Hair and urine lead, cadmium, nickel, and arsenic levels in children with attention‐deficit hyperactivity disorder: A case–control study in a tertiary care hospital in eastern India

Attention‐deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by inattention and/or hyperactivity–impulsivity. It is one of the most widespread neurodevelopmental conditions and has compound etiopathogenesis involving both environmental and genetic factors. Though the role of heavy metals on ADHD has been implicated but is less studied. Hair and urine are two non‐invasive methods which can substitute blood as a method of monitoring and assessing heavy metal levels. Twenty‐four cases of ADHD and their age matched healthy children (24) were taken as controls. Hair and urine samples were analyzed for lead, cadmium, nickel, and arsenic using inductively coupled plasma optical emission spectrometry (ICP‐OES) after acid digestion and extraction. The levels of heavy metals were significantly higher in cases; lead (p = .004, .003), cadmium (p = .020, <.001), and nickel (p = .016, <.001) of the hair and urine samples, respectively. Arsenic was below the limit of detection for all the samples. Hence, in conclusion, the heavy metal levels in hair and urine were significantly higher in ADHD cases as compared to their healthy counterparts.


| INTRODUCTION
In developing countries, the sociodemographic and epidemiological transition has altered the morbidity and mortality pattern among communities. As a result of these, the noncommunicable diseases (NCD) have risen to the top of the healthcare priority list. Neurological diseases are a major contributor to NCDs, affecting the quality of life, disability, morbidity, and mortality. 1 WHO states that neurodevelopmental disorders are the most potent health problems of the 21st century, and attentiondeficit hyperactivity disorder (ADHD) is one of the most diagnosed disorders in children. 2 It is a neurodevelopmental disorder characterized by inattention and/or hyperactivity-impulsivity, which hampers the normal activities and growth of the child. 3 This complex and heterogeneous condition, with well-defined neurological underpinnings, has been conceptualized over the last three decades as a chronic multifactorial disorder marked by symptoms of inattention, hyperactivity, and impulsivity. 4 The few of the challenges the ADHD children face are that they perform poorly in academic examinations, hence, are labeled as bad or slow learners by peers, and get ejected or suspended from classrooms.
Apart from these, there is a perceived decline in skill acquisition, which results in insufficient financial and work prospects as adults, causing them to have lower quality of physical and mental health outcomes than their healthy counterparts. 1,5,6 The genetic risks associated with ADHD that have been found thus far have small effect sizes or are infrequent.
They frequently increase the risk of developing a variety of different sorts of psychopathologies. The separation between genetic and environmental influences has been claimed to be erroneous, with ADHD being a multifactorial complex disorder in which multiple genes interact to influence the tendency. 7 By contrast, the environment contributes to the manifestation of the ADHD phenotype through interaction with the genotype. Metals enter the environment via a range of natural and artificial processes.
Metals' increasing circulation in the air, water, and soil, and their eventual entry into the human food chain, is a major environmental problem that offers unknown health risks to future generations. 8 Chemical elements are classified into trace elements (less than 100 mg/day) and minerals (more than 100 mg/day) according to their relative abundance in the human body. 9 Existing evidence suggests that in children with ADHD, a change in trace element and mineral status is linked to disease severity. 10 Lead poisoning is a public health issue, mainly in children, who are more vulnerable due to the norm of hand-using activity. Lead poisoning in gestation, infancy or early childhood can stifle mental growth and result in decreased intellect which can last into adulthood. 11 Similarly, cadmium has been known to be a neurotoxic agent with the most significant risk to human health. In children due to increased uptake and sequestration in tissues, as well as less effective excretory and detoxification potency it can lead to severe neurological damage in the early formative period. [12][13][14] Even elements like nickel and arsenic which have not been studied much in detail as to their effects on neurodevelopmental growth and associated disorders, have been known to cause serious neurological damage upon exposure in toxic amounts. [15][16][17][18] In contrast to occupational health, it is common in environmental health practice to find that young children and even some adults refuse blood sampling due to its invasive nature or emotional reasons, preferring to submit urine samples. Levels of these metals in the blood and urine are highly correlated. 19 According to the emerging data, determining heavy metal levels in hair samples could be a non-invasive method of analyzing concentrations of heavy metals accumulated over time. 20 In this study, we assessed the levels of heavy metals lead, cadmium, arsenic, and nickel in the hair and urine samples of ADHD children and to compare the same with healthy controls.

| MATERIALS AND METHODS
The study was carried out at the All India Institute of Medical Sciences (AIIMS), Bhubaneswar. Patients diagnosed with ADHD by a psychiatrist/ pediatrician were taken as cases and healthy children with no psychiatric or other chronic illness were taken as controls. The sample size was calculated to be 48, with an alpha-error of 5% and power of 80%. Accordingly, 24 cases and 24 controls were taken in as study participants between the ages of 3-16 years. The Institutional Ethics Committee of AIIMS, Bhubaneswar had granted the required ethical clearance. Participant's information sheet and informed consent form were provided to the parents, and assent was taken from the children in accordance with the Indian Council of Medical Research (ICMR), National Ethical Guidelines for Biomedical Research involving Children.
Hair samples were collected from the nape of the neck by a procedure adopted from International Atomic Energy Agency's assessment of at risk workers and the first morning urine sample was collected by a mid-stream clean catch technique. 21 The procedure for digestion of the hair and urine samples were as per the protocol prescribed in previous literature. 22 All samples were collected in containers pre-treated with nitric acid and stored at À80 C for extraction. Hair samples were digested nitric acid and hydrogen peroxide by heating at 180 C for 15 min, followed by cooling. 23 The extraction of heavy metals from urine samples was done using a solution containing 0.1% (V/V) Triton-X-100 (Sigma) and 1% ultrapure concentrated nitric acid. 24 Thirty milliliters of urine sample was taken in a beaker and heated at 60 C with few drops of perchloric acid and concentrated nitric acid for 10 min (until white fumes are produced) to remove any dissolved precipitates, infective agents or any solids, as well as most salts of in form of chlorides or nitrates. Five milliliters of this sample was added to 45 mL of a solution containing 0.1% (V/V) Triton-X-100 (Sigma) and 1% ultrapure concentrated nitric acid making the total volume to 50 mL. The extracted sample was then transferred to a seal-capped container after passing through a filter paper and stored at room temperature for heavy metal analysis. 24 Each hair sample (500 mg) was digested in a mixture of 6 mL of 65% Nitric Acid and 2 mL of 30% Hydrogen Peroxide by heating at 180 C for 15 min followed by cooling at room temperature for 15 min. The cooled digested samples and blanks were then transferred into a 50 mL beaker and diluted with deionized water till mark. The extracted sample was then transferred to a seal-capped container after passing through a filter paper and stored at room temperature for heavy metal analysis. 23 The measurement of heavy metal levels was done by induc-

| RESULTS
The data was found to be non-normal in distribution, hence all non-parametric tests have been used. The demographic characteristic of the study population was similar with no significant difference on comparison cases with controls ( Table 1). The subtypes of ADHD categories are summarized in Table 2.
T A B L E 2 Subgroup profile of cases of ADHD. The difference in the two groups was measured by Mann-Whitney U test in hair and urine samples as shown in Figures 1 and 2, respectively. The effect size for Mann-Whitney U test was expressed by Rank-Biserial correlation with a 95% CI and has been summarized in Table 3.
Arsenic was below the limits of detection for ICP-OES in all the samples and hence has not been analyzed subsequently. In ADHD cases the median lead level was 3.12 μg/g of hair as compared to Spearman's correlation was performed to assess the correlation between the deposition heavy metals in hair and their urinary excretion (Table 4). Lead levels in hair and urine had a positive correlation which was statistically significant in the control group only (r = .754, p < .001). There was a negative correlation of hair and urine cadmium levels I the ADHD cases (p = .509) and a positive correlation in the healthy controls which was statistically significant (r = .678, p = .000).
Nickel levels in hair and urine correlated negatively in cases and positively in controls but neither were statistically significant. Urinary nickel was found to correlate significantly with urine lead and cadmium. The unadjusted correlations have been tabulated in Table 5.
The correlation heatmap conditioned on adjusting for age, sex, and BMI has been summarized in Figure 2. It shows significant correlation of lead compared with other heavy metals.
Logistic regression was performed to determine the odds of heavy metals in their association with ADHD. The adjusted and unadjusted odds along with coefficient B has been tabulated in

| DISCUSSION
Our study was aimed to estimate the levels of heavy metals in hair and urine in ADHD cases and healthy controls and compare the two groups. As seen from the results, there exists a significant difference in lead, cadmium, and nickel levels in both the samples between the two groups. There have been very few studies inquiring into the concentration of heavy metals sequestered in hair and excreted in urine for ADHD children.
In both hair and urine samples, the median concentration of lead and cadmium were significantly higher in the ADHD group. have shown an increased serum concentration in ADHD children of lead and cadmium, respectively. [28][29][30] Similarly, significantly higher levels of nickel were found in hair and urine of ADHD children.
Though previous literature is lacking in this aspect, various developmental disorders have been attributed to nickel accumulation in children. 31 The brain is largely believed to be a primary organ of nickel neurotoxicity. Among the numerous potential pathways underlying Ni-induced neurotoxicity, oxidative stress is a critical factor. 32 As previously noted, disruptions in iron-sulfur cluster (ISC) lead to formation and usage of free radicals, shifting to impaired energy metabolism by increasing aberrant mitochondrial electron transport. 33 The fact that arsenic was not determined in any sample is a welcoming evidence of the fact that the potable water is relatively safe as otherwise it would have been a concern of major environmental pollution. 34 The study had certain limitations owing to the COVID-19 pandemic as the sample population was concentrated around a particular city and a certain socioeconomic segment which may not be a true