Assessment of Copper and Zinc Levels in Hair and Urine of Children With Attention Deficit Hyperactivity Disorder: A Case-Control Study in Eastern India

Background Attention deficit hyperactivity disorder (ADHD) is a neuro-developmental ailment diagnosed with inattention and hyperactivity-impulsivity. It is one of the most prevalent neurodevelopmental disorders and has complex aetiology, both genetic and environmental. There is a perceived decrease in skill acquirement, leading to insufficient income and job opportunities as adults, which drives them towards poor physical and mental outcomes compared to their contemporaries without ADHD. The impact of heavy metals on ADHD is a topic of interest but is much less studied. Copper has been implicated as a pro-oxidant and in the metal accelerated production of free radicals that may affect oxidative stress. Zinc also serves as an antioxidant, and changes in its concentrations may impact the homeostasis of oxidative stress. Methods Twenty-four children diagnosed with ADHD were taken as cases and matched with 24 healthy controls. Hair and urine samples were collected from all the study participants. The samples were collected in sterile containers according to established protocols. Acid digestion of hair samples was done using 65% nitric acid and 30% hydrogen peroxide. Urine samples were extracted by a solution of 0.1% Triton-X-100 and 1% ultrapure nitric acid. The levels of zinc and copper were determined in both samples by inductively coupled plasma optical emission spectrometry (ICP-OES). The copper/zinc ratio (Cu/Zn) was calculated from these values. Mann Whitney U Test and receiver operating characteristic (ROC) analysis were done to estimate statistical significance. Results The median age of the study population was eight years. Overall, 34 male and 14 female subjects participated. There was no significant difference in height, weight and BMI between the cases and controls. Hair zinc levels in the ADHD group (198.49 µg g(-1) of hair) was significantly lower than the control group (527.05 µg g(-1) of hair). However, hair copper levels were increased significantly in the ADHD children (14.01 µg g(-1) of hair) compared to the controls (7.43 µg g(-1) of hair). Urine zinc levels were significantly lower in cases than controls (525.7 µg g(-1) of spot urine creatinine vs 1374.09 µg g(-1) of spot urine creatinine). However, copper levels in urine were higher in the ADHD children (17.01 µg g(-1) of spot urine creatinine compared to 7.26 µg g(-1) of spot urine creatinine in controls). Both hair and urine copper to zinc ratio was significantly higher in the ADHD group. On ROC analysis, the hair Cu/Zn ratio had an area under the curve (AUC) of 0.920 (p-value <0.001), and the urine Cu/Zn ratio had an AUC of 0.967 (p-value <0.001). When used as a diagnostic classifier for ADHD based on the cut-off value determined by ROC, both hair and urine Cu/Zn ratio had high sensitivity and specificity. Conclusion Low zinc levels in the urine and hair of children and higher levels of copper may impact the aetiology of ADHD in these children. At an early stage, the Cu/Zn ratio in both hair and urine samples may be used as a precise biomarker to identify and monitor such children.