Leucovorin and calcium folinate in autism: Comprehensive review of research, mechanisms, and clinical applications

Leucovorin, also known as folinic acid or the reduced form of folic acid (5-formyltetrahydrofolate), has emerged as a promising therapeutic intervention for a subset of children with autism spectrum disorder (ASD). The clinical interest in leucovorin treatment stemmed from the discovery that cerebral folate deficiency (CFD), a condition characterised by inadequate folate levels in the central nervous system despite normal systemic folate concentrations, occurs frequently in children with ASD. This comprehensive review synthesises the current evidence on leucovorin use in autism, highlighting the key researchers, landmark studies, proposed biological mechanisms, and established dosing protocols that have shaped this emerging area of biomedical treatment for ASD.

Cerebral folate deficiency and folate receptor autoantibodies

The pathophysiological foundation for leucovorin treatment in autism rests primarily on understanding cerebral folate deficiency syndrome (CFDS) and its role in neurodevelopmental disorders. The cerebral folate receptor alpha (FR-?), a high-affinity transporter, is responsible for transporting the active form of folate, 5-methyltetrahydrofolate (5-MTHF), across the blood-brain barrier into the cerebrospinal fluid [1], [2]. When this transport system becomes compromised, brain folate concentrations fall precipitously while peripheral folate levels remain normal, creating the unique biochemical signature of CFD [3].

The primary cause of CFD in children with autism has been identified as the presence of serum autoantibodies directed against the folate receptor alpha (FRAAs), which block or bind to the folate receptor and impair its ability to facilitate folate transport [2], [3]. These folate receptor autoantibodies represent a form of autoimmune dysfunction, where the body’s own immune system produces antibodies that interfere with essential nutrient transport systems. The prevalence of FRAAs in children with autism is remarkably high; a landmark meta-analysis found that approximately 71% of children with ASD are positive for folate receptor autoantibodies [2], while other studies have reported prevalences ranging from 33.7% to 75.3% depending on the population studied [1], [4].

The clinical significance of this autoimmune mechanism was elegantly demonstrated through cerebrospinal fluid (CSF) analysis. In the foundational 2012 study by Richard Frye and colleagues, researchers measured serum FRA concentrations in 93 children with ASD and found a high prevalence (75.3%) of FRAs [1]. Crucially, in 16 children with detectable blocking FRAs, the concentration of these blocking autoantibodies significantly correlated with CSF 5-methyltetrahydrofolate concentrations, which were below the normative mean in every single case. This direct correlation provided compelling evidence that FRAAs were indeed responsible for depleting brain folate levels in affected children [1]. Furthermore, research has demonstrated that higher FRAA serum titers are inversely correlated with lower 5-MTHF CSF concentrations, establishing a dose-response relationship between antibody burden and brain folate depletion [2].

Beyond FRAs, another cause of cerebral folate deficiency that has been identified in autism is mitochondrial dysfunction, which was attributed to CFD in approximately 43% of cases in meta-analytic studies [2]. Soluble folate binding proteins (sFBPs) have also been detected in some patients with ASD, and these abnormal proteins appear to interfere with normal folate metabolism and have been associated with more severe autism symptoms and poorer adaptive functioning [5], [6].

Key researchers and landmark studies

The field of folate and autism research has been shaped by several pioneering investigators whose systematic investigations have provided the evidence base for clinical applications. Dr Richard Frye at the University of Arkansas and his colleagues have been instrumental in characterising the relationship between cerebral folate deficiency and autism. Frye’s 2012 publication in Molecular Psychiatry [1] is considered the seminal work that first demonstrated both the high prevalence of folate receptor autoantibodies in autism and the therapeutic responsiveness of FRAA-positive children to leucovorin treatment. In this open-label controlled study of 93 children with ASD, children with FRAs were treated with oral leucovorin calcium at 2 mg/kg/day (maximum 50 mg per day) and compared with a wait-list control group. Over a mean treatment period of 4 months, treated children showed significantly higher improvement ratings in verbal communication, receptive and expressive language, attention, and stereotypical behaviour compared with controls, with approximately one-third of treated children demonstrating moderate to much improvement [1].

Building on this initial work, Frye and colleagues conducted a rigorous double-blind, placebo-controlled trial published in Nature Molecular Psychiatry in 2016 [7]. This landmark study included 48 children with ASD and language impairment who were randomised to receive either high-dose folinic acid (2 mg/kg/day, maximum 50 mg/day) or placebo for 12 weeks. The results demonstrated that improvement in verbal communication was significantly greater in the folinic acid group compared to placebo, with a medium-to-large effect size (Cohen’s d=0.70). Importantly, FRAA status was predictive of treatment response; for FRAA-positive participants, the improvement was even more pronounced, with a large effect size (Cohen’s d=0.91) [7]. Additional improvements were observed across multiple behavioural domains, including the Vineland Adaptive Behaviour Scale, the Aberrant Behaviour Checklist, the Autism Symptom Questionnaire, and the Behavioural Assessment System for Children, with no significant differences in adverse effects between treatment groups [7].

Dr Vincent Ramaekers and Dr Edward Quadros, primarily working at institutions in Europe and the United States, respectively, have made significant contributions to understanding folate receptor autoimmunity in autism and other developmental disorders. Ramaekers and colleagues published an important self-controlled trial in 2019 examining the combined treatment of nutritional deficiencies along with high-dose folinic acid in 82 children with infantile autism [8]. The study found that 75.6% of children had folate receptor autoantibodies, and compared to untreated autistic children whose Childhood Autism Rating Scale (CARS) scores remained unchanged, the 2-year treatment with folinic acid decreased the initial CARS score from severe (mean 41.34) to moderate or mild autism (mean 34.35), with complete recovery achieved in 17 of 82 children (20.7%). The study also revealed that prognosis was less favourable with higher FR autoantibody titers or the presence of maternal FR autoantibodies [8].

Dr Derrick Rossignol has been instrumental in synthesising the evidence base through comprehensive systematic reviews and meta-analyses. A 2021 systematic review and meta-analysis by Rossignol and Frye examined 21 studies (including four placebo-controlled and three prospective, controlled trials) that treated individuals with ASD using d,l-leucovorin [2]. The meta-analysis found improvements with d,l-leucovorin in overall ASD symptoms (67%), irritability (58%), ataxia (88%), pyramidal signs (76%), movement disorders (47%), and epilepsy (75%) among individuals with ASD and CFD. Critically, d,l-leucovorin was found to significantly improve communication with medium-to-large effect sizes and to have positive effects on core ASD symptoms and associated behaviours such as attention and stereotypy in individual studies with large effect sizes [2].

Recent research has continued to refine the understanding of biomarkers predictive of treatment response. A 2024 retrospective analysis by Frye and colleagues examined data from 110 consecutive ASD clinic patients who underwent testing for folate receptor alpha autoantibodies and soluble folate binding proteins [6]. The study found that higher binding FRAA titres were associated with greater therapeutic response to leucovorin treatment, as measured by improvements in the Social Responsiveness Scale and Aberrant Behaviour Checklist, confirming that FRAA status serves as a biomarker for identifying which children with autism are most likely to benefit from leucovorin therapy [6].

Proposed biological mechanisms

The therapeutic mechanisms underlying leucovorin’s effects in autism involve multiple interconnected biochemical pathways that are disrupted in the disorder. Understanding these mechanisms is essential for rational treatment application and for identifying which children with autism are most likely to benefit from this intervention.

Folate-dependent one-carbon metabolism

Folate serves as a critical cofactor in one-carbon metabolism, a central metabolic process essential for DNA synthesis, DNA methylation, and neurotransmitter production [9]. When cerebral folate deficiency occurs due to FRAAs blocking the folate receptor, this entire metabolic pathway becomes compromised in the brain. The active form of folate, 5-methyltetrahydrofolate (5-MTHF), is the key one-carbon donor in these reactions. Leucovorin, being a reduced form of folate with the chemical structure 5-formyltetrahydrofolate, can bypass the defective folate receptor by utilising alternative, lower-affinity folate transporters such as the reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT) [10]. This alternative transport mechanism allows leucovorin to restore folate-dependent metabolic reactions in the brain despite the blockade of the primary folate receptor pathway.

The downstream consequences of restored folate metabolism are substantial. Adequate folate availability permits normal methylation reactions, including methylation of the phospholipids that form neuronal membranes, methylation of myelin proteins essential for nerve conduction, and epigenetic regulation of genes critical for brain development and function [11]. Furthermore, normal folate metabolism supports the production of neurotransmitters including serotonin, dopamine, and ?-aminobutyric acid (GABA), all of which are implicated in the pathophysiology of autism  [9].

Glutathione metabolism and oxidative stress

Oxidative stress, characterised by an imbalance between reactive oxygen species and antioxidant defences, has been consistently identified as a significant pathophysiological feature in autism. Glutathione, the brain’s most abundant antioxidant molecule, plays a central protective role against oxidative damage. Importantly, glutathione synthesis depends on adequate folate status through the methylation cycle; folate deficiency impairs methylation reactions and consequently compromises glutathione production and metabolism [12].

Research examining oxidative stress biomarkers in children with severe autism and folate receptor autoimmunity revealed that compared to controls, autistic children with FRAs demonstrated significant increases in oxidative DNA damage in lymphocytes, plasma ceruloplasmin and copper levels with a high copper/zinc ratio, thiol proteins, and superoxide dismutase (SOD) activity, while vitamin C levels were significantly diminished [12]. These findings indicate a state of dysregulated redox metabolism in autism with FRAA-associated cerebral folate deficiency. Treatment with folinic acid can restore glutathione metabolism by normalising one-carbon metabolism, thereby enhancing the cell’s capacity to synthesise and regenerate this critical antioxidant.

A landmark study examining the relationship between glutathione redox status and treatment response found that in 37 children with autistic disorder treated with methylcobalamin (a form of vitamin B12) combined with folinic acid, all Vineland Adaptive Behaviour Scale subscales significantly improved, with an average improvement of 7.7 months in skills and an average effect size of 0.59 [13]. Critically, greater improvement in glutathione redox status was associated with greater improvement in expressive communication, personal and domestic daily living skills, and interpersonal, play-leisure, and coping social skills. This demonstrates that improvements in behavioural outcomes from folate-based treatment are mechanistically linked to normalisation of glutathione metabolism [13].

Neurotransmitter synthesis and brain development

The critical role of folate in neurotransmitter synthesis provides another mechanism through which cerebral folate deficiency contributes to autism symptoms, and through which leucovorin treatment exerts therapeutic effects. Folate-dependent methylation reactions are required for the synthesis of serotonin, dopamine, and other monoamine neurotransmitters essential for normal brain function. Children with autism and cerebral folate deficiency due to folate receptor autoimmunity demonstrate significantly lower cerebrospinal fluid serotonin metabolites [12]. By restoring folate availability in the brain, leucovorin treatment normalises the production of these critical neurotransmitters, contributing to improvements in mood, social behaviour, and cognitive function.

Additionally, folate is essential for the methylation and maturation of proteins crucial for synaptic transmission and neuronal plasticity. Adequate folate status supports the normal development and function of the nervous system throughout the critical developmental windows when the brain is particularly vulnerable to nutritional and metabolic disruptions [11]. The relationship between critical periods of neurodevelopment and maternal or foetal folate status is particularly important, as maternal folate receptor autoantibodies can impair folate transport across the placenta to the developing foetus, potentially setting the stage for autism-spectrum neurodevelopmental pathology in offspring [11].

DNA methylation and epigenetic regulation

Folate-dependent methylation reactions control epigenetic regulation of gene expression through DNA methylation and histone modifications. These epigenetic processes are critical for regulating genes involved in neuronal development, synaptogenesis, and the establishment of neural circuits. Aberrant DNA methylation patterns have been documented in autism, and restoration of normal methylation capacity through improved folate metabolism represents a mechanism through which leucovorin could ameliorate neurodevelopmental dysfunction [9]. The ability of folinic acid treatment to improve adaptive functioning and communication in children with autism may reflect, in part, normalisation of epigenetically regulated genes essential for social-emotional processing and language development.

Dosing protocols and clinical applications

The establishment of appropriate dosing regimens for leucovorin in autism represents a critical practical consideration for clinical application. The doses investigated and recommended have been refined through multiple clinical trials and systematic experience with this treatment.

Standard dosing

The most commonly used and evidence-supported dosing regimen for leucovorin in autism is 2 mg/kg/day, with a maximum daily dose of 50 mg [1], [7], [14]. This dose has been consistently employed in the major double-blind, placebo-controlled trials and open-label studies that demonstrated therapeutic benefit. In the landmark 2016 double-blind study by Frye and colleagues, children received 2 mg/kg/day of high-dose folinic acid for a 12-week treatment period with measurable benefits in communication and adaptive behaviour [7]. More recently, a 2024 pilot study examined 2 mg/kg/day dosing and found it to be safe and feasible, with no adverse events reported in 10 autistic children aged 4-8 years [14].

In other treated populations with cerebral folate deficiency, including those with FOLR1 gene mutations, folinic acid is commonly recommended at 2–10 mg/kg/day. If oral therapy doesn’t produce an adequate clinical response, some reports suggest adding intravenous folinic acid (50–100 mg once weekly) and, in selected cases, intrathecal folinic acid alongside this approach [15]. Grapp et al. proposed that IV and/or intrathecal folinic acid may be more effective than oral treatment alone [15]. Published regimens vary, including 1.7 mg/kg/day oral folinic acid or 8.9 mg/kg/day orally plus 500 mg/week IV folinic acid [15]. Folic acid is not recommended because it binds strongly to FR? and may compete with 5MTHF for receptor transport [15].

 The choice between oral and intravenous administration, as well as dose optimisation, may depend on the specific aetiology of the cerebral folate deficiency (FRA-based versus genetic) and the clinical response trajectory.

Duration of treatment and monitoring

Treatment duration in the published studies has ranged from 12 weeks to 2 years, with most controlled trials employing treatment periods of at least 12 weeks to assess meaningful behavioural change. The longitudinal study by Ramaekers and colleagues followed children for 2 years and observed progressive improvement in autism severity scores [8]. This suggests that extended treatment durations may be necessary to achieve maximal benefit. A reasonable approach to clinical practice would be to implement a trial of leucovorin for at least 12 weeks, with re-evaluation of benefits at that time to determine whether continuation is warranted.

Regular monitoring of treatment response is essential. Standardised instruments employed in clinical trials and recommended for monitoring include the Social Responsiveness Scale (SRS), the Aberrant Behaviour Checklist (ABC), particularly the irritability subscale, the Childhood Autism Rating Scale (CARS), and the Vineland Adaptive Behaviour Scale (VABS) for assessment of communication and adaptive functioning [6], [7]. Parent and caregiver reports of changes in behaviour, communication, social interaction, and adaptability should be systematically tracked to determine whether treatment is producing clinically meaningful benefit.

Safety and adverse effects

One of the significant advantages of leucovorin treatment is its generally favourable safety profile. The meta-analysis of 21 studies examining leucovorin in autism found that adverse effects across studies were generally mild  [2]. The most commonly reported adverse effects in the meta-analysis included aggression (9.5%), excitement or agitation (11.7%), headache (4.9%), insomnia (8.5%), and increased tantrums (6.2%), with no significant difference in adverse effects between the leucovorin and placebo groups in the double-blind controlled trial  [2], [7].

Recent smaller studies have similarly reported favourable tolerability. In the 2024 pilot study involving 10 autistic children, all children successfully consumed oral folinic acid supplements without adverse events, though the small sample size limits the generalisability of this finding [14]. In studies of genetic cerebral folate deficiency using higher doses of intravenous folinate, adverse effects have been minimal, with the focus of adverse effects being primarily related to the route of administration rather than the medication itself.

Some caution has been raised regarding potential risks associated with very high folate supplementation, particularly during pregnancy. One cell culture study reported that high concentrations of 5-methyltetrahydrofolate (above 1.25 ?M) led to DNA damage in human foetal lung cells and cell cycle arrest in G1 phase [16]. However, this concentration is substantially higher than typical therapeutic dosing, and clinical data in actual treated children have not demonstrated such concerns. Nevertheless, this finding suggests that while the standard therapeutic doses of 2 mg/kg/day appear safe, excessively high doses should be avoided, and normal clinical practice should adhere to the established dosing guidelines.

Biomarkers for predicting treatment response

One of the most important developments in this field is the identification of biomarkers that predict which children with autism are most likely to respond to leucovorin treatment. This represents a shift toward precision medicine and personalised treatment approaches in autism.

The most robust biomarker identified is the presence and titre of folate receptor alpha autoantibodies (FRAAs). Multiple studies have demonstrated that FRAA-positive children, particularly those with higher FRAA-binding titers, show a greater treatment response than FRAA-negative children. In a double-blind, controlled trial, FRAA status was predictive of treatment response, with FRAA-positive participants showing a significantly greater effect size (Cohen’s d=0.91) for improvement in verbal communication than the overall effect size (Cohen’s d=0.70) [7]. A 2024 study examining 110 ASD patients found that higher binding FRAA titres were associated with greater improvements in Social Responsiveness Scale subscales and Aberrant Behaviour Checklist irritability with leucovorin treatment [6].

Soluble folate binding proteins (sFBPs) have also been identified as a biomarker. Patients with ASD who are positive for sFBPs were found to have more severe baseline ASD symptoms but also showed improvement with leucovorin treatment [5]. This suggests that testing for sFBPs may identify another subgroup of children with autism who would benefit from treatment.

Glutathione redox status has emerged as a mechanistic biomarker, with studies showing that the magnitude of improvement in behavioural outcomes correlates with improvements in glutathione metabolism [13]. While glutathione testing is not routinely employed as a screening biomarker for treatment candidacy in clinical practice, monitoring changes in glutathione status in response to treatment may help confirm that the proposed mechanism of action is operating in individual patients.

Cerebrospinal fluid 5-methyltetrahydrofolate levels represent the gold standard biomarker for confirming cerebral folate deficiency, but lumbar puncture is rarely justified solely for assessment of CSF folate in children with autism. In contrast, serum FRAA testing is minimally invasive and has become a standard screening tool for identifying children who may be responsive to leucovorin  [1], [6].

Special considerations and clinical context

FRAA-Negative Autism

An important limitation of leucovorin treatment is that it is specifically indicated for children with autism who have positive FRAA status or other identified folate metabolism abnormalities. The meta-analysis and individual studies make clear that FRAA status is predictive of response; FRAA-negative children with autism do not show the same degree of benefit. This emphasises the critical importance of pre-treatment screening for folate receptor autoantibodies and other biomarkers of folate metabolism dysfunction. Treatment of FRAA-negative children with leucovorin is not supported by available evidence and should be avoided to prevent unnecessary medication exposure [2], [17].

Combination treatments

Several studies have examined combination treatments incorporating leucovorin with other biomedically targeted interventions. Ramaekers and colleagues’ trial combined correction of multiple nutritional deficiencies identified through biochemical testing with the addition of high-dose folinic acid in children with autism [8]. This approach, testing for and correcting multiple metabolic abnormalities simultaneously, may enhance treatment efficacy. Similarly, the study of methylcobalamin combined with folinic acid demonstrated synergistic benefits on glutathione metabolism and adaptive behaviour [13]. A comparison of metabolic treatments suggested that methylcobalamin with low-dose folinic acid had significant effects on glutathione and cysteine metabolism, while treatment with high-dose folinic acid alone did not significantly influence biomarkers of methylation, glutathione, or chronic oxidative stress in one analysis [18]. This finding warrants further investigation to clarify optimal dosing strategies and potential additive benefits of combination treatments.

Maternal and prevention implications

An emerging area of investigation is the role of folate receptor autoimmunity in pregnancy and its potential implications for preventing autism. Maternal folate receptor autoantibodies can block folate transport across the placenta to the developing foetus, potentially contributing to foetal neurodevelopmental abnormalities and increased risk of autism in offspring [3]. Theoretically, early identification and treatment of women with FRAA-positive status during pregnancy could reduce the risk of autism in their children. However, this remains an area requiring further research before clinical recommendations can be made  [9]. The current evidence supports the idea that supplementing folate during pregnancy may reduce autism risk in offspring, but the optimal form of folate (folic acid versus folinic acid), the timing of supplementation, and the populations most likely to benefit remain unclear.

Relationship to other autism biomarkers

It is important to recognise that autism is a heterogeneous disorder with multiple underlying biological subtypes. Folate receptor autoimmunity represents one identified metabolic abnormality affecting approximately 70% of children with autism, but other children with autism have different underlying pathophysiological abnormalities, including mitochondrial dysfunction, immune dysregulation, oxidative stress from other aetiologies, and genetic variations. The identification of folate-related pathology as a biomarker has facilitated the development of a targeted treatment for a specific autism subgroup, representing an important advance in personalised and precision medicine approaches to autism [17], [19].

Current medical guidelines and clinical adoption

Despite accumulating evidence from multiple controlled trials, the American Academy of Paediatrics (AAP) currently does not formally recommend the use of leucovorin for the treatment of autism [17]. This conservative stance likely reflects the relatively small number of placebo-controlled trials, the heterogeneity of autism, and the fact that not all children with autism will benefit from this treatment. However, the evidence base continues to expand, with multiple recent publications adding to the data supporting efficacy in the FRAA-positive subgroup.

Many paediatricians and developmental medicine specialists have reported increased requests from families for leucovorin prescriptions, driven by growing awareness of this treatment option on social media and among parent advocacy groups [17]. In clinical practice, a reasonable approach involves screening interested families’ children for FRAA status, discussing the evidence base, likely benefits and risks, and implementing a trial of treatment with systematic monitoring of behavioural change using validated instruments.

Future directions and research gaps

Despite substantial progress, significant gaps remain in the understanding of folate-related autism pathophysiology and optimal treatment strategies. Further large-scale placebo-controlled trials with standardised outcome measures and long-term follow-up would strengthen the evidence base. Investigation of whether early intervention with leucovorin, potentially beginning in infancy for identified FRAA-positive children, could prevent or reduce the severity of autism symptoms would be valuable. The mechanisms underlying why some FRAA-positive children respond robustly while others show modest benefit require clarification. Additionally, research examining the relationship between folate metabolism and other proposed pathophysiological pathways in autism could identify synergistic treatment approaches. Finally, the potential for preventing autism through maternal folate supplementation in FRAA-positive women warrants rigorous prospective investigation.

Conclusion

Leucovorin and calcium folinate represent a targeted, evidence-based biomedical treatment option for children with autism spectrum disorder who have cerebral folate deficiency due to folate receptor alpha autoantibodies or other folate metabolism abnormalities. The extensive research conducted by pioneers including Richard Frye, Vincent Ramaekers, Edward Quadros, and Derrick Rossignol has established that approximately 70% of children with autism carry folate receptor autoantibodies that impair brain folate transport. Multiple placebo-controlled and open-label trials have demonstrated that treatment with leucovorin at a dose of 2 mg/kg/day (maximum 50 mg daily) for 12 weeks to 2 years produces improvements in communication, adaptive functioning, and associated behaviours, with the strongest improvements observed in children with high FRAA titres. The proposed mechanisms underlying treatment efficacy involve the restoration of folate-dependent one-carbon metabolism, normalisation of glutathione-mediated antioxidant defence systems, enhancement of neurotransmitter synthesis, and correction of epigenetic dysregulation. Safety data indicate that leucovorin is generally well-tolerated with mild and infrequent adverse effects. The identification of FRAA status as a biomarker enabling precise identification of treatment-responsive individuals represents an important advance in the development of personalised medicine approaches for autism. Continued research into optimising dosing strategies, identifying additional predictive biomarkers, and exploring combination treatment approaches will further refine the clinical application of folate-based therapies in autism.

Disclaimer: This article is for informational purposes only and does not constitute medical advice. Always consult a healthcare professional for diagnosis and treatment of medical conditions.

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