic review collates the studies using B12 as a treatment in ASD. A total of 17 studies were identified; 4 were double-blind, placebocontrolled studies (2 examined B12 injections alone and 2 used B12 in an oral multivitamin); 1 was a prospective controlled study; 6 were prospective, uncontrolled studies, and 6 were retrospective (case series and reports). Most studies (83%) used oral or injected methylcobalamin (mB12), while the remaining studies did not specify the type of B12 used. Studies using subcutaneous mB12 injections (including 2 placebo-controlled studies) used a 64.5–75 µg/kg/dose. One study reported anemia in 2 ASD children with injected cyanocobalamin that resolved with switching to injected mB12. Two studies reported improvements in markers of mitochondrial metabolism. A metaanalysis of methylation metabolites demonstrated decreased S-adenosylhomocysteine (SAH), and increased methionine, S-adenosylmethionine (SAM), SAM/SAH ratio, and homocysteine (with small effect sizes) with mB12. Meta-analysis of the transsulfuration and redox metabolism metabolites demonstrated significant improvements with mB12 in oxidized glutathione (GSSG), cysteine, total glutathione (GSH), and total GSH/GSSG redox ratio with medium to large effect sizes. Improvements in methylation capacity and GSH redox ratio were significantly associated with clinical improvements (with a mean moderate effect size of 0.59) in core and associated ASD symptoms, including expressive communication, personal and domestic daily living skills, and interpersonal, play-leisure, and coping social skills, suggesting these biomarkers may predict response to B12. Other clinical improvements observed with B12 included sleep, gastrointestinal symptoms, hyperactivity, tantrums, nonverbal intellectual quotient, vision, eye contact, echolalia, stereotypy, anemia, and nocturnal enuresis. Adverse events identified by meta-analysis included hyperactivity (11.9%), irritability (3.4%), trouble sleeping (7.6%), aggression (1.8%), and worsening behaviors (7.7%) but were generally few, mild, not serious, and not significantly different compared to placebo. In one study, 78% of parents desired to continue mB12 injections after the study conclusion. Preliminary clinical evidence suggests that B12, particularly subcutaneously injected mB12, improves metabolic abnormalities in ASD along with clinical symptoms. Further large multicenter placebo-controlled studies are needed to confirm these data. B12 is a promising treatment for ASD.

**Keywords:** autism spectrum disorder; cobalamin; glutathione; methylation; methylcobalamin; redox metabolism

#### **1. Introduction**

Autism spectrum disorder (ASD) is a common neurodevelopmental disorder affecting 2% of children in the United States [1]. ASD is defined behaviorally by reduced social communication and the existence of restrictive and repetitive behaviors and interests. Although

**Citation:** Rossignol, D.A.; Frye, R.E. The Effectiveness of Cobalamin (B12) Treatment for Autism Spectrum Disorder: A Systematic Review and Meta-Analysis. *J. Pers. Med.* **2021**, *11*, 784. https://doi.org/10.3390/ jpm11080784

Academic Editor: Farah R. Zahir

Received: 12 July 2021 Accepted: 8 August 2021 Published: 11 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

ASD is currently classified as a psychiatric disorder, a number of medical comorbidities and biochemical abnormalities are associated with ASD and may contribute to symptoms. These include immune disorders [2], mitochondrial dysfunction [3,4], oxidative stress [5], seizures [6], gastrointestinal problems [7], and impaired methylation capacity [8]. symptoms. These include immune disorders [2], mitochondrial dysfunction [3,4], oxidative stress [5], seizures [6], gastrointestinal problems [7], and impaired methylation capacity [8]. The methylation cycle recycles homocysteine to methionine, the precursor of S-ade-

communication and the existence of restrictive and repetitive behaviors and interests. Although ASD is currently classified as a psychiatric disorder, a number of medical comorbidities and biochemical abnormalities are associated with ASD and may contribute to

*J. Pers. Med.* **2021**, *11*, x FOR PEER REVIEW 2 of 23

The methylation cycle recycles homocysteine to methionine, the precursor of Sadenosylmethionine (SAM), which is the major methyl donor for many chemical processes in the body including DNA methylation. Importantly, the methylation cycle is intricately connected to redox metabolism through the contribution of homocysteine (Figure 1). Homocysteine is metabolized to cystathionine and then to cysteine, which is the rate-limiting precursor of glutathione (GSH), the major antioxidant in the body. nosylmethionine (SAM), which is the major methyl donor for many chemical processes in the body including DNA methylation. Importantly, the methylation cycle is intricately connected to redox metabolism through the contribution of homocysteine (Figure 1). Homocysteine is metabolized to cystathionine and then to cysteine, which is the rate-limiting precursor of glutathione (GSH), the major antioxidant in the body.

**Figure 1.** The connected folate, methylation, and redox cycles. Ovals represent enzymes and boxes represent metabolites. Red indicates metabolites and enzymes repeatedly noted to be consistently abnormal in ASD. Green highlights treatments that improve the metabolism of these cycles. Folic acid is shown in yellow as it is a suboptimal treatment because it is oxidized and has to be converted to active forms of folate. **Figure 1.** The connected folate, methylation, and redox cycles. Ovals represent enzymes and boxes represent metabolites. Red indicates metabolites and enzymes repeatedly noted to be consistently abnormal in ASD. Green highlights treatments that improve the metabolism of these cycles. Folic acid is shown in yellow as it is a suboptimal treatment because it is oxidized and has to be convertedto active forms of folate.

The first study to formally evaluate methylation and redox capacity in ASD reported significantly lower plasma levels of methionine, SAM, homocysteine, cystathionine, cysteine, and total GSH, and significantly higher concentrations of S-adenosylhomocysteine (SAH), adenosine, and oxidized GSH (GSSG) in 20 children with ASD compared to 33 controls. The lower SAM/SAH ratio is indicative of impaired methylation capacity and a The first study to formally evaluate methylation and redox capacity in ASD reported significantly lower plasma levels of methionine, SAM, homocysteine, cystathionine, cysteine, and total GSH, and significantly higher concentrations of S-adenosylhomocysteine (SAH), adenosine, and oxidized GSH (GSSG) in 20 children with ASD compared to 33 controls. The lower SAM/SAH ratio is indicative of impaired methylation capacity and a lower GSH with elevated GSSG represents impaired redox capacity [8]. These initial findings have been confirmed in several other prospective, controlled studies in blood [9–12] and brain samples [13–15], and a meta-analysis has confirmed the consistency of these

findings across multiple studies and multiple laboratories [5]. A meta-analysis of 22 studies confirmed a consistent finding of impaired DNA methylation in ASD [16] and impaired DNA methylation has been associated with mitochondrial dysfunction in ASD [17]. These biochemical abnormalities have even been proposed to be diagnostic of ASD as they could predict the presence of ASD with a 98% correct classification rate using Fisher discriminant analysis [18].

Impairments in the methylation cycle commonly lead to an accumulation in homocysteine, a finding which was reported in a number of studies in ASD [19–21], including a meta-analysis of 31 studies [22]. However, some studies also report lower homocysteine levels in some children with ASD [8,9]. This may be related to the fact that homocysteine is the precursor to two pathways. First, methionine synthase (MS), a B12 and folatedependent enzyme, converts homocysteine to methionine; one study reported lower MS mRNA in the brains of individuals with ASD (especially in younger children) compared to controls [23]. Lower MS mRNA would presumably result in lower production of the MS enzyme, which would slow the conversion of homocysteine to methionine and result in a build-up of homocysteine. However, homocysteine is also the precursor to cysteine, which is the rate-limiting substrate for the production of GSH. ASD is associated with lowered concentrations of GSH in blood [8,9,24], mitochondria [25], lymphoblasts [25], and brain tissue [15,26], and a recent meta-analysis found consistent depletion of GSH in ASD across 14 studies [5]. Since GSH is produced from homocysteine (and cysteine), GSH depletion may lead to the depletion of homocysteine by consuming it as a precursor. Impaired methylation can also weaken sulphation pathways; lower blood and higher urinary sulfate concentrations have been reported in ASD as early as 1997 [27–29].

Treatments that have been shown to improve methylation and GSH production in ASD include methylcobalamin (mB12), betaine (anhydrous trimethylglycine), and leucovorin (folinic acid) [8,11]. Cobalamin (B12) exists in several forms: cyanocobalamin (cB12, a synthetic form of B12 not found in a natural form; available by injection or orally) and three naturally occurring forms: mB12, hydroxycobalamin (hB12), and adenosylcobalamin (aB12); the latter three natural forms have better bioavailability compared to cB12 and are available in an injectable form or orally [30]. B12 is important for brain development, and B12 deficiency has been associated with regression in social interaction in one child [31] and in another who developed Childhood Disintegration Disorder [32].

Although a number of studies have used B12 as a treatment in children with ASD, these studies have not been systematically reviewed to date. This systematic review identifies and collates the studies using B12 as a treatment in individuals with ASD. When possible, this review lists out the type(s) of B12 used along with dosing, route of administration, types of studies, clinical outcomes, and adverse events (AEs). Meta-analysis was used to examine biochemical changes in methylation and redox metabolism as well as AEs.

#### **2. Materials and Methods**

#### *2.1. Search Process*

A prospective protocol for this systematic review was developed a priori, and the search terms and selection criteria were chosen in an attempt to capture all pertinent publications. A computer-aided search of PUBMED, Google Scholar, EmBase, Scopus, and ERIC databases from inception through June 2021 was conducted to identify pertinent publications using the search terms 'autism', 'autistic', 'Asperger', 'ASD', 'pervasive', and 'pervasive developmental disorder' in all combinations with the terms "MB12", "Methylcobalamin", "Cobalamin", "B12", "Cyanocobalamin", "Hydroxycobalamin", "Adenosylcobalamin", and "Vitamin B12." References cited in identified publications were also searched to locate additional studies.

#### *2.2. Study Selection and Assessment*

This systematic review and meta-analysis followed PRISMA guidelines [33]. The PRISMA Checklist is found in Supplementary Table S1, and the PRISMA Flowchart is

displayed as Figure 2. One reviewer screened titles and abstracts of all potentially relevant publications. Studies were initially included if they (1) involved individuals with ASD; and (2) reported on the use of B12 as a treatment in ASD. Articles were excluded if they: (1) did not involve humans (for example, cellular or animal models); and or (2) did not present new or unique data (such as review articles or letters to the editor). After screening all records, 17 publications met inclusion criteria (see Figure 2); two reviewers then independently reviewed these articles for inclusion and assessed factors such as the risk of bias. As per standardized guidelines [34], selection, performance detection, attrition, and reporting biases were considered. Two DBPC studies that used only injected mB12 were identified, but since only one of these studies provided detailed descriptive statistics of the clinical outcome measures [35], a meta-analysis could not be conducted on clinical outcome measures. displayed as Figure 2. One reviewer screened titles and abstracts of all potentially relevant publications. Studies were initially included if they (1) involved individuals with ASD; and (2) reported on the use of B12 as a treatment in ASD. Articles were excluded if they: (1) did not involve humans (for example, cellular or animal models); and or (2) did not present new or unique data (such as review articles or letters to the editor). After screening all records, 17 publications met inclusion criteria (see Figure 2); two reviewers then independently reviewed these articles for inclusion and assessed factors such as the risk of bias. As per standardized guidelines [34], selection, performance detection, attrition, and reporting biases were considered. Two DBPC studies that used only injected mB12 were identified, but since only one of these studies provided detailed descriptive statistics of the clinical outcome measures [35], a meta-analysis could not be conducted on clinical outcome measures.

"Adenosylcobalamin", and "Vitamin B12." References cited in identified publications

This systematic review and meta-analysis followed PRISMA guidelines [33]. The PRISMA Checklist is found in Supplementary Table S1, and the PRISMA Flowchart is

*J. Pers. Med.* **2021**, *11*, x FOR PEER REVIEW 4 of 23

were also searched to locate additional studies.

*2.2. Study Selection and Assessment* 

**Figure 2.** PRISMA 2020 flow diagram for this systematic review. **Figure 2.** PRISMA 2020 flow diagram for this systematic review.

#### *2.3. Meta-Analysis 2.3. Meta-Analysis*

MetaXL Version 5.2 (EpiGear International Pty Ltd., Sunrise Beach, Queensland, Australia) was used with Microsoft Excel Version 16.0.12827.20200 (Redmond, WA, USA) to perform the meta-analysis. The data from this meta-analysis is available upon request to the authors. Random-effects models, which assume variability in effects from both sampling error and study level differences [36,37], were used to calculate incidence across studies (AEs Meta-analysis) while pooled Cohen's d' (a measure of effect size) was calculated from the standardized mean difference of outcome measures using the inverse variance heterogeneity model since it has been shown to resolve issues with underestimation of the statistical error and spuriously overconfident estimates with the random effects model when analyzing continuous outcome measures (Biochemistry Meta-analysis) [38]. Effect sizes were considered small if Cohen's d' was 0.2; medium if Cohen's d' was 0.5; MetaXL Version 5.2 (EpiGear International Pty Ltd., Sunrise Beach, Queensland, Australia) was used with Microsoft Excel Version 16.0.12827.20200 (Redmond, WA, USA) to perform the meta-analysis. The data from this meta-analysis is available upon request to the authors. Random-effects models, which assume variability in effects from both sampling error and study level differences [36,37], were used to calculate incidence across studies (AEs Meta-analysis) while pooled Cohen's d' (a measure of effect size) was calculated from the standardized mean difference of outcome measures using the inverse variance heterogeneity model since it has been shown to resolve issues with underestimation of the statistical error and spuriously overconfident estimates with the random effects model when analyzing continuous outcome measures (Biochemistry Meta-analysis) [38]. Effect sizes were considered small if Cohen's d' was 0.2; medium if Cohen's d' was 0.5; and large if Cohen's d' was 0.8 or higher [39]. Cochran's Q was calculated to determine heterogeneity of effects across studies, and when significant, the I<sup>2</sup> statistic (Heterogeneity Index) was calculated to determine the percentage of variation across studies that was due to heterogeneity rather than chance [40,41], and the Luis Furuya-Kanamori (LFK) Index derived from Doi plots was reviewed for significant asymmetries (>±2) [42,43].

#### **3. Results**

This section will discuss the type(s), routes, and dosing parameters of B12 used along with the type of study and phenotypes of patients (Section 3.1), biochemical changes (Section 3.2), clinical outcome measures (Section 3.3), and AEs (Section 3.4) associated with B12 treatment studies. In the clinical outcomes section, the outcomes are presented and organized by study type.

### *3.1. B12 Administration: Type of B12, Route, Dosing, and Type of Study* 3.1.1. Type of B12

Five studies did not specify the type of B12 used [32,44–47]. Two studies used oral cB12 [48,49]. One study used a 50/50 oral mixture of mB12 and cB12 [50]. One study initially used cB12 injections and switched to mB12 injections due to worsening anemia from cB12 in 2 children and also used oral hB12 in one child [51]. The remaining 8 studies used mB12 [8,11,35,52–56]. Thus, of the 12 studies specifying the type of B12 used, 10 (83%) used mB12 by itself or in combination with cB12.

#### 3.1.2. Route Parameters of B12

Two studies did not specify the route of B12 administration [45,52]. Six studies used subcutaneously (SC) injected mB12 [8,11,35,53,55,56] and 4 studies used intramuscularly injected B12 [32,46,47,51]. Five studies used oral B12 [44,48–50,54]. One study used both injected cB12 and oral hB12 [51]. Thus, of the 15 studies specifying the route of administration, 10 (67%) used an injected form.

#### 3.1.3. Dosing of B12

Six studies (including two DBPC studies) used subcutaneous mB12 injections at a dose ranging from 64.5 to 75 µg/kg/dose [8,11,35,53,55,56]. One study used a lower dose of 25–30 µg/kg/dose (up to 1500 µg), but the route of administration was not specified [52]. One study used oral mB12 at a dose of 500 µg per day [54] while 2 studies used oral cB12 in doses of 500–1600 µg per day along with a multivitamin/mineral supplement (MVI) [48,49]. Another study used a 50/50 mixture of mB12 and cB12 500 µg per day combined in an MVI [50]. One study used oral B12 1.2 µg per day but did not specify the type of B12 given [44]. One study used 1000 µg of B12 (type not specified) intramuscularly for 5 days and then weekly for 8 weeks [32]. One study used mB12 intramuscularly at 1 mg once per week in 2 children and 10 mg of oral hB12 in another child [51]. Finally, 3 studies did not list the dose of B12 given [45–47].

#### 3.1.4. Types of B12 Studied

Of the 17 treatments studies, 2 were DBPC studies using mB12 injections alone [35,55], 2 were DBPC studies using oral cB12 in a MVI preparation [48,49], 1 was a prospective, controlled study [50], 6 were prospective, uncontrolled studies [8,11,44,45,52,53], and 6 were retrospective case series/reports [32,46,47,51,54,56]. Two studies reported on the same cohort of patients [11,53].

#### 3.1.5. Phenotypes of Patients in B12 Studies

Table 1 lists the phenotypes for the 17 studies. Six studies used DSM-4 criteria to diagnose ASD [8,11,44,52,55,56]. Six studies used autism diagnostic observation schedule (ADOS) and/or autism diagnostic interview (ADI) to confirm the diagnosis [35,45,50,51,54,55]. Five studies did not specify criteria for autism [32,46–49]. Two studies only enrolled patients with abnormal biochemical findings (such as methylation abnormalities or oxidative stress) [11,53].


**Table 1.** Phenotypes of autism in the 17 reviewed studies, by year published.

Autism Diagnostic Interview (ADI); Autism Diagnostic Observation Schedule (ADOS); Childhood Autism Rating Scale (CARS); childhood disintegrative disorder (CDD); Diag-

nostic and Statistical Manual of Mental Disorders, fourth edition (DSM-4); Gilliam Autism Scale (GARS); glutathione (GSH); Preschool Language scale, Fourth Edition (PSL4); oxidized glutathione (GSSG); Pervasive Developmental Disorder, Not Otherwise Specified (PDD-NOS); S-adenosylhomocysteine (SAH); S-adenosylmethionine (SAM); years old (yo).

#### *3.2. Biochemical Changes with B12 Treatment*

#### 3.2.1. Review of Studies

Eleven studies examined biochemical changes with B12 treatment and are outlined in Table 2. A 12-week randomized DBPC crossover study of 30 children with ASD (ages 3–8 yo) used mB12 64.5 µg/kg subcutaneous injections every 3 days for 6 weeks or a placebo. GSH and GSH/GSSG were measured before and after treatment. No significant changes in GSH related metabolites were observed comparing the treatment group to the placebo group. However, significant improvements in GSH and the GSH redox ratio were found in a subgroup of 9 children who were considered "responders" based on significant improvements on the CGI and 2 other behavioral outcomes, suggesting these may be biomarkers for identifying children who respond to mB12 treatment [55]. In another randomized DBPC study, 57 children with ASD received 75 µg/kg mB12 subcutaneous injections every 3 days for 8 weeks or placebo injections, and clinical improvements were positively associated with increased plasma methionine, decreased SAH, and improved methylation capacity as measured by the SAM/SAH ratio [35].

**Table 2.** Biochemical outcomes for treatment studies of cobalamin injections for autism spectrum disorder. Clinical global impression scale (CGI); subcutaneous (SC); years old (yo).



**Table 2.** *Cont.*

In a prospective study of 8 children with ASD, 75 µg/kg mB12 SC two times per week for one month was given along with folinic acid (800 µg twice a day orally) and betaine (1000 mg twice a day orally). This treatment led to significant decreases in adenosine and GSSG as well as significantly increased levels of methionine, cysteine, GSH, SAM/SAH, and GSH/GSSG [8].

In a study of 40 children with ASD, 75 µg/kg mB12 SC 2 times per week for 3 months given with folinic acid (400 µg twice per day) led to increased cysteine, cysteinylglycine, and GSH, and decreased GSSG [11]. Improvements in GSH redox status were associated with improvements in expressive communication, personal and domestic daily living skills, and interpersonal, play-leisure, and coping social skills [53].

In a case series of 3 patients with ASD with transcobalamin deficiency/transcobalamin II (TCN2) mutations (one patient had metabolic acidosis and pancytopenia), cB12 intramuscular injections (1 mg once or twice weekly) were started along with carnitine. However, both patients developed acute anemia on cB12 injections, which improved when switching to mB12 intramuscular injections 1 mg weekly; in the third child, homocysteine levels normalized with 10 mg per day of hB12 orally once per day [51].

In a randomized DBPC study of 141 children and adults with ASD, a MVI containing 500 mcg of oral cB12 led to significant improvements compared to placebo in total sulfate, SAM, reduced glutathione, GSSG:GSH, nitrotyrosine, adenosine triphosphate (ATP), NADH, and NADPH [49].

In a study of 13 patients with ASD, mB12 25–30 µg/kg/day (up to 1500 µg/day; route of administration not specified) was given for 6–25 months. Five patients had normal B12 serum levels and after the study, 4 cases had above normal serum B12 without any apparent AEs [52].

In another study of 30 children with ASD, oral B12 (type not specified) 1.2 µg per day given with vitamin B6 200 mg and folic acid 400 µg led to a significant reduction in urinary homocysteine [44]. In a prospective, uncontrolled study of 127 children with ASD, B12 treatment (type and route of administration not specified) was administered to 38% of the ASD group along with mitochondrial supplements. Analysis showed that B12 treatment improved complex I activity compared to not supplementing with B12. In addition, B12 treatment was associated with the better coupling of Complex I and Citrate Synthase mitochondrial enzymes [45].

Finally, one retrospective study examined 24 children with ASD (mean age 9.3 ± 3.5 years) who received SC mB12 injections (75 µg/kg, given every 1–3 days) and compared urinary and plasma cobalt levels to 48 children (mean age 8.9 ± 3.7 years) who did not receive mB12 injections. The mean plasma cobalt concentration in the mB12 group was 0.82 ± 0.19 µg/L

compared to 0.12 ± 0.10 in the untreated group (*p* < 0.001). The investigators noted that the study was limited as it could not determine what form of cobalt was present (free or bound in mB12), how the cobalt was distributed in the body, and what the tissue levels would be. This study did not report clinical outcomes [56].

#### 3.2.2. Meta-Analysis of Biochemical Changes Related to Methylcobalamin

Only three studies [8,11,35] provided enough detailed information about biochemical metabolites before and after treatment to be included in a meta-analysis. All studies used subcutaneously injected mB12. One study [35] was a DBPC study that used mB12 alone, and two studies [8,11] were uncontrolled prospective studies, which used mB12 with additional treatments. Of these latter two studies, one study [8] obtained biochemical measurements three months after starting daily 800 µg of leucovorin (folinic acid) and 1000 mg of betaine and then three months after adding mB12. In the meta-analysis, the biochemical measurements directly before and after adding mB12 were used rather than using the baseline measures obtained before any treatments were added. For the second study [11], mB12 was provided along with daily 800 µg of leucovorin (folinic acid). Common metabolites across all three studies were analyzed, and the results are outlined in Table 3. Even though these 3 studies used different dosing parameters and two used mB12 with folinic and one without, both mB12 and folinic have been shown to lead to biochemical changes in methylation metabolites and work in a synergistic fashion. Therefore, combining these studies to examine biochemical changes was felt to be advantageous.


**Table 3.** Meta-analysis of biochemical changes with subcutaneously injected methylcobalamin in children with ASD. Oxidized glutathione (GSSG); S-adenosylhomocysteine (SAH); S-adenosylmethionine (SAM); methylation capacity (SAM/SAH); total GSH (tGSH); total glutathione redox ratio (tGSH/GSSG) \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.0001.

Meta-analysis of the methylation metabolites demonstrated small effect sizes that were in the direction expected for improvement of methylation metabolism for the majority of the metabolites. Specifically, the weighted mean differences suggested an increase in Methionine, SAM, and SAM/SAH ratio, and a decrease in SAH. None of the effects were overall statistically significant, and the Cochran's Q statistic suggested that the variation was not due to heterogeneity across studies but rather due to random chance. However, for homocysteine, although the overall effect was not significant, the variation in the measurement was found to be largely due to variation across studies with a slight asymmetry in the Doi plots. This was due to one study [35] demonstrating a decrease in homocysteine with treatment and two studies [8,11] demonstrating an increase in homocysteine with treatment.

Meta-analysis of the redox metabolism metabolites demonstrated statistically significant medium to large effect sizes that were in the direction expected for an improvement in redox metabolism for all of the metabolites examined. Specifically, the weighted mean differences demonstrated an increase in cysteine, total GSH, and total GSH/GSSG redox ratio, and a decrease in GSSG. The I<sup>2</sup> statistic suggested that the variation was due to heterogeneity across studies, specifically one study [35] demonstrating more marginal effects as compared to the other two studies [8,11]. However, Doi plots did not demonstrate any significant asymmetries.

#### *3.3. Clinical Outcomes with B12 Treatment*

Twelve studies examined clinical outcomes and are outlined in Table 4. Improvements reported in these studies included sleep, hyperactivity, gastrointestinal problems, tantrums, nonverbal IQ, expressive, written and receptive communication skills, daily living skills, social skills domains, vision, eye contact, echolalia, stereotypy, anemia, and nocturnal enuresis.

**Table 4.** Studies of cobalamin injections for autism spectrum disorder with clinical outcomes. Childhood autism rating scale (CARS); clinical global impression scale (CGI); subcutaneous (SC); years old (yo).



**Table 4.** *Cont.*

3.3.1. Double Blind Placebo-Controlled Studies

Four DBPC studies examined B12 use in ASD, with two of the studies administering it alone as an injection in the form of mB12 and two studies using cB12 in an orally administered MVI.

In a 12-week randomized DBPC crossover study, 30 children with ASD (ages 3–8 yo) received mB12 64.5 µg/kg SC injections every 3 days for 6 weeks or placebo injections for 6 weeks; a 6-month extension study was also performed. No significant changes in clinical outcomes were observed comparing the treatment group to the placebo group. A total of 22 children entered the 6-month open-label extension portion of the study, but no clinical outcomes were reported for this time period in the publication. A subgroup of 9 children were considered "responders" based on significant improvements on the CGI and 2 other behavioral outcomes. The authors commented that the crossover design without a washout period between the two treatments might have made it more difficult to see a significant difference between groups [55]. In another randomized DBPC study by some of the same investigators, 57 children with ASD received 75 µg/kg mB12 subcutaneous injections every 3 days for 8 weeks or placebo injections. A significant improvement in CGI-I as rated by clinicians was observed with mB12 treatment, but no significant improvements in the treatment group were observed in the parent-rated aberrant behavior checklist (ABC) or social responsiveness scale (SRS). Notably, this study did not include the crossover design, which was felt to be a weakness in design from the first study [35].

In a randomized DBPC 3-month study of 20 children with ASD (ages 3–8 yo), a MVI, which also contained 1200–1600 mcg of oral cB12, led to significant improvements in sleep and gastrointestinal problems compared to placebo [48]. In another randomized DBPC study of 141 individuals with ASD, a MVI containing 500 mcg of oral cB12 led to significant improvements in several behavioral scales compared to placebo, including hyperactivity, tantrums, and receptive language [49].

#### 3.3.2. Prospective, Controlled Study

In a prospective, controlled, single-blind (clinical evaluators blinded) 12-month study, 37 individuals with ASD were treated with an oral MVI containing 500 mcg of B12 (50% as mB12 and 50% as cB12) along with other nutritional and dietary interventions and were compared to 30 control ASD individuals who were not treated. Significant improvements were observed in nonverbal IQ, communication, daily living skills, and social skills domains in the treated ASD group compared to controls [50].

#### 3.3.3. Prospective, Uncontrolled Studies

There were six prospective, uncontrolled studies of B12 in ASD [8,11,44,45,52,53], but no clinical outcomes were reported in four of these studies [8,11,44,45].

In a study of 13 patients with ASD, mB12 25–30 µg/kg/day (up to 1500 µg/day, route of administration unknown) given for 6–25 months led to significant improvements in IQ, developmental quotient, and Childhood Autism Rating Scale (CARS) score; improvements were similar in older children as in younger children, and in children with lower IQ compared to those with higher IQ [52].

In a study of 40 children with ASD, 75 µg/kg mB12 SC 2 times per week for 3 months given with folinic acid (400 µg twice per day) led to significant improvements on all subscales of the Vineland Adaptive Behavior Scale (VABS), including significant improvements in receptive, expressive, and written communication skills, personal, domestic, and community daily living skills, and interpersonal, play-leisure, and coping social skills. The average effect size of improvement was 0.59 (a moderate effect size), and the average improvement in skills development was 7.7 months [53]. This improvement over a 3-month period was consistent with the notion that the children started "catching up" in development.

#### 3.3.4. Retrospective Studies

Five retrospective studies examined B12 treatment in ASD [32,46,47,51,54]. The first three studies used intramuscular B12 (type not specified) to treat a B12 deficiency. Improvements in vision were found in a case series of 3 children with ASD and optic nerve atrophy [46]. In a 9-year-old child with ASD, pulmonary hypertension and nutritional deficiencies (including B12 deficiency), B12 along with other nutritional supplements led to improvements in pulmonary hypertension and musculoskeletal problems [47]. Improvements in eye contact, licking fingers, hyperactivity, pacing, echolalia, repetitive behaviors, and in the CARS score were observed in a 14-year-old vegetarian boy with Childhood Disintegrative Disorder (CDD) with daily injections of 1000 µg B12 for 5 days and then weekly for 8 weeks along with oral antioxidants and vitamins [32]. In another study, daily oral mB12 500 µg resolved nocturnal enuresis a 18-year-old child with ASD, with the enuresis reoccurring when mB12 was stopped [54]. Finally, in one child, "developmental milestones improved" with 10 mg per day of hB12 orally once per day [51].

#### *3.4. Adverse Effects of B12*

Here the potential AEs (see Table 4) of injected and oral forms of B12 are discussed separately, followed by the studies in which the route of administration is unknown.

Both DBPC studies [35,55] that used mB12 injections reported AEs. No serious AEs were reported in either study. Hyperactivity and increased mouthing of items were reported as AEs in one study [55], but the study did not indicate whether these AEs were significantly different between the treatment and placebo groups or what percentage of children had one of these side effects. The other DBPC study [35] reported 21 adverse events in the mB12 group compared to 24 in the placebo group (no significant difference). Adverse events in the mB12 group included a cold (11%), fever (7%), flu (4%), growing pains (4%), increased hyperactivity (7%), increased irritability (4%), lack of focus (4%), mouthing (19%), nosebleed (7%), rash (4%), stomach flu (4%), and trouble sleeping (4%); these were not significantly different compared to the placebo group. Two prospective, uncontrolled studies used mB12 injections [8,53], with one reporting AEs [53]. Adverse events in this study included hyperactivity (10%, improved when the folinic acid dose was lowered), sleep disruption (3%), difficulty falling asleep (3%), increased impulsiveness (3%), and irritability (3%). After study completion, 31/40 (78%) of parents indicated a desire to continue treatment (8 parents (20%) did not respond to this question) [11,53]. Of the five case reports using injected B12, only two reported AEs. Two patients who received cB12 injection developed acute anemia, which resolved by changing the cB12 form to mB12 injections [51]. In a child receiving oral mB12, increased motor stereotypy and hyperactivity were noted within 100 days of starting treatment [54]. Finally, one retrospective study reported higher plasma and urinary cobalt levels in patients who received SC mB12 compared to controls, but no apparent adverse events from this finding were observed [56].

Of the two DBPC studies that used B12 incorporated into an oral MVI [48,49], one reported no side effects in patients who followed the correct dosing parameters, but nausea and vomiting in 2 children (18%) in the treatment group when the MVI was taken on an empty stomach (against study protocol) [48]. The other study reported aggression (4%), night terrors (2%), trouble focusing (2%), moodiness (2%), nausea (2%), diarrhea (2%), mild behavioral problems (11% compared to 7% in the placebo group, *p* = ns), and diarrhea/constipation (11% compared to 7% in the placebo group, *p* = ns) with 2 participants withdrawing due to aggression and one withdrawing because of nausea and diarrhea [49]. In a prospective controlled single-blind study using an oral MVI, which included a 50/50 mixture of cB12 and mB12, AEs included moderate worsening of behaviors in 2 children (6%) found to have low levels of nutrients, particularly extremely low levels of cobalamin [50].

Three retrospective case series using intramuscular cobalamin with the type of B12 not specified did not report if any side effects occurred [32,46,47]. Two studies did not report the route of administration. One prospective uncontrolled study using mB12 (route not specified) reported there were no significant adverse events [52]. Another study did not examine AEs specifically [45].

#### Meta-analysis of Adverse Effects Related to Cobalamin

To better understand the AEs associated with cobalamin treatment, a meta-analysis was performed on the reported AEs across studies for studies using injected and oral B12 separately (see Table 5). Case reports were excluded from this analysis due to the potential bias of reporting in such studies, and only studies that included quantitative measures of the AEs in the samples studied were included. For injected B12, only two studies [11,35] met this criterion, with both studies using subcutaneously injected mB12. For oral B12, three studies [48–50] met this criterion, all using an MVI including B12 in various forms.


**Table 5.** Meta-analysis of adverse effects associated with cobalamin in children with ASD. Multivitamin (MVI) \* *p* < 0.05. Adverse effects that were statistically significant are in bold and italic.

> There was a low incidence (<5%) of most AEs for subcutaneously injected mB12 with only three AEs reaching significance: increased irritability (3.4%), trouble sleeping (7.6%) and increased hyperactivity (11.9%), suggesting that for most individuals with ASD, subcutaneously injected mB12 is well tolerated without AEs. There was also a low incidence (<5%) of most AEs for B12 included in an oral MVI. Unlike injected mB12, the

oral route was associated with gastrointestinal AEs, although these were also at a low incidence (<5%). Significant AEs for oral formulation included aggression (1.8%) and worsening behavior (7.7%), which is similar in incidence and character to the significant behavioral AEs seen in the injected route. However, again, most participants tolerated the B12 in a MVI without any AEs, suggesting that, in general, it was well tolerated.

#### **4. Discussion**

This review identified 17 studies using B12 as a treatment for ASD. Table 6 summarizes the major findings of these studies. Two studies were DBPC controlled studies using mB12 injections and two were DBPC studies using cB12 combined in an MVI. Most studies that specified the type of B12 used mB12 (10/12, 83%). Overall, the treatment was well tolerated with minimal AEs, and the majority of the studies reported positive effects on ASD symptoms, although some studies found that these improvements were limited to a subgroup of children with baseline unfavorable biochemistry.

**Table 6.** Summary of studies on cobalamin treatment for ASD with clinical outcomes. Childhood Autism Rating Scale (CARS); Clinical Global Impression Scale (CGI); glutathione (GSH); oxidized glutathione (GSSG); intelligence quotient (IQ); multivitamin (MVI); S-adenosylhomocysteine (SAH); S-adenosylmethionine (SAM); methylation capacity (SAM/SAH); subcutaneous (SC); total GSH (tGSH); total glutathione redox ratio (tGSH/GSSG).


#### *4.1. Improvements in Autism Symptoms with Cobalamin*

Cobalamin treatment was found to result in improvements in both core and associated ASD symptoms. Three of the prospective studies examined changes in biochemistry and gave subcutaneously injected mB12 with two of the studies demonstrating overall clinical improvements and all studies suggesting improvements in a subgroup with unfavorable biochemistry. The study that did not show an overall effect used a DBPC crossover design

but not a washout period between the crossover, potentially making it more difficult to observe a significant difference between treatment groups [55]. The other DBPC study did not use a crossover design and was able to document improvements as ranked by clinicians compared to placebo [35]. The other prospective study was open-label but differed in two critical ways from the others in its design: first, subcutaneous mB12 was combined with oral folinic acid, and second, only participants with unfavorable biochemical profiles consisting of decreased methylation capacity and decreased GSH redox ratio were entered into the study. This latter study demonstrated significant improvements on all subscales of the VABS with a moderate effect size (0.59) and an average improvement in skills development of 7.7 months during the three-month treatment period. This suggests that mB12 treatment with folinic acid might help some children with ASD "catch up" in development [53]. Other retrospective studies of injected B12 are consistent with these prospective studies, demonstrating improvement in physical health as well as core (e.g., eye contact, echolalia, and repetitive behaviors) and associated (e.g., hyperactivity) ASD behaviors [32,46,47].

Oral B12 combined with a MVI has been studied in several prospective controlled studies with these studies demonstrating improvements in physical medical issues such as gastrointestinal problems and sleep [48], as well as in behaviors such as hyperactivity and tantrums [49], and in cognitive skills, including language [49], non-verbal IQ and social skills [50]. Oral mB12 alone improved nocturnal enuresis in one study [54]. One study in which the route of treatment was not known found that mB12 gave similar improvements in both older and younger children as well as those with lower IQ. This suggests that even older and more severely affected individuals with ASD can improve with mB12 treatment [52].

#### *4.2. Biochemical Effects of Cobalamin*

Studies have reported a wide variety of improvements in biochemistry associated with B12 treatment, including improvements in methylation with injected [8,35] and oral [44,49] B12, redox metabolism with injected [8,11] and oral [49] B12 and mitochondrial function with oral [49] or unspecified [45] B12 treatment.

The meta-analysis examined whether there were consistent changes in biochemistry with B12 treatment. Changes in methylation were not found to be consistent when combining studies. However, homocysteine at baseline was markedly lower in the studies that demonstrated an increase in homocysteine with treatment (baseline homocysteine 6.7 ± 0.7 [8] and 4.8 ± 1.8 [11]) as compared to the study that demonstrated a decrease in homocysteine (baseline homocysteine 8.9 ± 1.0 [35]). Furthermore, even after treatment, in the studies where homocysteine started out lower, the post-treatment homocysteine concentrations were still lower than the post-treatment homocysteine in the study where homocysteine started out higher. This was most likely driven by differences in the design of the studies. In the two studies in which homocysteine was low, entry into the study required an unfavorable biochemistry profile [8,11], whereas, in the study with the higher homocysteine, no such criterion was implemented [35]. This may reflect that mB12 can drive homocysteine to an optimal concentration with the direction of change dependent on the starting concentration for the particular participant. Clearly, larger studies, which are sensitive to the baseline biochemical abnormalities, are needed to examine the complexity of the effect of mB12 on methylation. The meta-analysis did demonstrate significant improvements in transsulfuration and redox metabolism across studies with a medium to large effect size, suggesting that mB12 may be important for improving redox metabolism independent of the abnormalities in methylation metabolism.

The strongest evidence that the effect of B12 on biochemistry is clinically important is reflected in the studies which demonstrate that changes in biochemistry are associated with positive changes in clinical symptoms. Although one DBPC study of subcutaneously injected mB12 did not find an overall effect of mB12 on clinical outcomes, it did find that a subgroup of clinical "responders" showed improvements on the clinical global impression scale and at least two additional behavioral measures; these responders had significant improvements in GSH and the GSH redox ratio, suggesting these biomarkers could be used to predict response to mB12 treatment in children with ASD [55]. The second DBPC study of subcutaneously injected mB12 demonstrated that clinician-rated improvements were significantly and positively associated with improvements in methylation metabolism [35]. Finally, in another prospective study of subcutaneously injected mB12, significant improvements in the GSH redox ratio were significantly associated with clinical improvements, including expressive communication, personal and domestic daily living skills, and interpersonal, play-leisure, and coping social skills [53]. These findings further support the notion that certain biochemical abnormalities might predict treatment response to mB12 injections.

#### *4.3. Biological Mechanisms of Actions*

There are several potential biological mechanisms of action of B12 treatment. First, as demonstrated by the biochemical meta-analysis, subcutaneously injected mB12 consistently and significantly improves redox metabolism, including increasing Cysteine, GSH, and the GSH redox ratio as well as decreasing GSSG with medium to large effect sizes. A systematic review and meta-analysis have demonstrated that individuals with ASD overall demonstrate low GSH and Cysteine and increased GSSG across multiple research groups in multiple countries [57]. These redox metabolism abnormalities have been documented in multiple tissues in individuals with ASD, including in post-mortem brain [15,58]. In addition, these redox abnormalities have been shown to result in oxidative damage to DNA, protein, and lipids, as well as mitochondrial dysfunction and inflammation in the brain of individuals with ASD [15,59]. Thus, improving redox abnormalities can have widespread positive effects on the physiological function of the brain and other key important organs such as the immune system and GI tract in individuals with ASD.

Second, the brain of individuals with ASD has been repeatedly shown to have an excitatory/inhibitory imbalance such that the cortex is overexcited and underinhibited [60]. Glutamate is the major excitatory neurotransmitter of the cortex, thus decreasing glutamate neurotransmission is believed to be potentially therapeutic in ASD [61]. Glutamate is one of the three key precursors of GSH, along with cysteine and glycine. Thus, improved production of GSH, in part driven by mB12, may have other positive effects in neurotransmitter metabolism by reducing glutamate concentrations in the cortex (See Figure 3).

Third, polymorphisms in TCN2, the cobalamin binding protein, are associated with an increased risk of ASD [9], and ASD is one of the characteristics of some individuals with a mutation in TCN2 [51]. Thus, higher concentrations of B12 may be required in the blood in those with defects in the cobalamin binding protein in order to obtain sufficient levels of B12 into the tissues, including the brain.

Fourth, one controlled study reported a more than 3-fold reduction in mB12 concentration in brain tissue from individuals with ASD [58]. The reason for this finding is not clear but it is very possible that, such as the case with folate, some individuals with ASD may have a defect in the transport mechanism for B12 into the brain. Indeed, in one study, it was found that the folate receptor alpha autoantibody was associated with higher levels of blood cobalamin concentrations [62]. These higher blood concentrations of B12 could potentially be caused by B12 being blocked from uptake into the tissues. If such a transportation mechanism is present, a high blood concentration of B12 may be needed to overcome the blockage in the transportation mechanism, similar to the use of high dose folinic acid as a therapy for dysfunction of the folate receptor alpha [63]. Notably, one study reported that higher than normal serum concentrations of B12 was not associated with any observable adverse effects in individuals with ASD [52].

Fifth, methionine synthase, the key B12 dependent enzyme required for the proper functioning of the methylation cycle may be dysfunctional in the brain of individuals with ASD as one study observed that the mRNA for this enzyme is underexpressed in brain tissue of individuals with ASD [23]. Thus, high concentrations of B12 may be important to allow the limited amount of this enzyme to function optimally in the brain.

**Figure 3.** The production of glutathione results in the consumption of glutamate, the major excitatory neurotransmitter of the cortex, thus reducing glutamate in the brain. This diagram shows the connected folate, methylation, and redox metabolic pathways in the brain. Ovals represent enzymes and boxes represent metabolites. Red indicates metabolites and enzymes repeatedly noted to be consistently abnormal in ASD. Green highlights treatments that improve the metabolism of these cycles. Folic acid is shown in yellow as it is a suboptimal treatment because it is oxidized and has to be converted to active forms of folate. **Figure 3.** The production of glutathione results in the consumption of glutamate, the major excitatory neurotransmitter of the cortex, thus reducing glutamate in the brain. This diagram shows the connected folate, methylation, and redox metabolic pathways in the brain. Ovals represent enzymes and boxes represent metabolites. Red indicates metabolites and enzymes repeatedly noted to be consistently abnormal in ASD. Green highlights treatments that improve the metabolism of these cycles. Folic acid is shown in yellow as it is a suboptimal treatment because it is oxidized and has to be converted to active forms of folate.

Third, polymorphisms in TCN2, the cobalamin binding protein, are associated with an increased risk of ASD [9], and ASD is one of the characteristics of some individuals with a mutation in TCN2 [51]. Thus, higher concentrations of B12 may be required in the blood in those with defects in the cobalamin binding protein in order to obtain sufficient levels of B12 into the tissues, including the brain. Fourth, one controlled study reported a more than 3-fold reduction in mB12 concentration in brain tissue from individuals with ASD [58]. The reason for this finding is not clear but it is very possible that, such as the case with folate, some individuals with ASD may have a defect in the transport mechanism for B12 into the brain. Indeed, in one study, Sixth, B12 could be helpful in improving mitochondrial function. Two studies reported improvements in mitochondrial-related markers in children with ASD. One prospective, controlled, single-blind 12-month study reported improvements in mitochondrial related markers including ATP, NADH, and NADPH with an oral MVI containing 500 mcg of B12 (50% as mB12 and 50% as cB12) along with other nutritional treatments [49]. In another prospective study in 127 children with ASD, B12 treatment was associated with the better coupling of Complex I and Citrate Synthase mitochondrial enzymes [45]. Since mitochondrial dysfunction is a common medical comorbidity in children with ASD [3,4], the use of B12 appears important in treating this problem.

it was found that the folate receptor alpha autoantibody was associated with higher levels of blood cobalamin concentrations [62]. These higher blood concentrations of B12 could potentially be caused by B12 being blocked from uptake into the tissues. If such a transportation mechanism is present, a high blood concentration of B12 may be needed to overcome the blockage in the transportation mechanism, similar to the use of high dose folinic acid as a therapy for dysfunction of the folate receptor alpha [63]. Notably, one study reported that higher than normal serum concentrations of B12 was not associated with any observable adverse effects in individuals with ASD [52]. Fifth, methionine synthase, the key B12 dependent enzyme required for the proper functioning of the methylation cycle may be dysfunctional in the brain of individuals with ASD as one study observed that the mRNA for this enzyme is underexpressed in brain tissue of individuals with ASD [23]. Thus, high concentrations of B12 may be important Lastly, some individuals with ASD have lower intake and blood levels of B12 compared to controls. For example, a meta-analysis of 29 studies reported children with ASD had a significantly lower dietary intake of B12 compared to controls [64], while another meta-analysis of 16 studies found plasma B12 concentrations significantly lower in ASD compared to controls, although evidence of potential publication bias was found [5]. These finding may be due to feeding difficulties, which are common in individuals with ASD [65]. Thus, B12 treatment may be correcting a deficiency in some children with ASD. In addition, because the absorption of B12 orally may be dependent on adequate calcium intake [66], future studies examining the effect of calcium intake in conjunction with B12 might be helpful in ASD. Since some patients with ASD take multiple nutritional supplements, the potential interaction between B12 and other nutritional supplements also warrants further studies.

to allow the limited amount of this enzyme to function optimally in the brain.

#### *4.4. Formulation*

Most studies (67%) that specified the route of administration used an injected form of B12. All studies that used the injected formulation and specified the type of B12 used mB12, except for one study, which initially used cB12 and changed to mB12. All studies that have linked changes in biochemistry with improvements in autism symptoms used subcutaneously injected mB12. The injected formulation is preferred by some practitioners because individuals with ASD may not be able to successfully take B12 orally due to GI disorders. Indeed, gastritis and enterocolitis may prevent optimal absorption while sensory aversions or esophagitis may prevent ASD children from easily swallowing a supplement [67,68]. Furthermore, as mentioned above, if there is a defect in B12 transportation in the blood or across the blood–brain barrier, higher blood concentrations of B12 may be needed to optimally deliver B12 into the organs, particularly the brain. It is possible that the GI tract is not even designed to absorb the necessary quantity of B12 that might be needed if indeed a problem with B12 transportation is present. Injected B12 may help raise the blood level higher than that achieved with oral B12.

A few studies combined B12 with other vitamins and/or minerals, thus these studies may reflect the combination of B12 with other supportive treatments. One study examined changes in biochemical markers of methylation and redox metabolism before and after adding betaine and folinic acid, and then after adding subcutaneously injected mB12 and found that the addition of mB12 to betaine and folinic acid both had a positive effect on methylation and redox metabolism [8]. Thus, in studies that have used other supportive treatments, B12 may have worked in concert with these other treatments. A recent randomized, single-blind study using a combination of various dietary and nutritional treatments in individuals with ASD, including essential fatty acids, Epsom salt baths, digestive enzymes, carnitine, and a MVI containing a combination of mB12 and cB12 reported significant improvements over one year compared to a control group that was not treated, suggesting a combination of treatments, which affect methylation and redox metabolism might lead to more robust improvements [50]. Further research will be needed to understand the optimal combination of treatments.

#### *4.5. Adverse Events*

The meta-analysis of AEs demonstrated a low incidence of AEs with most children tolerating the treatment without any AEs. Only one study described a potentially serious AE, anemia when using injected cB12. This AE resolved with changing to injected mB12. mB12 given by injection or orally has not been associated with any severe or serious AEs, perhaps suggesting that it is the preferred form of B12 for individuals with ASD at this time. After the completion of one study, 31/40 (78%) of parents indicated a desire to continue mB12 treatment [11,53]. This suggests that parents observed enough clinical improvements and a low enough rate of adverse effects to continue mB12 injections. Interestingly, although the majority of significant AEs reported pertained to worsening behavior and sleep, some studies have demonstrated significant improvements in such symptoms, including hyperactivity [32,49] and sleep [48], suggesting that overall many children have improvements in these key symptoms, and that having such symptoms at baseline is not necessarily a contraindication for starting B12 treatment. One study reported some individuals with ASD had above normal serum B12 levels with B12 treatment without any apparent AEs [52]. Finally, one retrospective study examined 24 children with ASD who received mB12 injections and compared urinary and plasma cobalt levels to 48 children who did not receive mB12 injections. These children were treated with the standard dose of mB12 used in the SC mB12 studies (75 µg/kg, injected every 1–3 days). The mean plasma cobalt concentration in the mB12 group was significantly elevated (0.82 ± 0.19 µg/L) compared to the untreated group (0.12 ± 0.10). The investigators noted that the study was limited as it could not determine what form of cobalt was present (free or bound in mB12), how the cobalt was distributed in the body, and what the tissue levels would be. Furthermore, no apparent adverse events from this finding were reported in the study [56]. Notably, adverse

effects from serum cobalt are unlikely to occur with a blood cobalt concentration under 100 µg/L (using a conservative estimate), which is over 100 times the level reported in this aforementioned study [69]. Furthermore, no study has reported AEs consistent with cobalt toxicity (e.g., iron deficiency, pernicious anemia, cardiomyopathy, and polycythemia).

#### *4.6. Limitation of Published Studies*

Many studies demonstrated detection bias as only a few studies used standardized outcomes. This made it impossible to perform a meta-analysis across clinical outcomes. Second, although some studies were prospective, they were uncontrolled and unblinded and not randomized, opening up the possibility of selection and performance bias. Finally, the retrospective studies had the potential drawback of being open to attrition and reporting bias.

#### **5. Conclusions**

Overall, B12 appears to have evidence for effectiveness in individuals with ASD, particularly in those who have been identified with unfavorable biochemical profiles. In general, B12 appears to be very well-tolerated and safe. Two types of B12 have been studied in controlled and/or prospective clinical trials: (1) subcutaneously injected mB12 has evidence for improving clinical symptoms of ASD and improving methylation and redox metabolism, especially in those with unfavorable biochemistry and when combined with folinic acid (aka leucovorin) and (2) a mixture of cB12 and mB12 included in a MVI also appears to be associated with improvements in clinical symptoms, biochemistry, and physical medical disorders.

As mentioned above, the current set of studies have their limitations and should be used to design and implement well-controlled blinded randomized clinical trials in the future. Additionally, the ASD population is very heterogeneous, making it important to understand the subset of children with ASD in which B12 treatment may be most effective. There is evidence that B12, particularly subcutaneously injected mB12, may be particularly helpful for a subset of individuals with ASD with unfavorable biomarkers, suggesting that biomarkers need to be studied alongside clinical outcomes with a prior hypothesis regarding subgroups that may optimally respond to treatment. Thus, including reliable biomarkers that can guide treatment will be helpful to optimize this potentially important well-tolerated safe treatment.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/jpm11080784/s1, Supplementary Table S1: PRISMA Checklist.

**Author Contributions:** Conceptualization, methodology, formal analysis, writing—original draft preparation, review and editing, were performed by both D.A.R. and R.E.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** The review did not receive any financial or grant support from any sources.

**Institutional Review Board Statement:** Not applicable. This review is not human research.

**Informed Consent Statement:** Not applicable. This review is not human research.

**Data Availability Statement:** All data are presented within the article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Ratings of the Effectiveness of Nutraceuticals for Autism Spectrum Disorders: Results of a National Survey**

**James B. Adams 1,\*, Anisha Bhargava <sup>2</sup> , Devon M. Coleman <sup>1</sup> , Richard E. Frye <sup>3</sup> and Daniel A. Rossignol <sup>4</sup>**


**Abstract:** Autism spectrum disorder (ASD) often involves a wide range of co-occurring medical conditions ("comorbidities") and biochemical abnormalities such as oxidative stress and mitochondrial dysfunction. Nutritional supplements ("Nutraceuticals") are often used to treat both core ASD symptoms and comorbidities, but some have not yet been formally evaluated in ASD. The potential biological mechanisms of nutraceuticals include correction of micronutrient deficiencies due to a poor diet and support for metabolic processes such as redox regulation, mitochondrial dysfunction and melatonin production. This paper reports on the results of the National Survey on Treatment Effectiveness for Autism, focusing on nutraceuticals. The Survey involved 1286 participants from across the United States. Participants rated the overall perceived benefits and adverse effects of each nutraceutical, and also indicated the specific symptoms changed and adverse effects. From these ratings the top-rated nutraceuticals for each of 24 symptoms are listed. Compared to psychiatric and seizure medications rated through the same Survey, on average nutraceuticals had significantly higher ratings of Overall Benefit (1.59 vs. 1.39, *p* = 0.01) and significantly lower ratings of Overall Adverse Effects (0.1 vs. 0.9, *p* < 0.001). Folinic acid and vitamin B12 were two of the top-rated treatments. This study suggests that nutraceuticals may have clinical benefits and favorable adverse effect profiles.

**Keywords:** autism; autism spectrum disorder; nutraceuticals; survey; vitamins; minerals; B12; folinic acid

#### **1. Introduction**

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder involving core problems in social communication and repetitive behaviors and affects about 2% of children in the United States [1]. A number of medical conditions cooccur with ASD (termed "comorbidities"), including intellectual disability [2], epilepsy [3], gastrointestinal disorders (such as constipation and diarrhea) [4], sleep disorders [5], attention deficit disorder [5], anxiety [5], and irritability, self-injurious behavior, and depression [6]. Other studies have reported biochemical abnormalities, including problems with methylation pathway insufficiency [7,8], insufficient production of melatonin for sleep [9], mitochondrial dysfunction [10,11] and oxidative stress [12,13]. Currently, there are no FDA-approved medications for treating the core symptoms of ASD (social communication and restricted/repetitive behaviors), although there are two FDA-approved medications for treating the associated symptom of irritability [14].

Compared to typically developing (TD) children, feeding difficulties are common in children with ASD and include food refusal, eating a limited variety of foods and

**Citation:** Adams, J.B.; Bhargava, A.; Coleman, D.M.; Frye, R.E.; Rossignol, D.A. Ratings of the Effectiveness of Nutraceuticals for Autism Spectrum Disorders: Results of a National Survey. *J. Pers. Med.* **2021**, *11*, 878. https://doi.org/10.3390/jpm11090878

Academic Editor: Elizabeth B. Torres

Received: 27 July 2021 Accepted: 29 August 2021 Published: 31 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

having more problems with mealtime behavior [15], and they may have nutrient-poor diets [16]. One meta-analysis of 17 prospectively controlled studies reported significantly more feeding problems in children with ASD compared to controls (odd ratios 5.11, 95% CI 3.74–6.97) and significantly lower intake of calcium and protein in the ASD group [17]. A systematic review of 29 studies reported that feeding problems were associated with impaired sensory processing, perception, more rigidity and challenging behaviors [18]. A prospective, randomized controlled trial of a comprehensive dietary and nutritional intervention found that a combination of six treatments (vitamins/minerals, essential fatty acids, Epsom salts, carnitine, digestive enzymes, and a healthy low-allergen diet) led to significant improvements over one year in autism symptoms, developmental age, and non-verbal IQ compared to controls [19].

Because of potential deficiencies of nutrients (often related to feeding problems) and biochemical abnormalities (e.g., oxidative stress, mitochondrial dysfunction, methylation problems, among others) reported in individuals with ASD, a number of studies have investigated the use of vitamins, minerals, and other nutritional supplements (hereafter termed "Nutraceuticals"). The use of nutraceuticals is typically considered a form of complementary and alternative medicine (CAM), although many nutraceuticals are based on the science of nutritional biochemistry and target deficiencies and biochemical problems. Owen-Smith et al. (2015) conducted a survey of 42 CAM treatments used in ASD and reported 88% of participants had been treated with at least one CAM treatment [20]. Frye et al. (2011) surveyed the effectiveness of seizure treatments (including nutraceuticals) in 733 children with ASD and seizures compared to 290 controls and reported some CAM treatments (such as vitamin B6, magnesium, taurine, and vitamin B12) were rated as helpful for treating seizures [21]. In addition, a large online survey was conducted by the Autism Research Institute (the "Parent Ratings of Behavioral Effects of Biomedical Interventions Survey") [22]. This survey of 27,000 parents of individuals with ASD rated the effectiveness of 84 various medications, supplements, and diets, using a six-point scale from "made worse" to "made better"; a number of treatments were reported as beneficial, including methylcobalamin (MB12), melatonin, digestive enzymes, fatty acids, cod liver oil, vitamin B6, zinc, magnesium, folic acid, vitamin C, and vitamin A [23]. Although these surveys focused on the overall effectiveness ratings for medications and nutraceuticals used in ASD individuals, most of these studies did not utilize a separate rating scale for the benefits and adverse effects (AEs) and did not obtain information on the effects of these treatments on specific symptoms of ASD.

Some of the medical comorbidities and biochemical abnormalities reported in individuals with ASD might improve with nutraceuticals. For example, randomized clinical trials for ASD have demonstrated the efficacy of melatonin supplementation [9], folinic acid [24–26], vitamin/mineral supplements [27,28], comprehensive nutritional interventions [19], N-acetyl cysteine (NAC) [29], and sulforaphane [30].

This paper presents the results of a national survey (the "National Survey on Treatment Effectiveness for Autism") in individuals with ASD and contains more extensive assessments of the treatment effects on specific behaviors and AEs of nutraceuticals in ASD. A previous paper from this Survey reported on the results for psychiatric and seizure medications [31].

#### **2. Materials and Methods**

The research team created the "National Survey on Treatment Effectiveness for Autism" (from now on referred to as "the Survey") and obtained reviews by families of children/adults with ASD and experts in a variety of fields who treat individuals with ASD. This study was approved by the Institutional Review Board of Arizona State University (STUDY00003766). The Survey was advertised to families of individuals with ASD across the country with the assistance of over 50 autism organizations (see Acknowledgements). A full explanation of the Survey creation and distribution can be found in the previous paper [31]. The Survey obtained general medical history and the use of

psychiatric and seizure medications, general medications, nutraceuticals, diets, therapies, and information on Kindergarten through grade 12 education. This paper reports data only on the nutraceutical section from Survey responses from 1710 people (of which 1286 (75.2%) rated the effects of nutraceuticals); additional responses were collected since the analysis reported in the previous paper. The exact diagnosis of the individual with ASD was queried using the following categories: autism, Asperger's syndrome, autism spectrum disorder, high-functioning autism, pervasive developmental disorder not otherwise specified (PDD-NOS), no current diagnosis but was previously on the autism spectrum, and "other" in order to capture both DSM-IV and DSM-5 diagnostic categories. These diagnoses were reported by the participant, but not verified in this study since it was an anonymous survey.

The Survey was divided into sub-sections for various types of nutraceuticals (amino acids, vitamins, etc.). At the beginning of each sub-section, the Survey asked what nutraceuticals the participant had taken (from a list of 123 nutraceuticals found in Table S1). For each nutraceutical taken, the Survey asked the participant to rate the overall perceived benefit of the nutraceutical (no benefit = 0, slight benefit = 1, moderate benefit = 2, good benefit = 3, great benefit = 4), the primary symptoms benefited (if any), the overall AE of the nutraceutical (no adverse effect = 0, mild adverse effect = 1, moderate adverse effect = 2, severe adverse effect = 3), and the specific symptoms that were adversely affected (if any). Table 1 shows the symptom list from which participants could select (they could select one or more for each treatment). Finally, the Survey asked for the overall average effect of all nutraceuticals (on a 7-point scale ranging from "much better" to "much worse"). Only treatments with 20 or more responses were included in this analysis. It should be noted that the ratings are the perceived benefit of the evaluator (primarily a caregiver or sometimes the person with ASD), and not ratings by a clinician or physician, which is a limitation of the study.

For each treatment, the top 3 benefits were reported as well as any other benefits with over 20% of participants reporting a benefit. For AEs, the top 3 AEs were reported and any other AEs which were reported by 15% or more of participants. These were arbitrary cut-offs to limit table entries to the most relevant symptoms; a slightly lower cut-off for AEs was chosen since they were so rare.

The top-rated treatments for each symptom were calculated by multiplying the overall net benefit by the percentage of participants who had improvements in that symptom. For each symptom, the three top-rated treatments are reported, as well as any other treatments with a score of 0.2 or higher (equivalent to 10% of participants reporting a moderate benefit).

In order to determine if any of the nutraceuticals were related to changes in ASD severity, two questions were asked on the Survey. Specifically, the ASD severity rated at 3 years of age (which would be close to most patient's diagnosis) was compared to the currently rated ASD severity. The categories of severity were coded on a five-point scale with increasing numeric values corresponding to increasing severity. Specifically, no symptoms (0), very mild symptoms (1), mild symptoms (2), moderate symptoms (3), and severe symptoms (4). The current ASD severity was subtracted from the severity at baseline (3 years of age) such that a decrease in severity would indicate an improvement. The generalized linear model performed in IBM SPSS PASW Release 18.0.0 (Armonk, New York) was used to analyze change in severity. The model included gender (male, female), developmental profile, number of antibiotic treatments in the first 3 years of life (since that has been reported higher in ASD), and baseline ASD severity. In general, only treatments that were used by 100 respondents or more were analyzed to ensure generalizability and a wide range of ASD severity changes. Two different approaches were used. First, it was determined whether use of the nutraceutical was associated with improvements in ASD symptoms by comparing those who used the nutraceutical to those who did not. Second, the association between the perceived benefits of the nutraceutical with the change in ASD symptoms was examined by comparing current ASD severity versus severity at 3 years old. This later analysis including an interaction between treatment and severity at 3 years

of age in order to determine whether the change in severity associated with the treatment was affected by the severity of ASD at age 3 years of age. A one-way analysis of variance was also used to determine whether severity at 3 years of age was related to the use of any treatment studied.


**Table 1.** All Symptom Options.

#### **3. Results**

#### *3.1. Demographics and Medical History*

The characteristics of the 1286 participants and their medical history are outlined in Table 2. The majority of the surveys were completed by the primary caregiver of an individual with ASD (85%). More than half of the surveys were for children under 13 years old (54%), with 21% for teenagers and 16% for young adults (18 years or older). Seventy six percent of participants were male, and 24% were female. Autism was the most frequent diagnosis (43%), followed by Autism Spectrum Disorder (22%) and Asperger's syndrome (14%). The most common developmental history was "Abnormal development from early infancy, with no major regression or plateau in development" (32%). Furthermore, most participants received antibiotics during their first 3 years of life, with a median of 3 rounds. Most participants had moderate autism-related symptoms at age 3 years old (38%) and currently (38%).


**Table 2.** Completion, Age, Gender, Medical Diagnosis, Developmental History, and Antibiotic Use.


**Table 2.** *Cont.*

1 . Grandparents were taken from those responded with "other" and noted they were grandparents. Numbers may not add up to 100% due to rounding.

#### *3.2. Nutraceuticals*

Of the 123 nutraceuticals included in the Survey (found in Supplemental Table S1), 58 had 20 or more responses and are reported here. These nutraceuticals are reported in eight general categories—the categories and the number of nutraceuticals for each category are: amino acids (4), essential fatty acids (7), glutathione-related nutraceuticals (4), individual minerals (9), individual vitamins/vitamin-like nutraceuticals (22), multivitamins (3), sleep treatments (3), and others (5). The most commonly used treatments were generic child/adult multivitamin (34%), melatonin (29%), omega 3 fatty acids (15%), vitamin C (14%), krill oil (13%), fish oil (13%), vitamin D (12%), magnesium (12%), Epsom salts (11%), and zinc (10%).

#### 3.2.1. Amino Acids

Amino Acids were rated as having a slight to moderate (1.1 to 1.6) overall perceived benefit with minimal AEs (0.1 to 0.4). For the amino acid blend, glutamine and taurine, the primary benefit was general benefit (43–57%) with small benefits in other symptoms. For tryptophan, the primary benefits were helping with falling asleep and staying asleep (see Table S2 and Figure 1).

#### 3.2.2. Fatty Acids

Fatty Acids (FA) were rated as having a moderate to good benefit (1.2 to 2) with minimal overall AEs (0 to 0.2). For all FAs, the primary benefit was general benefit (32% to 59%), with secondary benefits in attention and cognition. See Table S3 and Figure 2.

**Figure 1.** Overall Benefit Score and Adverse Effect Score for Amino Acid Treatments from Highest Overall Benefit to Lowest Overall Benefit.

**Figure 2.** Overall Benefit Score and Adverse Effect Score for Fatty Acid Treatments from Highest Overall Benefit to Lowest Overall Benefit.

3.2.3. Glutathione-Related Nutraceuticals

Glutathione-related nutraceuticals (including NAC) were rated as having a slight to moderate benefit (1.1 to 1.7) with minimal AEs (0 to 0.3). The most common benefit was general benefit (4% to 56%). See Table S3 and Figure 3.

**Figure 3.** Overall Benefit Score and Adverse Effect Score for Glutathione-Related Treatments from Highest Overall Benefit to Lowest Overall Benefit.

#### 3.2.4. Individual Minerals

Individual minerals were rated as having a slight to moderate benefit (1.3 to 2.1) with minimal AEs (0–0.3). The most common benefit was general benefit (15% to 70%). Lithium also helped with anxiety (24%), and magnesium helped with constipation (27%). Iron caused some gastrointestinal adverse effects in 17%. See Table S4 and Figure 4.

**Figure 4.** Overall Benefit Score and Adverse Event Score for Individual Minerals from Highest Overall Benefit to Lowest Overall Benefit.

#### 3.2.5. Individual Vitamins/Vitamin-like Nutraceuticals

Individual vitamins and vitamin-like nutraceuticals were rated as having slight to moderate overall benefits (1.0 to 2.2) with minimal AEs (0 to 0.3). The most common benefit was general overall benefit (14% to 62%). High dose folinic acid (above 5 mg/day) improved cognition (33%), attention (29%), and language/communication (24%). Moderate dose folinic acid (below 5 mg/day) also improved language/communication (20%). P5P improved anxiety (20%) and TMG improved language/communication (29%). Injected vitamin B12 improved language/communication (30%), cognition (28%), and attention (20%). Oral vitamin B12 improved cognition (25%) and language/communication (18%). Vitamin C also improved overall health (27%). See Table S6 and Figure 5.

**Figure 5.** Overall Benefit Score and Adverse Event Score for Individual Vitamins/Vitamin-like Nutraceuticals from Highest Overall Benefit to Lowest Overall Benefit.

#### 3.2.6. Multivitamins

Multivitamins were rated as having a slight to moderate benefit (1.4 to 1.9) with minimal AEs (0.0 to 0.2). The most common benefit was general benefit (50–55%). High dose multivitamin also improved general health (26%), and a high dose multivitamin, specifically designed for ASD, improved cognition (21%). See Table S7 and Figure 6.

#### 3.2.7. Sleep-Related Nutraceuticals

Sleep-related nutraceuticals were rated as having slight to moderate benefit (1.2–2.1), with minimal AEs (0.1 to 0.3). The primary benefit was falling asleep (36–74%), followed by staying asleep (27–35%). For 5-HTP, there was also a general benefit (27%). See Table S8 and Figure 7. It is noteworthy that melatonin had the highest overall benefit score and was used by a very high number of participants.

**Figure 6.** Overall Benefit Score and Adverse Event Score for Multivitamins from Highest Overall Benefit to Lowest Overall Benefit.

**Figure 7.** Overall Benefit Score and Adverse Event Score for Sleep Treatments from Highest Overall Benefit to Lowest Overall Benefit.

#### 3.2.8. Other Miscellaneous Nutraceuticals

For other miscellaneous nutraceuticals, the general benefit ranged from 1.3 to 2.2 (slight to moderate benefit) with minimal AEs (0.0 to 0.2). All of these nutraceuticals had improvements in general benefit (22% to 67%). Epsom salts improved aggression/agitation (35%) and attention (26%). A fruit/vegetable powder concentrate also improved consti-

pation (24%) and general health (24%). GABA improved anxiety (26%). See Table S9 and Figure 8.

**Figure 8.** Overall Benefit Score and Adverse Effect Score for Other Miscellaneous Nutraceuticals from Highest Overall Benefit to Lowest Overall Benefit.

#### 3.2.9. Average of All Nutraceuticals

Averaging all the nutraceuticals reported in this paper, the average Overall Benefit and Overall AE was 1.6 (SD = 0.3) and 0.1 (SD = 0.1), respectively, reflecting that participants reported on average slight to moderate benefits with minimal adverse effects.

#### 3.2.10. Top Nutraceuticals by Symptom

Table 3 presents the top-rated nutraceuticals for 24 different symptoms. For most symptoms, nutraceuticals were moderately effective (net benefit scores >0.25), including aggression/agitation, anxiety, attention, cognition, constipation, diarrhea, general benefit, health, hyperactivity, irritability, language/communication, falling asleep, staying asleep, and social interaction/understanding. Other symptoms were only slightly affected (net benefit scores between 0.10 and 0.25) such as depression, eczema/skin problems, lethargy, obsessive-compulsive symptoms, reflux/vomiting, sensory sensitivity, stimming and tics/involuntary movements (Table 3).

It is important to note that less common problems, such as seizures, might receive lower scores since fewer individuals have these problems. These ratings should be interpreted cautiously, as they are averages, but they suggest which treatments families sensed were most helpful for a given symptom, which can potentially help guide treatment selection and future research.



3.2.11. Overall Effects of Nutraceuticals

As a final part of this Survey, participants were asked to rate the overall effect of nutraceuticals (Table 4). A total of 77% of participants reported that nutraceuticals had a positive effect, ranging from slightly better (24%) to much better (27%), with 23% reporting no effect, and no reports that they generally resulted in worsened symptoms.


**Table 4.** Rating of the Overall Effects of Nutraceuticals.

#### *3.3. Analysis of the Effect on Specific Supplements on Change in Severity*

To study the change in ASD severity related to nutraceuticals, nutraceuticals with at least 100 responses were selected in order to ensure there were enough cases to provide an adequate range of change in ASD severity. Since there were multiple categories of Omega 3 fatty acids (Fish Oils, Omega 3 Fatty Acids, Krill Oil) and B12 (oral and injected) these nutraceuticals were combined into categories. Thus, nutraceuticals selected included B12 (*n* = 170), Omega 3 fatty acids (*n* = 276), Epsom salt baths (*n* = 141), calcium (*n* = 110), magnesium (*n* = 153), zinc (*n* = 124), Vitamin C (*n* = 182), Vitamin D (*n* = 159), generic multivitamin (MVI) (*n* = 436), autism specific MVI (*n* = 103), and melatonin (*n* = 367). Because two other MVIs were being studied, high dose MVI (*n* = 45) was also included in the analysis. Because of the interest in the difference between injected vs. oral B12, the analysis was conducted on the separate groups of oral B12 (*n* = 127) and injected B12 (*n* = 76) as well as any B12 use. The analysis adjusted for baseline severity at age 3 years of age, developmental profile, number of rounds of antibiotic used in infancy, and gender.

#### 3.3.1. Specific Nutraceutical Use

First, the analysis determined whether the changes in severity from 3 years of age to the current age was related to taking a nutraceutical regardless of the reported specific beneficial response. The uses of any B12 [χ(1)<sup>2</sup> = 11.79, *p* < 0.001], injected B12 [χ(1)<sup>2</sup> = 5.58, *p* = 0.01] or oral B12 [χ(1)<sup>2</sup> = 11.48, *p* = 0.001], Calcium [χ(1)<sup>2</sup> = 8.29, *p* < 0.01], Magnesium [χ(1)<sup>2</sup> = 5.83 *p* = 0.01], Zinc [χ(1)<sup>2</sup> = 20.46 *p* < 0.001], Vitamin D [χ(1)<sup>2</sup> = 6.66 *p* = 0.01], or a multivitamin specifically formulated for ASD [χ(1)<sup>2</sup> =7.00 *p* < 0.01] were significantly related to a positive improvement in ASD symptoms (a reduction in ASD severity) as seen in Figure 9.

The change in ASD severity was also related to baseline severity at 3 years of age in all of the analyses, which included taking B12 [χ(1)<sup>2</sup> = 336, *p* < 0.001], B12 injections [χ(1)<sup>2</sup> = 332, *p* < 0.001], oral B12 [χ(1)<sup>2</sup> = 341, *p* < 0.001], Omega 3 Fatty Acids [χ(1)<sup>2</sup> = 336, *p* < 0.001], Epsom Salt Baths [χ(1)<sup>2</sup> = 329, *p* < 0.001], Calcium [χ(1)<sup>2</sup> = 343, *p* < 0.001], Magnesium [χ(1)<sup>2</sup> = 338, *p* < 0.001], Zinc [χ(1)<sup>2</sup> = 350, *p* < 0.001], Vitamin C [χ(1)<sup>2</sup> = 335, *p* < 0.001], Vitamin D [χ(1)<sup>2</sup> = 327, *p* < 0.001], Generic MVI [χ(1)<sup>2</sup> = 327, *p* < 0.001], high dose MVI [χ(1)<sup>2</sup> = 337, *p* < 0.001], autism specific MVI [χ(1)<sup>2</sup> = 321, *p* < 0.001], and Melatonin [χ(1)<sup>2</sup> = 355, *p* < 0.001]. In all models, a higher baseline severity was associated with a larger positive change in development as might be expected as higher severity patients have more potential for improvements.

Almost all of the associations shown in Figure 9 demonstrate that treatment was associated with greater improvements. The exceptions were generic multi-vitamin, presumably because that meant participants did not take a multi-vitamin specific for ASD, and melatonin, probably because it treats a specific problem and is given to children with sleep disorders who may require additional non-nutraceutical treatments.

**Figure 9.** Relationship between nutraceuticals and change in autism severity from 3 years of age to the current age.

#### 3.3.2. Perceived Benefit and Change in Autism Severity

A positive change in ASD severity was associated with the perceived benefit of any B12 supplement [χ(1)<sup>2</sup> = 10.14, *p* = 0.001] and the baseline ASD severity [χ(1)<sup>2</sup> = 94.85, *p* < 0.001] (Figure 10A), as well as the perceived benefit of injected B12 supplement [χ(1)<sup>2</sup> = 27.45, *p* < 0.001] and the baseline ASD severity [χ(1)<sup>2</sup> = 61.34, *p* < 0.001] (Figure 10B). Interestingly, the pattern of the child's development also affected the change in ASD severity when controlling for the benefit of injected B12 [χ(1)<sup>2</sup> = 24.32, *p* < 0.001]. This was due to the children with early onset ASD demonstrating significantly greater benefit (1.5) as compared to those who had a clinical regression and then a developmental plateau (−0.31), those with only a plateau (0.69) or those with a major developmental regression (0.46) when controlling for the perceived benefit of B12 injections.

A positive change in ASD severity was associated with the perceived benefit for Omega 3 Fatty Acids [χ(1)<sup>2</sup> = 6.10, *p* = 0.01] and the baseline ASD severity [χ(1)<sup>2</sup> = 148.38, *p* < 0.001] (Figure 10C). A positive change in ASD severity was associated with the perceived benefit in zinc supplementation [χ(1)<sup>2</sup> = 7.25, *p* < 0.01] and the baseline ASD severity [χ(1)<sup>2</sup> = 86.29, *p* < 0.001]. However, the effect of baseline ASD severity resulted in the perceived benefit only being obvious in the most severely affected patients (Figure 10D). Finally, a positive change in ASD severity was associated with the perceived benefit in Epsom salts [χ(1)<sup>2</sup> = 6.59, *p* = 0.01] and the baseline ASD severity [χ(1)<sup>2</sup> = 66.80, *p* < 0.001] (Figure 10E).

**Figure 10.** Association between change in ASD severity with the perceived benefit of the nutraceutical (**A**) Overall methylcobalamin; (**B**) Injected methylcobalamin; (**C**) Omega 3 Fatty Acids; (**D**) Zinc; (**E**) Epsom Salt Baths.

We also compared whether the severity of the diagnosis was related to starting any supplement. Those that took injected B12 [F(1710) = 4.244, *p* = 0.04], Epsom salt baths [F(1710) = 9.630, *p* < 0.01], Vitamin D [F(1710) = 7.184, *p* < 0.01], or MVI specific for ASD [F(1710) = 13.752, *p* < 0.001] had a higher severity at age 3 years of age whereas those that took a standard MVI [F(1710) = 16.640, *p* < 0.001] had a lower severity at age 3 years of age. The interaction with severity at 3 years of age and treatment was included in the linear model to determine if this effected the change in severity with treatment. For injected B12 [χ(1)<sup>2</sup> = 7.77, *p* < 0.01], Oral B12 [χ(1)<sup>2</sup> = 3.71, *p* = 0.05], Epsom salt baths [χ(1)<sup>2</sup> = 3.70, *p* = 0.05], Calcium [χ(1)<sup>2</sup> = 4.56, *p* < 0.05], Magnesium [χ(1)<sup>2</sup> = 3.93, *p* < 0.05], and Zinc [χ(1)<sup>2</sup> = 13.16, *p* < 0.001], the severity of autism at age 3 affected response to the treatment such that more severe individuals demonstrated a slightly lower response to some treatments.

#### **4. Discussion**

This study presents the Survey results of participants' reports of the perceived effectiveness and potential AEs of a wide range of nutraceuticals used in individuals with ASD. Nutraceuticals were generally reported to have a higher benefit compared to their AEs, with an average of 1.6 (slight/moderate benefit) and 0.1 (minimal AE), respectively. Reported benefits were generally in the slight/moderate range, and AEs were minimal.

The results of this study found significant benefits for many nutraceuticals with minimal adverse effects and are consistent with the findings of a number of clinical trials studying nutraceuticals in ASD. For example, double-blind, placebo-controlled studies, and/or meta-analyses have reported improvements in children with ASD using Lcarnitine [32,33], Coenzyme Q10 (ubiquinone) [34], digestive enzymes [35,36], high dose folinic acid (1–2 mg/kg/day) [24–26], MB12 injections [37], melatonin [9,38–42], a multivitamin/mineral supplement designed specifically for ASD [26,27], NAC [29,43–45], omega 3 fatty acids [46,47], vitamin C [48], vitamin D3 [49,50], and possibly B6/Mg [51,52]. Openlabel studies in ASD have also reported benefits for B vitamins [53,54], biotin [55], folic acid [56], an herbal formula [57], glutathione [58], iron [59], vitamin A [60] and zinc [61,62].

Some of the nutraceuticals in this Survey have not been previously studied in ASD including an amino acid blend, glutamine, taurine, tryptophan, evening primrose oil, flax seed oil, krill oil, calcium, chromium, iodine, lithium, potassium, selenium, vitamin E, vitamin K, valerian root, Epsom salts, GABA, and milk thistle. Thus, this Survey provides preliminary data on the effects (both beneficial and adverse) of these unstudied treatments which can help guide researchers to choose the most promising treatments to study in the future.

Some of the treatments reviewed may not only help certain symptoms of ASD but also treat underlying metabolic abnormalities associated with ASD. For example, mitochondrial dysfunction is relatively common in individuals with ASD [10,63] and is potentially treated with carnitine, Coenzyme Q10, B vitamins, and vitamin C [64]. Oxidative stress is also commonly associated with ASD [13] and is potentially treatable with antioxidants such as folinic acid, MB12, vitamin C, vitamin E, glutathione, ribose, and NADH. Melatonin is also an antioxidant and has positive effects on mitochondrial function [65].

Furthermore, children with ASD have been found to have multiple abnormalities related to one-carbon metabolism, including lower plasma levels of methionine, S-adenosylhomocysteine (SAM), homocysteine, cystathionine, cysteine, and total glutathione (GSH), as well as significantly higher concentrations of S-adenosylhomocysteine (SAH), adenosine, and oxidized glutathione (GSSG) [7,8]. Some studies have demonstrated that many children with ASD have a partial blockage in the transportation of folates into the brain due to an autoantibody to the folate receptor alpha, the primary mechanism which transports folate across the blood-brain barrier [66,67]. High dose folinic acid (1–2 mg/kg/day) has been shown to be an effective treatment for children with ASD with primary improvements in language in a double-blind placebo-controlled study [24], consistent with the findings of this Survey. Also consistent with this Survey, an open-label study found that high-dose folinic acid is effective for improving attention in children with ASD who possess the folate receptor alpha antibody [66], and two other placebo-controlled studies have also reported improvements with folinic acid in ASD [25,26]. These positive studies on the benefits of folinic acid are consistent with the results of Table 3, which demonstrates that folinic acid and vitamin B12 are two of the top-rated treatments for many ASD-related symptoms.

These abnormalities in one-carbon metabolism often result in problems in methylation and transsulfuration in ASD, resulting in a reduction in the production of glutathione [68]. In fact, these abnormalities appear to be so prevalent that they may be diagnostic for ASD [69]. Several studies [70,71] have addressed treatment of these linked pathways by providing cobalamin and folate derivatives to supplement the linked methylation-folate pathway in order to enhance the production of glutathione, while other studies have supplemented glutathione directly [58]. The findings of these studies of the benefits of cobalamin, folate, and glutathione are consistent with the results of this Survey.

It is interesting to compare the results of this Survey for nutraceuticals versus the results of this Survey for pharmaceuticals reported previously [31]. Averaging all nutraceuticals and all pharmaceuticals, the nutraceuticals had significantly higher Overall Benefit (1.59 vs. 1.39, *p* = 0.01) and significantly lower Overall Adverse Effect (0.1 vs. 0.9, *p* < 0.0001), based on a 2-sided *t*-test of the medications that had 20 or more responses [31]. Caution is needed in interpreting these results, since there are substantial variations in ratings for individual treatments. However, in general, these findings suggest that nutraceuticals may

be important treatment options for ASD, and more research into nutraceuticals and how they affect metabolism is warranted.

#### *4.1. Strengths of This Study*

One strength of this study is that some of these nutraceuticals have not been formally studied to date; therefore, this is the first data available on these treatments for ASD. Another advantage is that a uniform rating scale was used for all treatments, so that direct comparisons between different treatments could be made—this is often not possible for comparing data from clinical trials, since different assessment tools are typically used. Finally, another strength is the large number of participants in this study.

#### *4.2. Limitations of This Study*

There are several limitations of this Survey to consider. One limitation is that it is based on survey data, so there may be a significant placebo effect, especially since one of the most common benefits reported was "general benefit—no specific symptom". The ratings are based on perceived benefit (primarily by caregivers) and not by medical professionals. Age at which treatment was administered was not collected, which is a limitation of the study. Furthermore, there was no data collected on the dosages or durations of treatments (other than high versus low dose folinic acid). Therefore, various doses and durations of treatments may have been used by participants. Another limitation is the ASD-related diagnoses were not confirmed with standardized testing but were gathered by participant self-report. Finally, there is the potential for recall bias, where participants may not completely remember the effects of certain treatments. This may be reflected by the fact that no participants listed any of the nutraceuticals as causing worsening in ASD-related symptoms.

#### **5. Conclusions**

This Survey provides important information on the overall and specific benefits and adverse effects of 58 of the most commonly used nutraceuticals in ASD. The Overall Benefits were rated slightly higher for the nutraceuticals than for the most commonly used pharmaceuticals reported in the previous paper, with significantly lower ratings of adverse effects. The perception of participants of slight/moderate benefit with minimal adverse effects potentially explains why nutraceuticals were used by 75.2% of individuals with ASD in the Survey. This is consistent with the growing number of positive randomized clinical trials of nutraceuticals in ASD. Further research into nutraceutical treatments for treating biochemical differences and ASD symptoms is warranted.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/jpm11090878/s1, Table S1. List of All Nutraceuticals in Survey. Table S2. Amino Acids. Table S3. Fatty Acids. Table S4. Glutathione-related Nutraceuticals. Table S5. Individual Minerals. Table S6. Individual Vitamins/Vitamin-like Nutraceuticals. Table S7. Multivitamins. Table S8. Sleep Treatments. Table S9. Other Miscellaneous Nutraceuticals.

**Author Contributions:** Conceptualization, J.B.A. and D.M.C.; methodology, J.B.A., D.M.C. and R.E.F. software, D.M.C.; validation, D.M.C. and A.B.; formal analysis, J.B.A., A.B. and R.E.F.; investigation, J.B.A. and D.M.C. resources, J.B.A. data curation, D.M.C. writing—original draft preparation, J.B.A. and A.B.; writing—review and editing, J.B.A., R.E.F. and D.A.R. visualization, A.B.; supervision, J.B.A.; project administration, J.B.A.; funding acquisition, J.B.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded in part by the Autism Research Institute and the Zoowalk for Autism Research.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of Arizona State University (protocol code STUDY00003766 approved 26 January 2016).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to plans for additional analysis.

**Acknowledgments:** We thank the following organizations for helping promote the Survey: Age of Autism, ASU Autism/Asperger's Research Program, Autism Academy for Education and Development, Autism Canada, Autism Conferences of America, Autism File, Autism Free Brain, Autism Nutrition Research Center, Autism Research Institute, Autism Society of Alabama, Autism Society of Bayou, Autism Society of Central Ohio, Autism Society of Central Texas, Autism Society of Dayton, Autism Society of El Paso, Autism Society of Emerald Coast, Autism Society of Greater Akron, Autism Society of Greater Harrisburg, Autism Society of Greater New Orleans, Autism Society of Greater Phoenix, Autism Society of Hawaii, Autism Society of Indiana, Autism Society of Inland Empire, Autism Society of Iowa, Autism Society—Kern Autism Network, Autism Society of Massachusetts, Autism Society of Michigan, Autism Society of Minnesota, Autism Society of Northern Virginia, Autism Society of Northwestern Pennsylvania, Autism Society of Oregon, Autism Society of Pittsburgh, Autism Society of Pennsylvania, Autism Society of San Diego, Autism Society of Southern Arizona, Autism Society of Southeastern Wisconsin, Autism Society of Treasure Valley, Autism Society of Western New York, Autism Society of Westmoreland, Autism Society of West Virginia, Autism Society of Wisconsin, Autism Speaks, Autism Spectrum Therapies, Autism Tennessee, Autism Treatment Network, East Valley Autism Network, Generation Rescue, GOALS for Autism, Inc., Guthrie Mainstream Services, Hope Group, Independent Living Experience, National Autism Association, North Bridge College Success Program, Organization for Autism Research, Southwest Autism Research and Resource Center (SARRC), SEEDs for Autism, S.E.E.K Arizona, STARS, Talking About Curing Autism (TACA), Unlocking Autism, US Autism and Asperger's Association (USAAA). We thank Steve Edelson for his detailed review of the Survey. We especially thank the >1000 participants who participated in the Survey, and those who provided initial feedback on the early versions of the Survey.

**Conflicts of Interest:** J.B.A. serves as President of the Autism Nutrition Research Center, but does not receive any salary or royalties from them. The other authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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