Metallomics analysis for early assessment and individualized intervention of neurodevelopmental disorders

Abstract

The children today are in the midst of the epidemic of neurodevelopmental disorders. In this metallomics study for the scalp hair samples of total 2550 children with autistic disorders (2108 males and 442 females aged 0–15 years), it was demonstrated that near one-half of the infantile individuals aged 0–3 years are suffering from zinc deficiency and toxic metal burdens. Zinc level correlated closely to the index of zinc/iron ratio more than zinc/copper ratio. Furthermore, there were significant relationships between zinc deficiency and toxic metal burdens such as lead and aluminum, which were inversely associated with not only zinc level but also zinc/iron ratio with higher regression coefficients of r = −0.486 and −0.551 (P < 0.00001), respectively. High-significant inverse association was detected between zinc and molybdenum concentration (r = −0.509) and also between zinc/iron ratio and molybdenum (r = −0.548). These findings suggest that infantile zinc deficiency relates to the high burdens of not only toxic but also some essential metals such as molybdenum, iron, and manganese and that these various mineral imbalances play principal roles in the etiology of neurodevelopmental disorders. We expect that the early assessment and intervention of the mineral imbalances (or dis-homeostasis) in individual child open an avenue for evidence-based individualized treatment of neurodevelopmental disorders and also of the comorbid immune disorders, in near future.

Graphical Abstract

Infantile Zn and Mg deficiency and high burdens of toxic metals.

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Introduction

The children today in developed countries are in the midst of the epidemic of neurodevelopmental disorders such as autism spectrum disorder (ASD) and attention-deficit/hyperactivity disorder (ADHD). These disorders are a group of neurodevelopmental disorders heritable with a strong genetic basis of 65 or more candidate genes, and their prevalence has been increasing to 13%.^1–4 However, the key genetic determinants are still unclarified and the interaction of hereditary factors with some environmental factors seems play a critical role in the pathogenesis.³ Thus, the elucidation of the etiology and effective treatment of neurodevelopmental disorders is one of the challenges today.

Great advances in a high-sensitive and reliable trace element analysis method using inductively coupled plasma mass spectrometry (ICP-MS) have enabled us to apply it to medical researche and estimate the chronic toxic metal burden and mineral deficiency in the human body.^5–7

In the previous studies, we have reported that some restricted mineral deficiency (e.g. zinc and magnesium) and toxic metal burdens, especially during the restricted time window of 0–3 year old, epigenetically play principal roles as environmental factors in autistic disorders and that metallomics approach lead to early screening and prevention of the neurodevelopmental disorders.^6–9

This infantile zinc-deficiency-related epigenetic hypothesis of neurodevelopmental disorders has been supported and reinforced by many research groups^10–17 and has been expected to lead to approaching its early intervention/treatment.^9,17–20

In this metallomics study for total 2550 children with autistic disorders, it is demonstrated that many children with the neurodevelopmental disorders are suffered from high burdens of some essential metals such as molybdenum, iron, and manganese, not only toxic metal burdens. Furthermore, it is suggested that the index of zinc/iron ratio is another useful biomarker for assessment of the Japanese children suffered from neurodevelopmental disorders. We expect that the early assessment and intervention of various mineral imbalances open an avenue for evidence-based individualized therapy for neurodevelopmental disorders and also their comorbid allergic immune disorders.

Materials and methods

Samples and trace element analysis

Human scalp hair, a kind of cellular body, is widely used as a non-invasive, stable, and useful biospecimen for assessment of environmental exposure of toxic metals, for evaluation of nutritional status, and in forensic science.^5,7,8,16,20

On the basis of informed consent, scalp hair samples from 2550 (male: 2108; female: 442) autistic Japanese subjects aged 0–15 years were collected in the period from June 2005 to September 2015, although 0-year-old subject was only two (10- and 11-month-old female). These subjects were comprised of the children diagnosed with ASD by their physicians. Hair sampling was recommended to cut as close to the scalp of the occipital area as possible.

Hair sample of 75 mg was weighed into a 50 ml plastic tube and washed with acetone and then with a 0.01% triton solution, as recommended by the Hair Analysis Standardization Board. The washed hair sample was mixed with 10 ml 6.25% tetramethyl ammonium hydroxide (TMAH, Tama Chemical, Kawasaki, Japan) and 50 μl 0.1% gold solution (SPEX Certi Prep, Metuchen, NJ, USA), and then dissolved at 75 C with shaking for 2 h. After cooling the solution to room temperature, internal standard (scandium, gallium, and indium) solution was added, and after adjusting its volume gravimetric, the obtained solution was used for multi-mineral analysis. The trace element concentrations were determined with inductively coupled plasma mass spectrometry (ICP-MS; 7500ce, Agilent Technologies, Santa Clara, CA, USA) as reported previously^6–8 and expressed as ng/g hair (ppb) or μg/g hair (ppm). Human hair-certified reference material (NIES CRM No. 13) was used to check for the accuracy of the analysis. The inter-daily variation of zinc, magnesium, calcium, aluminum, cadmium, lead, mercury, and arsenic determination was 2.2, 9.6, 6.3, 8.2, 6.9, 11.1, 9.4, and 6.9%, respectively. The control geometric mean value and reference range for each trace element were obtained from the data for 436 male healthy subjects aged 21–40 year old, as previously reported.^6,7,19,20

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Ethics Committee of La Belle Vie Research Laboratory. All of the data obtained are held securely in such a form as to ensure anonymity.

Statistical analysis

Because each trace element concentration in scalp hair was almost log-normally distributed, the mineral concentration was converted to a logarithm, and the geometric rather than arithmetic mean is used as a representative of its hair concentration. The relation between high-burdened metal and zinc concentration or zinc/iron ratio of the subjects was examined by Pearson's correlation coefficient test.

Results

Zinc and magnesium deficiency in children with autistic disorders

Total 2550 children with autistic disorders (2108 males and 442 females aged 0–15 year old) were investigated. The histogram of hair logarithmic zinc concentrations for the autistic subjects was non-symmetric with tailing in the lower range, and 790 in 2550 subjects (31.0%) were found to have lower zinc concentration than −2 SD (standard deviation) level of the reference range [86.3–193 μg/g hair (ppm); geometric mean = 129 ppm], defined as zinc deficiency (Fig. 1). The incidence rate of zinc deficiency in the age group of 0–3 year old was in the high range of 42–58%, with the mean rate of 43.9 and 55.8% in male and female, respectively (Fig. 2). In the 4–9-year-old group, zinc deficiency rate is decreasing from over 40 to 10% or less. In the group aged 10–15 year old, zinc deficiency rate is remaining at a lower level, and the mean rate in male and female was 3.7 and 6.6%, respectively, indicating that infants are more susceptible to zinc deficiency than elder children. The minimum zinc concentration of 10.7 ppm detected in a 2-year-old boy corresponded to ∼1/12 of the mean reference level.

Following to zinc deficiency, magnesium and calcium deficiency was observed in 477 (18.7%) and 137 (5.4%) individuals in the autistic children (Table 1), and for the other essential metals, such as iron, chromium, copper, manganese, and molybdenum, their incidence rates of deficiency were 1% or less. The incidence rate of magnesium deficiency in the age groups of 0–3, 4–9, and 10–15 years old is 26.3, 17.9, and 3.7% in male and 23.1, 16.4, and 1.3% in female subjects, respectively, suggesting that infants are also liable to magnesium deficiency than elder children. The minimal magnesium concentration of 3.88 ppm detected in a 2-year-old girl corresponds to almost 1/10 of the mean reference level (39.5 ppm). Calcium deficiency was observed in lower age groups <10 year old.

Toxic metal burdens in children with autistic disorders

In contrast, some toxic metals such as aluminum, cadmium, and lead highly accumulated over their +2 SD levels, in the individuals of 444 (17.4%), 221 (8.7%), and 114 (4.5%), and their incidence rates were higher than that of arsenic and mercury (2.5 and 1.6%, respectively) (Table 2). The detected maximal level of aluminum, cadmium, and lead was 79.4 ppm in a 4-year-old boy, 5.47 ppm in a 5-year-old boy and 24.9 ppm in a 5-year-old girl, respectively, corresponding to 21-, 782- and 57-fold of each mean reference level. The maximal burden level of mercury and arsenic of 40.1 ppm and 1.7 ppm, respectively, corresponds to 10- and 33-fold of the mean reference level.

Essential metal burdens in children with autistic disorders

It is surprising that some essential heavy metals were highly burdened in the autistic children, as well as toxic metals. High molybdenum burdens over their +2 SD reference level were observed in 922 autistic individuals (36.2%), and the maximal concentration of 1341 ppb was detected in a 7-year-old boy, corresponding to 48-fold of the mean reference level (Table 3). Next to molybdenum, iron, vanadium, manganese, zinc, and copper exhibited high burden over their +2 SD reference levels in the autistic individuals of 318 (12.5%), 272 (10.7%), 190 (7.5%), 69 (2.7%), and 55 (2.2%), respectively. The maximum concentration of iron (39.9 ppm), vanadium (2.6 ppm), manganese (14 ppm), zinc (495 ppm), and copper (346 ppm) corresponds to 6.5-, 212-, 130-, 3.8-, and 18-fold of each mean reference level, respectively.

For the other burdened metals, even though their prevalence was <2.0%, the maximal levels detected were over 10-fold of the mean reference level, and some elements such as chromium, cobalt, and lithium reached to an extra-ordinal high level of 39-, 679-, and 78-fold of the mean reference level, respectively, suggesting the possibility of their adverse effects even though essential elements.

Relationships between zinc deficiency and high-burdened metals

It is notable that there were significant relationships between zinc deficiency and some toxic metal burdens: the burden levels of lead, aluminum, cadmium, and arsenic inversely correlated to zinc concentration (r = −0.339, −0.247, −0.198, and −0.143, respectively; P < 0.001) (Fig. 3). Furthermore, a more intimate inverse relationship was observed between zinc and molybdenum concentration (r = −0.509, P < 0.00001) (Fig. 4).

As well as zinc, magnesium also exhibited a significant inverse correlation with some burdened metals such as not only arsenic, aluminum, and lead (r = −0.273, −0.159, and −0.142, respectively; P < 0.001) but also molybdenum (r = −0.387) (Table 4). There was a mild inverse correlation of zinc with chromium and iron concentration (r = −0.205 and −0.154, respectively; P < 0.001) observed, but no inverse correlation either between zinc and copper (r = 0.081) or between molybdenum and copper (r = 0.058) was observed in the autistic subjects. It is emphasized that zinc is only one heavy essential metal exhibiting inverse correlations with various toxic and heavy metals, suggesting that this element can compete and antagonize toxic metals and plays the pivotal or gatekeeper role in bio-metal homeostasis.

One case in only two 0-year-old individuals (10-month-old female) in 2550 subjects was assessed as suffering from zinc and magnesium deficiency of 79.1 ppm (0.6-fold of the mean reference level) and 15.0 ppm (0.4-fold), with a high molybdenum burden of 220 ppb (7.8-fold), corresponding to nearly 2-fold of its +3 SD level (Fig. 5).

Zinc/iron and zinc/copper ratio

As well as the zinc level itself, the ratio of zinc/iron (Zn/Fe ratio) seems to be a useful biomarker for assessing neurodevelopment disorders in Japanese autistic children examined: 671 individuals (26.3%) exhibited a lower ratio than its −2 SD level (Zn/Fe = 9.90), and the minimum ratio of 0.78 corresponded to 1/27 of the mean reference level of 21.0 (Fig. 6). Furthermore, zinc/iron ratio exhibited a high-significant inverse-relationship with aluminum (r = −0.551), lead (r = −0.486), cadmium (r = −0.369, P < 0.00001), and molybdenum (r = −0.548) concentrations (Fig. 7).

In contrast, on the zinc/copper ratio (Zn/Cu) reported as a good blood plasma biomarker for autism, there was less close correlation to the disorders in this study for Japanese subjects: the incidence rate of the individuals exhibiting lower ratios than 1.83 of its −2 SD level was as a few as 4.5% (115 in the 2550 autistic subjects), compared to the incidence of 26.3 and 31.0% based on the zinc/iron ratio and zinc concentration itself, respectively. One rare case suffering from a high copper burden of 200 ppm, with a low zinc/copper ratio of 0.48, is shown in Fig. 8, in which co-burdens of chromium, molybdenum, lithium, etc. were detected.

Discussion

Metallomics analysis for early assessment of neurodevelopmental disorders

Zinc is well accepted as an essential trace element, which functions as the active center in 300 kinds of enzymes and regulates the structure and function of ∼10% in the total gene-coded proteins named ‘zinc-finger proteins’, contributing to the interaction of genetic and environmental factors and transcriptional regulation. Thus, zinc is necessary for normal neurogenesis and migration, myelination, synaptogenesis, and regulation of neurotransmitter release in the fetal cortex, hippocampus, cerebellum, and the autonomic nervous system.^21–23 Recently, zinc has been shown to regulate the function of the brain Shank family proteins, ‘Shank2 and Shank3’, and also prenatal zinc deficiency reduced the expression levels of these proteins, suggesting that these Shank proteins act as mediators of zinc effects on synaptic function.^10,11,14 Furthermore, it is hypothesized that zinc is well positioned to serve as a dynamic regulator to activate Shank-dependent pathways in synapse formation and plasticity in developing neurons¹⁴ and that zinc deficiency is a key epigenetic factor in the etiology of ASD.^{6–9,10–20}

In addition, dietary restriction-induced zinc deficiency has been reported to up-regulate intestinal zinc-importer (Zip4) and induce the increase in Zip4 protein located in the plasma membrane of enterocytes.^24,25 This adoptive response to zinc deficiency is known to lead to increasing in the risk of high-uptake of toxic metals such as cadmium and lead. Thus, infants with zinc deficiency are liable to be exposed to an increased risk of absorbing a high amount of toxic metals and retaining them in their body, as shown by the inverse relationships (Fig. 2). These findings suggest that the increased risk of toxic metal burdens attendant on the infantile zinc deficiency seem also synergistically contribute to the pathogenesis of neurodevelopmental disorders.^7–12

Early diagnosing neurodevelopmental disorders such as ASD and ADHD was difficult since there was no clinical biochemical test, like a blood test, to diagnose the disorders. Therefore, autism was not usually diagnosed until after age 2, when delays in a child's social behavior and language skills become apparent, and many children cannot receive a final diagnosis and treatment until much older. This delay means that children with neurodevelopmental disorders fail to get the help needed and fail to have the chance of intervention/treatment in the narrow time window of mal-neurodevelopmental and for therapy.

Thus, neuroscientists and psychologists use terms such as ‘critical or sensitive period’, ‘infantile time window’, or ‘the first 1000 days’ to describe time epoch of neurodevelopment/vulnerability and opportunity for intervention/therapy.^{6–9,26–28} This critical period is typically conceptualized as an early life epoch when alterations to brain structure/function by several environmental epigenetic factors (e.g. zinc and magnesium deficiency and various toxic metal or chemical substance burdens) result in irreversible long-term consequences.^6–9 The critical term ‘sensitive period’ or ‘infantile time window’ can also be used in a positive manner to describe the time window when the brain tissue is particularly receptive to positive nutritional or social intervention.^9,28 Both concepts rely on the observation that the younger, rapidly developing brain is more vulnerable than the elder brain, and also retains a greater degree of plasticity (e.g. recoverability). Although the distinction may become less meaningful, either concept emphasizes the need for pediatricians to focus on making sure the child is receiving adequate nutrition to promote normal brain development in a timely fashion.^6–9,12,28

Thus, Insel proposed ‘The sooner we are able to identify early markers for autism, the more effective our treatment/interventions can be.’²⁹ Policymakers also have recently placed a great deal of emphasis on the ‘first 1000 days’ and ‘0–3 years’ as golden opportunities to influence child outcomes.

In an exploring study using validated tooth-matrix biomarkers, Arora et al. and Curtin et al. suggested that essential mineral deficiencies such as manganese and zinc and toxic metal burden of lead during specific developmental windows (both fetal and infantile) increase ASD risk and severity, supporting the hypothesis of systemic mineral imbalances in ASD and suggesting critical roles of fetal and postnatal metal dysregulation in the brain mal-development, and suggesting that altered zinc-copper rhythmicity precedes the emergence of ASD.^12,13

As shown in Table 2, aluminum is the most high-burdened toxic metal in the autistic subjects examined. On this toxic metal, the group of Exley reported that the aluminum content of brain tissue in autism was consistently high, as well as in the patients with Alzheimer’s disease and multiple sclerosis.^30–33 Furthermore, Lyons-Weiler and Ricketson reported that infants receive 17 times more aluminum than would be allowed if doses were adjusted per body weight, and several critical mistakes have been made in the consideration of pediatric dosing of aluminum contained in vaccines.³⁴

It is notable that not only toxic but also some essential metals are highly burdened in the autistic children, as shown in Table 3. The high burdens of ‘redox metals’ such as iron, manganese, and copper, which have a characteristic of producing a reactive oxygen species ‘hydroxyl radical’ by the Fenton reaction, probably play critical roles in the pathogenesis of neurodevelopmental disorders, as well as in neurodegeneration diseases such as Parkinson's, Alzheimer diseases, multiple sclerosis, and amyotrophic lateral sclerosis.^35–42

High molybdenum burden and its close inverse relationship with zinc in autistic children (Table 3 and Fig. 3) suggest that the overloading of molybdenum is probably another factor inducing zinc deficiency. And these findings accord with the fact that the patients with ASD and ADHD are comorbid with atopic dermatitis and food allergy, and they are inevitable to take soybean foods rich in molybdenum, as protein sources, in place of milk and eggs. In fact, in a large population-based survey of children's health in the USA and Taiwan, a striking association between atopic dermatitis and autism (OR: 3.04 and Hazard ratio: 16.6, respectively) is demonstrated.^43,44 These findings suggest that assessment of mineral imbalances (or dysregulation) in the child population is helpful for early diagnosis and open a novel therapeutic pathway for treating children suffering from not only neurodevelopmental disorders but also allergic diseases such as atopic dermatitis, food allergy, and asthma.

Faber et al. reported that the mean plasma Zn/Cu ratio in 230 children with autistic disorder is below the cut-off value of the lowest 2.5% of healthy children, and the zinc/copper ratio has been used as a helpful biomarker of heavy metal toxicity in children with ASD.^38,39–41 While, in our study for 2550 Japanese autistic children, the zinc/copper ratio was not closely correlated to autistic disorders: only just 4.5% (115 individuals) showed lower ratios than its cut-off (−2 SD) level, compared to the incidence rate of 31.0% based on the zinc deficiency itself. Instead of the zinc/copper ratio, the ratio of zinc/iron was found to be a good biomarker candidate for assessing autistic children: 26.3% (671 individuals) exhibited lower ratios than its −2 SD level (Fig. 5), and the minimum was 0.78 corresponding to as low as 1/27 of the mean reference ratio of 21.0. Furthermore, the zinc/iron ratio is more high-significantly inversely correlated with aluminum (r = −0.551), lead (r = −0.486), cadmium (r = −0.369), and molybdenum (r = −0.548, P < 0.00001) concentration (Fig. 6), suggesting that this Zn/Fe ratio is a useful biomarker as well as zinc itself for Japanese children with the neurodevelopmental disorders. These findings suit the reports on risk of iron-fortified infant formula and supplements in pregnancy and childhood.^45,46

The gap of zinc/copper ratio data between in the Faber study and in our study is probably explained by the following two circumstances: that is, the first is the difference in the ingestion of inorganic copper from drinking water and taking a high-fat diet and cacao bean products such as cocoa and chocolates,⁴¹ and the second is the difference in the zinc and copper contents in the follow-up milk products used in the USA and Japan, which are given to the infants over 6-month. In the milk products supplied in the USA and also Europe, zinc, and copper (5.5 and 0.4 mg/100 g powder milk) are co-supplemented with iron (7 mg/100 g), but in the Japanese products the mean iron content is as high as 9 mg/100 g (8.3–9.6 mg/100 g) and neither zinc nor copper is supplemented.^47,48 Therefore, it is supposed that the infants given Japanese follow-up milk products are suspected liable to zinc deficiency and iron excess, and the infants given US/European products are liable to copper excess or zinc/copper ratio decrease. Thus, many of the Japanese autistic children, especially infants, exhibit zinc deficiency and marginal iron excess with zinc/iron ratio decreasing, with less zinc/copper ratio decreasing, as shown in Fig. 6. In contrast, in US/European countries taking copper-rich foods and water and giving zinc and copper co-supplemented follow-up milk to babies, zinc/copper ratio decreasing has been reported useful and estimated as a good biomarker for autism.^38–41

Early intervention/therapy: metallomics-based supplementation of nutrients

Zinc deficiency has been remarked in not only developing but also developed countries, through imbalanced intake of nutrients and dieting.^6,19,21,49 However, there are a few controlled clinical studies on the supplementation of deficient minerals for the children with neurodevelopmental disorders. A valuable controlled clinical study on zinc supplementation for autistic elder children has been reported by Russo and Devito.³⁹ They report that high plasma copper levels in the patients with autism and pervasive developmental disorder (PDD) (mean age: 11.7 years) decreased to the normal level, and the severity of symptoms (e.g. awareness, receptive language, focus and attention, and hyperactivity, etc.) significantly decreased in autistic individuals following zinc and B-6 therapy. The beneficial effect of zinc on infantile neurodevelopment has been reported in a randomized, controlled trial of Peruvian infants aged 6–18 months, where zinc supplementation has been shown to sustain normative neurodevelopment.⁵⁰

Arnold et al. report that the mean serum zinc level in children was significantly lower in ADHD group, and that serum zinc level correlated inversely with parent- and teacher-rated inattention in ADHD children.^51,52 Furthermore, zinc treatment was reported significantly superior to a placebo in reducing symptoms of hyperactivity, impulsivity, and impaired socialization in ADHD patients.^53,54 Another preliminary human study showed that many children with ADHD have lower zinc concentration in comparison to healthy children, and zinc supplement as an adjunct to methylphenidate has favorable effects in the treatment of ADHD children, pointing to the possible association between zinc deficiency and ADHD pathophysiology.⁵⁵

Kozielec et al. have reported that in 116 hyperactive children with ADHD, magnesium deficiency was found in 95% of the subjects, most frequently in hair (77.6%), next in red blood cells (58.6%), and in blood serum (33.6%).⁵⁶ Furthermore, they reported that in the group of ADHD children given 6 months of magnesium supplementation, a significant decrease in hyperactivity and increase in hair magnesium contents has been achieved.⁵⁷ Mousain-Bosc et al. also reported that 52 hyper-excitable children have low intra-erythrocyte magnesium levels with normal serum magnesium values and that magnesium/vitamin B6 supplementation can restore the erythrocyte magnesium levels to normal and improve their abnormal behaviors.⁵⁸ They also reported that 33 children with clinical symptoms of PDD or autism exhibit significantly lower red blood cell magnesium values, and that the combination therapy with magnesium/vitamin B6 for 6 months significantly improved PDD symptoms in 23/33 children (P < 0.0001) with concomitant increases in intra-erythrocyte magnesium values.⁵⁹

We also have experienced considerable cases of autistic children improved by the nutritional intervention supplementing deficient nutrients, on the basis of metallomics analysis (unpublished). The therapeutic efficacy of the evidence-based micronutrient supplementation for children with neurodevelopmental disorders remains to be demonstrated by a large double-blinded controlled study.

Conclusion and perspective

In this metallomics study for 2550 autistic children, we demonstrate that many of them are suffering from zinc and magnesium deficiency and marked high burdens of not only toxic but also some essential metals such as molybdenum, iron, manganese, and copper, especially in the early life of 0–3 years old, a critical period of a narrow ‘infantile time window’ in mal-neurodevelopment. Furthermore, it is notable that many of the highly burdened heavy metals detected in the autistic children have close relationships with the deficiency of the restricted essential elements (zinc and magnesium), and these findings suggest that supplementation of these deficient nutrients is helpful for intervention/therapy.

Thus, the early assessment of various profiles of body mineral imbalances, especially during the critical time window is useful for early diagnosis of neurodevelopmental disorders and probably for evidence-based individualized therapy of the patients during the therapeutic time window.

We hope that early assessment of mineral imbalances of individual children opens an avenue of evidence-based personalized precision medicine for neurodevelopmental disorders and also for the comorbid such as allergic immune diseases, in this decade.

Acknowledgements

The authors appreciate the subjects and their parents for their cooperation. They thank the laboratory team for their technical contributions to the trace element analysis.

Funding

None declared.

Conflicts of interest

The authors declare no conflict of interest.

Data availability statement

The datasets underlying this article cannot be shared publicly due to for the privacy of individuals that participated in the study. The data will be shared on reasonable request to the corresponding author.

References