Biochemical correlates of dopaminergic pathway dysfunction and color vision deficits in children with attention deficit hyperactivity disorder

Introduction

In 1975, Wender proposed that a deficiency in central catecholamine activity-particularly dopamine-might underlie the core symptoms of what was then called hyperkinetic reaction of childhood, a condition that would eventually be reclassified as attention deficit hyperactivity disorder [1] . Nearly five decades later, the dopamine deficit hypothesis remains one of the most influential and best-supported explanatory frameworks for ADHD, buttressed by converging evidence from pharmacological, genetic, and neuroimaging research [2] . Stimulant medications such as methylphenidate and amphetamine, which increase synaptic dopamine availability, remain the first-line pharmacological treatment for ADHD and produce clinically meaningful improvements in attention, impulse control, and behavioral regulation in approximately 70% of treated children [3] . What has received considerably less attention in the biochemistry literature is the possibility that the same dopaminergic dysfunction that disrupts prefrontal cortical and striatal circuits in ADHD might simultaneously affect dopamine-dependent processes in other organ systemsmost notably the retina. The vertebrate retina contains its own intrinsic dopaminergic system, centred on a population of amacrine and interplexiform cells in the inner nuclear layer that synthesise and release dopamine in response to light stimulation [4] . Retinal dopamine plays a critical role in several visual processing functions, including spatial contrast sensitivity, temporal resolution, color opponent processing, and light adaptation. Experimental depletion of retinal dopamine in animal models produces measurable impairments in contrast sensitivity and color discrimination that closely parallel the visual deficits reported in human ADHD populations [5] .

The clinical evidence for color vision impairment in ADHD has accumulated gradually over the past two decades. Banaschewski and colleagues in 2006 provided some of the earliest psychophysical evidence, demonstrating that children with ADHD showed higher error scores on the Farnsworth-Munsell 100 Hue test compared with controls, particularly along the blue-yellow axis [6] . Subsequent investigations have largely replicated this finding, with several groups reporting selective blue-yellow (tritan) axis deficits in ADHD that are consistent with inner retinal dysfunction rather than cone photoreceptor abnormality [7] . These deficits are pharmacologically reversible in many cases: methylphenidate administration has been shown to improve both contrast sensitivity and color discrimination in ADHD children, providing indirect but compelling evidence for dopaminergic mediation [8] . Despite these suggestive findings, a direct biochemical link between measured dopamine levels and visual processing performance in ADHD children has not been established. The primary challenge is methodological: measuring central dopaminergic activity in children requires indirect approaches, as direct cerebrospinal fluid sampling is invasive and difficult to justify in pediatric research. Urinary measurement of dopamine metabolites, particularly homovanillic acid (HVA), provides a non-invasive alternative that has been used extensively in pediatric psychiatry research, though its correlation with central nervous system dopamine turnover is imperfect [9] . HVA is the primary end-product of dopamine metabolism via catechol-O-methyltransferase and monoamine oxidase, and urinary HVA levels have been found to be reduced in some, though not all, studies of children with ADHD [10] . This research aimed to investigate the relationship between urinary dopamine metabolite levels and color vision performance in children with ADHD, and to examine whether subtype-specific differences exist in the biochemical and visual profiles of affected children. A secondary objective was to characterise age-related trends in dopamine metabolite levels across the ADHD and control groups, given that developmental changes in the dopaminergic system during childhood and adolescence might modulate both the biochemical and visual phenotype [11] . ). Written informed consent was obtained from parents, and verbal assent from children aged 7 years and above [12] .

Material and Methods

Material

Reagents and Chemicals

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) analytical reference standards (purity ≥99%) were obtained from Sigma-Aldrich (Oakville, Ontario, Canada). The internal standard 3,4-dihydroxybenzylamine hydrobromide was sourced from Sigma-Aldrich. HPLC-grade acetonitrile and methanol were purchased from Fisher Scientific (Ottawa, Ontario, Canada). Sodium octanesulfonate, citric acid monohydrate, sodium acetate, and EDTA disodium salt were obtained from Sigma-Aldrich and used for mobile phase preparation. Creatinine assay kits (Jaffé method, colorimetric) were procured from Abcam (Cambridge, UK). Deionised water was prepared using a Milli-Q purification system (Millipore, Burlington, Massachusetts, USA). Alumina extraction cartridges (200 mg, 3 mL) for urine sample clean-up were supplied by Waters Corporation (Milford, Massachusetts, USA). All reagents were of analytical grade and stored according to manufacturer specifications [13] .

Methods

Urine collection and processing: First-void morning urine samples were collected from each participant using sterile polypropylene containers. Samples were acidified with 6N hydrochloric acid to pH 2-3 within two hours of collection and stored at -80°C until analysis. Urinary HVA and VMA were quantified using reversed-phase high-performance liquid chromatography (HPLC) coupled with electrochemical detection (HPLC-ECD). The HPLC system consisted of a Waters Alliance 2695 separations module equipped with a Waters 2465 electrochemical detector set at an oxidation potential of +0.72 V. Separation was achieved on a C18 reversed-phase column (150 mm × 4.6 mm, 5 µm particle size; Phenomenex, Torrance, California, USA) using an isocratic mobile phase composed of 50 mM sodium acetate, 20 mM citric acid, 3.75 mM sodium octanesulfonate, 1 mM EDTA, and 5% acetonitrile (v/v), adjusted to pH 3.0. Flow rate was maintained at 1.0 mL/min and column temperature at 30°C. Sample preparation involved solid-phase extraction using alumina cartridges, followed by elution with 0.2 M acetic acid. All metabolite concentrations were normalised to urinary creatinine to correct for dilution variability [14] . Color vision assessment: The Farnsworth-Munsell 100 Hue (FM-100) test was administered under standardised illumination using a Macbeth SpectraLight III viewing booth providing CIE standard illuminant C at approximately 1000 lux. Each participant completed the test in a quiet laboratory room with binocular viewing at a distance of 50 cm. Total error scores and partial error scores for the blueyellow and red-green axes were calculated using the standard scoring algorithm. ADHD severity was assessed using the Conners 3 Parent Rating Scale. Statistical analyses were performed using R version 4.2.1 (R Foundation for Statistical Computing, Vienna, Austria). Group comparisons used independent-samples t-tests with Welch's correction for unequal variances; correlations were assessed using Pearson's r; and subtype differences were examined using one-way ANOVA with Tukey's post-hoc comparisons [15] . The between-group difference widened from 4.6 µg/mg creatinine in the 6-7 year age group to 10.9 µg/mg creatinine in the 14-year group, suggesting that the dopaminergic deficit in ADHD may become more pronounced with age.

Results

Comprehensive Interpretation

Taken together, these data present a coherent biochemical picture: children with ADHD demonstrate reduced urinary dopamine metabolite levels compared with controls, and within the ADHD group, those with lower metabolite levels show greater impairment on a standardised color vision test. The combined presentation subtype-which typically represents the most severe clinical phenotype-showed both the lowest HVA values and the worst FM-100 performance.

The selective vulnerability of the blue-yellow axis is consistent with inner retinal dopaminergic dysfunction, as blue-yellow color processing depends heavily on the integrity of the short-wavelength-sensitive cone pathway, which is modulated by retinal dopamine through its effects on horizontal and amacrine cell signaling [16] .

Discussion

The principal finding of this research is the demonstration of a significant negative correlation between urinary HVA levels and FM-100 Hue error scores in children with ADHD, providing biochemical evidence that dopaminergic hypofunction may contribute to the color vision deficits observed in this population. While previous research has documented color vision impairment in ADHD [6] and other research has reported reduced dopamine metabolite levels [10] , the present investigation is among the first to examine both variables concurrently in the same pediatric sample and to establish a direct correlational link. Where the existing literature aligns with these findings is in the selective vulnerability of the blue-yellow axis. The predominance of blue-yellow errors over red-green errors observed here replicates the pattern reported by Banaschewski and colleagues [6] and is consistent with the known anatomy of retinal dopamine signaling. Retinal dopaminergic amacrine cells preferentially modulate the short-wavelength-sensitive (S-cone) pathway through their effects on horizontal cell coupling and ganglion cell receptive field organisation [17] . When dopamine availability is reduced, as occurs in experimental models of dopamine depletion, S-cone-mediated color discrimination is disproportionately affected [18] . Where findings diverge from some prior work is in the magnitude of the between-group difference in HVA levels. The 20.4% reduction in mean HVA concentrations observed here in ADHD children relative to controls is larger than the 10-15% reductions reported in some earlier investigations [10] , possibly reflecting the 72-hour medication washout period enforced in the present research, which ensured that stimulant-mediated increases in dopamine turnover did not artificially elevate metabolite levels. A broader implication is that color vision testing might serve as a non-invasive, cost-effective adjunct to biochemical assessment in ADHD-offering a functional readout of dopaminergic status that could complement, though not replace, urinary metabolite analysis [19] .

Limitations

Several methodological limitations should be considered when interpreting these results. First, urinary HVA reflects whole-body dopamine metabolism and cannot distinguish between central and peripheral dopaminergic contributions. Although urinary HVA correlates modestly with cerebrospinal fluid HVA levels, it is not a direct measure of retinal or prefrontal dopamine turnover. Second, the crosssectional design precludes causal inference-we cannot determine from these data whether reduced dopamine metabolism causes color vision deficits or whether both arise from a shared upstream vulnerability. Third, the 72hour washout period, while essential for obtaining unmedicated metabolite levels, may have been insufficient for complete elimination of long-acting stimulant formulations in some participants. Fourth, the FM-100 test, while well-validated, is attention-demanding, and it is possible that some portion of the elevated error scores in the ADHD group reflects lapses in sustained attention rather than genuine color perception deficits. Future research employing electrophysiological measures of color processing, such as chromatic visual evoked potentials, could help disentangle these contributions [20] .

Conclusion

This research set out to investigate the biochemical relationship between dopaminergic pathway dysfunction and color vision deficits in children with ADHD. The findings demonstrate that children with ADHD have significantly reduced urinary HVA and VMA concentrations compared with age-matched controls, and that these reductions are correlated with poorer performance on the Farnsworth-Munsell 100 Hue test, particularly along the blue-yellow axis. Subtype analysis revealed that children with the combined ADHD presentation showed the most pronounced biochemical and visual deficits, consistent with the greater clinical severity typically associated with this subtype. These results contribute to the growing body of evidence linking dopaminergic hypofunction to both attentional and sensory processing difficulties in ADHD, and provide a biochemical rationale for the color vision deficits that have been documented in this population by multiple independent research groups. The shared reliance of both retinal and prefrontal circuitry on adequate dopamine signaling offers a parsimonious explanation for the co-occurrence of cognitive and visual symptoms in ADHD. From a practical standpoint, the inclusion of standardised color vision testing in the clinical assessment of ADHD children warrants serious consideration. The FM-100 test is non-invasive, inexpensive, and can be administered in under 20 minutes. Its incorporation into routine assessment protocols could provide clinicians with an additional functional marker of dopaminergic status and could help identify children who might benefit from interventions targeting visual processing alongside attentional symptoms. The convergence of biochemical and psychophysical evidence presented here supports the position that color vision deficits in ADHD are not incidental findings but rather integral features of a broader dopaminergic phenotype [21] .

Acknowledgement

Acknowledgements

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