Effect of Leucovorin on Mitochondrial Respiration and One-Carbon Metabolism in Autism Spectrum Disorder: A Double-Blind Placebo-Controlled Substudy
4 affiliations
- 1College of Nursing, The University of Tennessee Health Science Center, Memphis, TN 38163, USA
- 2Arkansas Children’s Research Institute, Little Rock, AR 72202, USA
- 3Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
- 4Autism Discovery and Treatment Foundation, Phoenix, AZ 85050, USA
Abstract
Mitochondrial dysfunction and impaired methylation are implicated in autism spectrum disorder (ASD). Leucovorin, a reduced folate that bypasses both dihydrofolate reductase and folate receptor alpha, has shown clinical benefit in small trials in children with ASD, but the underlying biology is incompletely understood. In this pre-specified biomarker substudy of a completed double-blind placebo-controlled trial (NCT02000284; NCT01602016), children with ASD received leucovorin (2 mg/kg/day, maximum 50 mg/day) or a matched placebo for 12 weeks. Peripheral blood mononuclear cell respiration was measured by extracellular flux with a graded 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) challenge (38 participants; 17 leucovorin, 21 placebo) and plasma thiol-methylation metabolites were measured pre- and post-treatment (42 participants; 18 leucovorin, 24 placebo). Leucovorin increased basal, ATP-linked, proton-leak, maximal, and reserve respiration relative to the placebo (Group × Visit p ≤ 0.012, all q ≤ 0.012; Cohen’s d 0.48–0.92); there was no evidence that the treatment effect varied across the tested DMNQ concentrations. In exploratory moderator analyses, folate receptor alpha autoantibody (FRAA) status modified the effect on maximal respiratory capacity and reserve capacity (both p < 0.001), with the largest gains in FRAA-positive children receiving leucovorin, and a history of neurodevelopmental regression modified the effect on basal, proton-leak and reserve respiration. The plasma SAM/SAH ratio increased (d = 0.77) and S-adenosylhomocysteine decreased (d = −0.67) on leucovorin; both were nominally significant (p = 0.020 and 0.050) but neither survived false-discovery-rate correction within the thiol family. The findings support the folate–mitochondrial–redox axis as a candidate therapeutic target in ASD and warrant replication in larger independent cohorts.
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BibTeX
@article{rose2026effect,
title = {{Effect of Leucovorin on Mitochondrial Respiration and One-Carbon Metabolism in Autism Spectrum Disorder: A Double-Blind Placebo-Controlled Substudy}},
author = {Shannon Rose and Stepan Melnyk and S. Jill James and Richard E. Frye},
journal = {International Journal of Molecular Sciences},
year = {2026},
month = oct,
doi = {10.3390/ijms27209101},
url = {https://www.mdpi.com/1422-0067/27/20/9101},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Effect of Leucovorin on Mitochondrial Respiration and One-Carbon Metabolism in Autism Spectrum Disorder: A Double-Blind Placebo-Controlled Substudy
AU - Shannon Rose
AU - Stepan Melnyk
AU - S. Jill James
AU - Richard E. Frye
PY - 2026
DA - 2026/10/01
JO - International Journal of Molecular Sciences
DO - 10.3390/ijms27209101
UR - https://www.mdpi.com/1422-0067/27/20/9101
AB - Mitochondrial dysfunction and impaired methylation are implicated in autism spectrum disorder (ASD). Leucovorin, a reduced folate that bypasses both dihydrofolate reductase and folate receptor alpha, has shown clinical benefit in small trials in children with ASD, but the underlying biology is incompletely understood. In this pre-specified biomarker substudy of a completed double-blind placebo-controlled trial (NCT02000284; NCT01602016), children with ASD received leucovorin (2 mg/kg/day, maximum 50 mg/day) or a matched placebo for 12 weeks. Peripheral blood mononuclear cell respiration was measured by extracellular flux with a graded 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) challenge (38 participants; 17 leucovorin, 21 placebo) and plasma thiol-methylation metabolites were measured pre- and post-treatment (42 participants; 18 leucovorin, 24 placebo). Leucovorin increased basal, ATP-linked, proton-leak, maximal, and reserve respiration relative to the placebo (Group × Visit p ≤ 0.012, all q ≤ 0.012; Cohen’s d 0.48–0.92); there was no evidence that the treatment effect varied across the tested DMNQ concentrations. In exploratory moderator analyses, folate receptor alpha autoantibody (FRAA) status modified the effect on maximal respiratory capacity and reserve capacity (both p < 0.001), with the largest gains in FRAA-positive children receiving leucovorin, and a history of neurodevelopmental regression modified the effect on basal, proton-leak and reserve respiration. The plasma SAM/SAH ratio increased (d = 0.77) and S-adenosylhomocysteine decreased (d = −0.67) on leucovorin; both were nominally significant (p = 0.020 and 0.050) but neither survived false-discovery-rate correction within the thiol family. The findings support the folate–mitochondrial–redox axis as a candidate therapeutic target in ASD and warrant replication in larger independent cohorts.
SN - 1422-0067
ER -