Attention Deficit Hyperactivity Disorder (ADHD) is one of the most common neurodevelopmental disorders of childhood and adolescence, characterized by persistent patterns of inattention, hyperactivity, and impulsivity that interfere with functioning and development. According to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5), symptoms should be present before the age of 12 years, occur in more than one setting, and result in significant impairment in social, academic, or occupational functioning (1). ADHD represents a major public health concern because of its impact on educational achievement, social relationships, emotional well-being, and quality of life. Children affected by ADHD frequently experience academic underachievement, poor peer relationships, low self-esteem, and behavioral difficulties. Furthermore, untreated ADHD may persist into adolescence and adulthood, leading to increased risks of substance abuse, psychiatric comorbidities, and occupational dysfunction (2). Globally, the prevalence of ADHD is estimated to be approximately 5–7% among school-aged children. In India, reported prevalence rates vary widely from 1.3% to 12.2%, depending on diagnostic criteria, study population, and assessment methods employed (3,4). A systematic review conducted among Indian children reported a pooled prevalence of approximately 7.1%, highlighting ADHD as a significant paediatric and mental health problem in the country (5). Although genetic factors account for nearly 70–80% of ADHD susceptibility, increasing attention has been directed toward environmental, nutritional, and biochemical factors that may influence the development and severity of the disorder (6). Among these, micronutrient deficiencies, particularly Vitamin D deficiency and iron deficiency, have emerged as potential contributors to ADHD pathogenesis. Vitamin D, traditionally known for its role in calcium homeostasis and bone metabolism, is now recognized as a neuroactive steroid hormone with significant functions in brain development and neurological processes. Vitamin D receptors and Vitamin D-activating enzymes are widely distributed throughout the central nervous system, particularly in regions involved in attention, executive functioning, memory, and behavioral regulation, including the prefrontal cortex, hippocampus, cerebellum, and substantia nigra (7). Experimental studies have demonstrated that Vitamin D influences neuronal differentiation, axonal growth, neurotrophic factor production, neurotransmitter synthesis, synaptic plasticity, and neuroprotection (8). It has also been shown to regulate dopamine synthesis through activation of tyrosine hydroxylase, an enzyme critical in dopaminergic pathways implicated in ADHD pathophysiology (9). Consequently, Vitamin D deficiency during critical periods of brain development may adversely affect cognitive and behavioral outcomes. Vitamin D deficiency has become a widespread health concern worldwide and is particularly prevalent in India despite abundant sunshine. Studies conducted across various regions of India have consistently reported a high prevalence of Vitamin D deficiency among children and adolescents. Research by Harinarayan et al. demonstrated that more than 70% of apparently healthy Indian children exhibited inadequate Vitamin D levels (10). Similarly, Marwaha et al. reported Vitamin D deficiency in a substantial proportion of school-going children residing in urban India despite adequate sunlight availability (11). Several international studies have reported significantly lower serum Vitamin D levels in children diagnosed with ADHD compared to healthy controls. A systematic review and meta-analysis by Wang et al. found that children with ADHD had significantly reduced serum 25-hydroxy Vitamin D concentrations, suggesting a possible role of Vitamin D deficiency in ADHD pathogenesis (12). Similar findings have been reported by Khoshbakht et al., who observed an inverse association between Vitamin D levels and ADHD symptom severity (13). Another micronutrient receiving considerable attention in ADHD research is iron. Iron is an essential trace element required for multiple neurobiological processes, including myelination, energy metabolism, neurotransmitter synthesis, and neuronal development. Iron serves as a cofactor for tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Since dopamine dysregulation is considered a central mechanism in ADHD, disturbances in iron metabolism may contribute to symptom development and severity (14). Ferritin is the primary intracellular iron storage protein and is considered the most reliable indicator of body iron stores. Low serum ferritin levels reflect depleted iron reserves even before the development of overt anemia. Emerging evidence suggests that reduced ferritin levels may be associated with behavioral disturbances, cognitive deficits, impaired attention, and hyperactivity in children (15). The relationship between ferritin levels and ADHD was first highlighted by Konofal et al., who demonstrated significantly lower serum ferritin concentrations among children with ADHD compared to healthy controls (16). Subsequent studies have reported similar observations and suggested that reduced ferritin levels may correlate with increased symptom severity, particularly hyperactivity and impulsivity (17). Iron deficiency may impair dopaminergic neurotransmission within the basal ganglia and prefrontal cortex, thereby contributing to ADHD manifestations (18). Iron deficiency remains a major nutritional problem in India. According to the National Family Health Survey (NFHS-5), a substantial proportion of Indian children continue to suffer from iron deficiency and anemia despite various national nutritional interventions (19). Considering the high burden of iron deficiency in the Indian paediatric population, evaluation of iron stores through serum ferritin estimation may have significant implications in children with ADHD. Recent evidence indicates that Vitamin D deficiency and iron deficiency may coexist and exert synergistic effects on neurodevelopment and behavioral regulation. Both nutrients influence dopaminergic pathways, neuroinflammation, oxidative stress, and neuronal maturation. Deficiencies of either nutrient may adversely affect attention, executive function, learning, and behavioral control (20). Despite increasing global interest in nutritional factors associated with ADHD, relatively few studies have explored the combined relationship of serum Vitamin D and ferritin levels among Indian children with ADHD. India faces a dual burden of widespread Vitamin D deficiency and iron deficiency among children. Given the potential role of these micronutrients in brain development and behavior, identifying their association with ADHD could have important clinical and public health implications. Early detection and correction of these nutritional deficiencies may provide a simple, cost-effective adjunct to conventional ADHD management strategies. Therefore, the present study was undertaken to evaluate serum Vitamin D and ferritin levels in children diagnosed with ADHD and to assess their relationship with ADHD severity. The findings of this study may contribute to a better understanding of the nutritional aspects of ADHD and help formulate evidence-based recommendations for screening and management in Indian paediatric populations.

Study Design The present study was designed as a hospital-based observational case-control study conducted to evaluate serum Vitamin D and ferritin levels among children diagnosed with Attention Deficit Hyperactivity Disorder (ADHD) and to compare them with age- and sex-matched healthy controls. Study Setting The study was conducted in the Departments of Paediatrics of a tertiary care teaching hospital in India. Children attending the Paediatric Outpatient Department (OPD), and Child Guidance Clinic were screened for eligibility during the study period. Study Duration The study was carried out over a period of 12 months from Dec 2022 to Nov 2023. Study Population The study population comprised children aged 6–12 years. Study Groups Cases Group: Children diagnosed with ADHD according to Diagnostic and Statistical Manual of Disorders, Fifth Edition (DSM-5) criteria. Control Group: Age- and sex-matched healthy children attending the Paediatric OPD for minor ailments or routine health check-ups and having no history of neurological disorders. Sample Size The sample size was calculated using the formula for comparison of means between two independent groups: [n=\frac{(Z_{\alpha/2}+Z_{\beta})^2 (SD_1^2+SD_2^2)}{d^2}] Based on previous studies reporting significant differences in serum Vitamin D levels between ADHD children and controls, a minimum sample size of 50 cases and 50 controls was determined to achieve a power of 80% and confidence level of 95%. Thus, the total study population consisted of: • ADHD Cases = 50 • Healthy Controls = 50 Total Sample Size = 100 Sampling Technique Consecutive sampling technique was employed. Eligible participants fulfilling inclusion criteria were enrolled until the required sample size was achieved. Inclusion Criteria Cases 1. Children aged 6–12 years. 2. Diagnosed with ADHD according to DSM-5 criteria. 3. Newly diagnosed or untreated ADHD cases. 4. Parents/guardians willing to provide written informed consent. Controls 1. Healthy children aged 6–12 years. 2. Age- and sex-matched with cases. 3. No history of psychiatric illness. 4. Parents/guardians willing to provide written informed consent. Exclusion Criteria Children with any of the following conditions were excluded: 1. Chronic systemic illness (renal, hepatic, cardiac disease). 2. Intellectual disability. 3. Autism Spectrum Disorder. 4. Epilepsy or other neurological disorders. 5. Genetic syndromes. 6. Severe malnutrition. 7. Acute infection within the preceding four weeks. 8. Iron supplementation during the previous three months. Ethical Considerations Prior approval was obtained from the Institutional Ethics Committee before commencement of the study. Written informed consent was obtained from parents or legal guardians prior to enrollment. Assent was obtained from children whenever appropriate according to institutional guidelines. Confidentiality of participant information was maintained throughout the study. Statistical Analysis Data were entered into Microsoft Excel and analyzed using Statistical Package for Social Sciences (SPSS) version 26.0. Descriptive Statistics • Mean ± Standard Deviation (SD) • Frequency • Percentage Inferential Statistics Student's t-test Used for comparison of continuous variables between cases and controls. Chi-square Test Used for comparison of categorical variables. Pearson Correlation Coefficient Used to assess correlation between: • Vitamin D levels and ADHD severity score.

Table 1: Demographic Characteristics of Study Participants Variable ADHD Cases (n=50) Controls (n=50) p-value Age (years) 8.9 ± 1.8 9.1 ± 1.7 0.62 Gender Male, n (%) 38 (76.0) 37 (74.0) 0.81 Female, n (%) 12 (24.0) 13 (26.0) Residence Urban, n (%) 28 (56.0) 30 (60.0) 0.68 Rural, n (%) 22 (44.0) 20 (40.0) Socioeconomic Status* Upper 4 (8.0) 5 (10.0) 0.74 Upper Middle 10 (20.0) 12 (24.0) Lower Middle 22 (44.0) 21 (42.0) Upper Lower 11 (22.0) 9 (18.0) Lower 3 (6.0) 3 (6.0) Body Mass Index (kg/m²) 17.3 ± 2.1 17.8 ± 2.3 0.28 Parental Education Primary School 8 (16.0) 6 (12.0) 0.59 Secondary School 19 (38.0) 17 (34.0) Higher Secondary 15 (30.0) 16 (32.0) Graduate & Above 8 (16.0) 11 (22.0) Family History of ADHD 9 (18.0) 2 (4.0) 0.03 Values are expressed as Mean ± Standard Deviation or Number (%). The mean age of ADHD cases was 8.9 ± 1.8 years and that of controls was 9.1 ± 1.7 years, with no statistically significant difference (p = 0.62). Males constituted the majority of participants in both groups (76% in cases and 74% in controls). Distribution of residence, socioeconomic status, body mass index, and parental education was comparable between the groups (p > 0.05). A significantly higher proportion of ADHD children had a positive family history of ADHD compared to controls (18% vs 4%; p = 0.03). Table 2: Comparison of Serum Vitamin D Levels Between ADHD Cases and Controls Serum Vitamin D Level (ng/mL) ADHD Cases (n = 50) Controls (n = 50) p-value Mean ± SD 18.4 ± 6.2 29.8 ± 7.5 <0.001 Median 17.5 30.2 Range 8.2 – 34.5 15.6 – 46.8 Statistical test used: Student's t-test Level of significance: p < 0.05 considered statistically significant. The mean serum Vitamin D level in children with ADHD was 18.4 ± 6.2 ng/mL, which was significantly lower than that observed in healthy controls (29.8 ± 7.5 ng/mL). The difference between the two groups was statistically highly significant (p < 0.001). Children with ADHD had significantly lower serum Vitamin D levels compared to healthy controls, suggesting a possible association between Vitamin D deficiency and ADHD. This finding supports the hypothesis that Vitamin D may play a role in the neurobiological mechanisms underlying ADHD. Table 3: Comparison of Serum Ferritin Levels Between ADHD Cases and Healthy Controls Serum Ferritin Level (ng/mL) ADHD Cases (n = 50) Controls (n = 50) p-value Mean ± SD 24.6 ± 10.5 42.1 ± 12.8 <0.001 Median 22.8 40.5 Range 8.5 – 52.4 18.6 – 68.7 Statistical test used: Student's t-test Level of significance: p < 0.05 considered statistically significant. The mean serum ferritin level among children with ADHD was 24.6 ± 10.5 ng/mL, whereas the mean serum ferritin level among healthy controls was 42.1 ± 12.8 ng/mL. The difference between the two groups was statistically highly significant (p < 0.001). Children diagnosed with ADHD had significantly lower serum ferritin levels compared to healthy controls. Since ferritin reflects body iron stores and iron is essential for dopamine synthesis and normal neurodevelopment, reduced ferritin levels may contribute to the pathophysiology of ADHD. These findings suggest a possible association between depleted iron stores and ADHD in children. Table 4: Distribution of Vitamin D Status Among ADHD Cases and Healthy Controls Vitamin D Status ADHD Cases (n = 50) Controls (n = 50) Total (n = 100) p-value Deficient (<20 ng/mL) 31 (62.0%) 9 (18.0%) 40 (40.0%) <0.001 Insufficient (20–29 ng/mL) 14 (28.0%) 18 (36.0%) 32 (32.0%) Sufficient (≥30 ng/mL) 5 (10.0%) 23 (46.0%) 28 (28.0%) Total 50 (100%) 50 (100%) 100 (100%) Statistical test used: Chi-square test (χ²) Level of significance: p < 0.05 considered statistically significant. Among the ADHD cases, 62.0% of children were Vitamin D deficient, 28.0% had insufficient Vitamin D levels, and only 10.0% had sufficient Vitamin D levels. In contrast, among the healthy controls, only 18.0% were Vitamin D deficient, while 46.0% had sufficient Vitamin D levels. The distribution of Vitamin D status differed significantly between the two groups (p < 0.001). Vitamin D deficiency was considerably more prevalent among children with ADHD than among healthy controls. The significantly higher proportion of Vitamin D deficiency in ADHD cases suggests that inadequate Vitamin D status may be associated with the development or severity of ADHD symptoms. These findings support the need for routine assessment of Vitamin D levels in children diagnosed with ADHD. Table 5: Correlation of Serum Vitamin D and Ferritin Levels with ADHD Severity Scores Parameter Mean Value Correlation Coefficient (r) p-value Serum Vitamin D (ng/mL) 18.4 ± 6.2 -0.48 0.001 Serum Ferritin (ng/mL) 24.6 ± 10.5 -0.42 0.003 Statistical test used: Pearson's Correlation Analysis Level of significance: p < 0.05 considered statistically significant. A statistically significant negative correlation was observed between serum Vitamin D levels and ADHD severity scores (r = -0.48, p = 0.001). Similarly, serum ferritin levels showed a significant negative correlation with ADHD severity scores (r = -0.42, p = 0.003). The negative correlation coefficients indicate that as serum Vitamin D and ferritin levels decrease, ADHD severity scores tend to increase. Children with lower Vitamin D and ferritin levels exhibited more severe symptoms of inattention, hyperactivity, and impulsivity. Both serum Vitamin D and ferritin levels were inversely associated with ADHD severity. These findings suggest that deficiencies in Vitamin D and body iron stores may contribute not only to the occurrence of ADHD but also to the severity of clinical manifestations. Monitoring and correcting these deficiencies may potentially improve symptom control and overall clinical outcomes in children with ADHD.

The present study was undertaken to evaluate serum Vitamin D and ferritin levels in children with Attention Deficit Hyperactivity Disorder (ADHD) and to assess their relationship with ADHD severity. The study demonstrated significantly lower serum Vitamin D and ferritin levels in ADHD cases compared to healthy controls. In addition, both parameters showed a significant inverse correlation with ADHD severity scores. These findings support the hypothesis that micronutrient deficiencies may play a contributory role in the pathophysiology and severity of ADHD. In the present study, serum Vitamin D levels were significantly lower in ADHD cases (18.4 ± 6.2 ng/mL) compared to controls (29.8 ± 7.5 ng/mL) (p < 0.001). Vitamin D deficiency (<20 ng/mL) was observed in 62% of ADHD children, indicating a high burden of hypovitaminosis D in this group. Similar findings have been reported in previous Indian studies. Harinarayan et al. highlighted that Vitamin D deficiency is highly prevalent among Indian children despite adequate sunlight exposure, mainly due to lifestyle factors, reduced outdoor activity, and dietary insufficiency (10). Marwaha et al. also reported widespread Vitamin D deficiency among urban school children in India, emphasizing that hypovitaminosis D is a major public health concern (11). International evidence also supports this association. Khoshbakht et al. reported in a systematic review and meta-analysis that children with ADHD have significantly lower serum Vitamin D levels compared to controls (13). Wang et al. similarly demonstrated that Vitamin D deficiency is associated with increased risk of ADHD (12). The biological plausibility of this association is well established. Vitamin D receptors (VDR) are widely distributed in brain regions involved in attention, executive function, and behavior regulation such as the prefrontal cortex, hippocampus, and cerebellum (7). Vitamin D plays an important role in neuronal differentiation, neurotrophic factor regulation, and neurotransmitter synthesis, particularly dopamine modulation, which is central to ADHD pathophysiology (9). Deficiency of Vitamin D during neurodevelopmental stages may therefore contribute to behavioral and cognitive dysfunction. In the present study, serum ferritin levels were significantly lower in ADHD cases (24.6 ± 10.5 ng/mL) compared to controls (42.1 ± 12.8 ng/mL) (p < 0.001), indicating depleted iron stores in ADHD children. These findings are consistent with earlier studies. Konofal et al. first demonstrated significantly reduced ferritin levels in children with ADHD and suggested a link between iron deficiency and ADHD symptoms (16). Subsequent studies by Cortese et al. confirmed that low ferritin levels are associated with greater severity of ADHD symptoms, particularly inattention and cognitive impairment (17). In the Indian context, iron deficiency remains a major nutritional problem. According to NFHS-5, a significant proportion of Indian children suffer from iron deficiency anemia due to inadequate dietary intake and poor nutritional status (19). This high background prevalence makes ferritin evaluation particularly relevant in Indian children with ADHD. Iron is essential for brain development and dopamine metabolism. It acts as a cofactor for tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Dopaminergic dysfunction in the prefrontal cortex and basal ganglia is a key mechanism in ADHD pathophysiology (14,18). Therefore, iron deficiency may impair attention, executive functioning, and behavioral control. The present study demonstrated a significant inverse correlation between serum Vitamin D levels and ADHD severity scores (r = -0.48, p = 0.001), and between ferritin levels and ADHD severity scores (r = -0.42, p = 0.003). This indicates that lower levels of these micronutrients are associated with more severe ADHD symptoms. Similar observations have been reported in earlier studies. Villagomez and Ramtekkar suggested that micronutrient deficiencies, including Vitamin D and iron, may influence both the occurrence and severity of ADHD symptoms (20). Eyles et al. also emphasized the role of Vitamin D in neurodevelopmental regulation and brain maturation (7). The stronger correlation observed with Vitamin D compared to ferritin may reflect its broader role in neurodevelopment, including neuroprotection, immune modulation, and synaptic plasticity. Ferritin, in contrast, is more specifically involved in dopaminergic neurotransmission. An important finding of the present study is the coexistence of Vitamin D deficiency and low ferritin levels in a substantial proportion of ADHD children. This suggests that multiple micronutrient deficiencies may act synergistically in affecting neurodevelopment and behavior. Both Vitamin D and iron influence dopaminergic pathways, oxidative stress regulation, and neuroinflammatory processes. Disruption of these pathways during critical brain development periods may contribute to the manifestation and severity of ADHD symptoms. India faces a dual burden of Vitamin D deficiency and iron deficiency among children. National surveys and community-based studies have consistently reported high prevalence of both conditions across different regions of the country (10,11,19). Factors such as poor dietary intake, limited sun exposure, urban lifestyle, and socioeconomic constraints contribute significantly to this burden. In this context, the findings of the present study have important clinical implications. Routine screening of Vitamin D and ferritin levels in children diagnosed with ADHD may help identify correctable nutritional deficiencies. Early intervention through supplementation and dietary modification may serve as a cost-effective adjunct to pharmacological and behavioral therapy. Limitations The present study has certain limitations. It is a single-center hospital-based study with a relatively small sample size, limiting generalizability. Dietary intake, sunlight exposure, and other micronutrients such as zinc and magnesium were not assessed. Longitudinal studies are required to further explore these associations.

Children with Attention Deficit Hyperactivity Disorder had significantly lower serum Vitamin D and ferritin levels compared to healthy controls. Both parameters showed an inverse relationship with ADHD severity. Routine screening for Vitamin D deficiency and depleted iron stores may be considered in children with ADHD. Further multicentric longitudinal studies are required to establish causality and evaluate the therapeutic benefits of nutritional supplementation.

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