Effect of Leucovorin on Mitochondrial Respiration and One-Carbon Metabolism in Autism Spectrum Disorder: A Double-Blind Placebo-Controlled Substudy

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.