Spectrofluorimetric determination of serum homovanillic acid using horseradish peroxidase and its association with autism spectrum disorder

Dopaminergic dysfunction during neural development correlates with developmental disorders, including autism spectrum disorder. Homovanillic acid, a dopamine metabolite, serves as a biomarker for central dopaminergic activity. The aim of the present study is to establish a validated spectrofluorimetric method for exploring the association between serum homovanillic acid level and autism spectrum disorder. The spectrofluorimetric method is based on the transformation of homovanillic acid into a highly fluorescent dimer via catalytic oxidation facilitated by horseradish peroxidase in the presence of hydrogen peroxide. The resultant dimer of homovanillic acid demonstrated a pronounced emission peak at 430 nm subsequent to excitation at 335 nm. The methodology underwent validation in accordance with ICH M10 guidelines, and endogenous homovanillic acid concentrations were determined utilizing the standard addition technique. The method was successfully applied for estimating the homovanillic acid level in the serum of children diagnosed with autism spectrum disorder, as well as in healthy children. The findings indicated that the concentration of homovanillic acid was markedly elevated in the serum of children with ASD (mean value of 89 µg/L, n = 24) in comparison to that of their healthy counterparts (mean value of 56 µg/L, n = 15), P < 0.01.