2025 Volume 5 Issue 2 Pages reg01-reg10
Elucidation of the aetiology and effective treatment of neurodevelopmental disorders is one of the challenges today. In this metallomics study for 2,550 children with autistic disorders aged 0–15 years, we demonstrate a remarkable molybdenum overload correlated to zinc deficiency. Histogram of scalp hair molybdenum concentration was log-normal distributed with the geometric mean of 62.0 ng/g (ppb), and 921 individuals (36.1 %) were found to have high molybdenum concentrations over 72.0 ppb that is the +2 S.D. threshold level in the reference range. In particular, the incidence rate of molybdenum overload in the infantile group aged 0–3 years was estimated 54.0 % (430 in 797) in male and 62.6 % (92 in 147) in female. Higher molybdenum concentrations over 116 ppb of the +3 S.D. level were detected at the rate of 18.7 and 21.8 % in male and female infant group aged 0–3 years, respectively. Interestingly, a high significant inverse relationship between molybdenum and zinc concentration (r = −0.510, p < 0.0001) was observed, with no significant association between molybdenum and copper (r = 0.058, p > 0.05). These findings suggest that the molybdenum overload in the children with autistic disorders is closely associated with zinc deficiency and maybe contributes to the aetiology of the neurodevelopmental disorders. We hope that evidence-based supplementation of the deficit minerals to individual leads to a precision intervention/treatment of the children with neurodevelopmental disorders.
The children today in developed countries are in the midst of the epidemic of a category of neurodevelopmental disorders such as autism spectrum disorder (ASD) and attention deficit/hyperactivity disorder (ADHD). These neurodevelopmental disorders are heritable with a strong genetic basis of more than 100 candidate genes, and their prevalence has been increasing to 13% [1-3]. However, the key genetic determinants are still unclarified and the interaction of hereditary factors with some environmental factors plays a critical role in the pathogenesis1, 2. Thus, the elucidation of the aetiology and effective treatment of the neurodevelopmental disorders is one of the challenges today.
In the previous studies [4-8], we have demonstrated that some restricted mineral deficiency (e.g. zinc and magnesium) and toxic metal burdens, especially during the restricted time window of the first 3 years of life, play principal epigenetic roles as environmental factors in neurodevelopmental disorders, and that metallomics analysis is a hopeful strategy leading to the early diagnosis and prevention of neurodevelopmental disorders. Thus, epigenetic interactions of the genetic factors with some environmental factors such as zinc deficiency and toxic metal’s burdens seem play a crucial role in the pathogenesis [4-17]. This infantile zinc-deficiency-based epigenetic aetiology of neurodevelopmental disorders has been supported and reinforced by many research groups [9-24] and has been expected to lead to approaching the personal early intervention/treatment [7, 8, 10, 14, 16-19].
The essentiality of molybdenum in human nutrition and health has been recognized since the early 1970s [25-27], but there was neither assessment of its nutritional status nor information on its physiological levels in serum or urine, because of its too low concentrations in the biospecimens to determine exactly.
In this metallomics study for 2,550 autistic children aged 0–15 years, we demonstrate that many of them, especially in the infantile group aged 0–4 years, are suffering from high molybdenum overload.
On the basis of informed consent, scalp hair samples from 2,550 (male: 2,108; female: 442) subjects with symptoms like autistic disorders aged 0–15 years were collected in the period from June 2005 to September 2015 (Table 1).
Hair sample of 75 mg was washed with acetone and then with 0.01% Triton solution, and then dissolved in 10 ml 6.25% tetra methyl ammonium hydroxide (TMAH), as previously reported [4-6, 8]. After internal standard solution was added, the obtained solution was used for the determination of 26 bio-trace elements (Li7, Be9, B10, Na23, Mg24, Al27, P31, K39, Ca43, V51, Cr53, Mn55, Fe56, Co59, Ni60, Cu63, Zn66, Ge72, As75, Br79, Se82, Mo95, Cd111, I127, Hg202, Pb208) with inductively coupled plasma mass spectrometry (ICP-MS; 7500ce, Agilent Technologies, Santa Clara, CA, USA), and expressed as ng/g hair (ppb) or micro-g/g hair (ppm). The method detection limit for molybdenum determination was 2.6 ppb, and the inter-daily variation of molybdenum determination was calculated 4.0 %. The control reference geometric mean (27.9 ppb) and reference range (10.8–72.0 ppb: −2 S.D – +2 S.D.) for molybdenum were obtained from the data for 436 male healthy subjects aged 21–40 year old, as previously reported.
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.
| Age (years) | Male Numbers | Female Numbers |
|---|---|---|
| 1 | 68 | 17 |
| 2 | 314 | 59 |
| 3 | 415 | 71 |
| 4 | 311 | 67 |
| 5 | 234 | 55 |
| 6 | 187 | 34 |
| 7 | 125 | 27 |
| 8 | 111 | 19 |
| 9 | 74 | 17 |
| 10 | 82 | 21 |
| 11 | 52 | 17 |
| 12 | 51 | 10 |
| 13 | 29 | 7 |
| 14 | 31 | 13 |
| 15 | 24 | 8 |
| Total | 2,108 | 442 |
Because each trace element in scalp hair is almost log-normally distributed, the molybdenum concentration was converted to logarithm, and the geometric rather than arithmetic mean is used as representative of its hair concentration. The relation between high-burdened metal and zinc concentration or age of the subjects was examined by Pearson’s correlation coefficient test, with the Excel 2019 (Bell Curve, Japan).
The histogram of scalp hair molybdenum concentrations for 2,550 autistic children (2,108 males and 442 females) is shown in Figure 1. The distribution of the logarithmic molybdenum concentration was near normally distributed with a geometric mean of 62.0 ng/g hair (ppb), and 924 individuals (36.2 %) in the 2,550 subjects were found to have higher molybdenum concentrations over 72.0 ppb (log Mo = 1.857), which is the upper threshold level of +2 S.D. in the reference range, and defined as molybdenum overload.
The molybdenum concentrations in the autistic subjects showed a significant inverse correlation with age (r = −0.463, p < 0.0001) (Figure 2), and more than one half of the infantile group aged 0–3 years (54.0 % in male and 62.6 % in female) were estimated suffering from high overload of this heavy essential metal (Figure 3). In the elder groups aged 4–9 and 10–15 years, the overload rate was downed to 29.2 and 7.8 % and to 33.3 and 5.3 % in male and female, respectively. There was little marked gender difference observed in the molybdenum concentration and overload rate in the autistic children.
Interestingly, a high significant inverse relationship between zinc and molybdenum concentration (r = −0.510, p < 0.0001) was observed in the children with autistic disorders (Figure 4), as well as between zinc and high-burden toxic metals. Whereas no significant association between molybdenum and copper level was observed in this study (r = 0.058, p > 0.05) (Table 2).
The maximum level of high molybdenum burden of 1,341 ppb was observed in a 7-year-old boy (Figure 5A), corresponding to about 48-fold of the mean reference level. In contrast, its minimum concentration of 7.3 ppb was observed in a 14-year-old boy and defined as deficient, corresponding to about 1/4th of the mean reference level (27.9 ppb) (Figure 5B), although the deficiency rate of molybdenum was as low as 0.04 %.
In one of the only two 0-year-old cases (11 months female), a high molybdenum level of 220 ppb near to 8-fold of the mean reference level was detected with high iodine and low magnesium, calcium and zinc level (Figure 6A). In another 0-year-old case (11 months female), a molybdenum overload of 104 ppb over the upper threshold level was observed with co-suffering from high burdens of lead, aluminium, nickel and others (Figure 6B).
The numbers on the abscissa indicate the logarithms of scalp hair molybdenum concentrations (ng/g: ppb). The height of each rectangle represents the frequency in the class interval in logarithmic hair molybdenum level. Two dotted vertical lines represent the −2 and +2 S.D. level of the reference range of hair molybdenum concentrations. The histogram of scalp hair molybdenum concentrations for 2,550 autistic subjects (2,108 males and 442 females) aged 0-15 years is shown in the logarithm.
The association of hair logarithmic molybdenum concentration with age in autistic subjects (N = 2,550) is shown. Each spot represents the corresponding age and logarithmic molybdenum concentration of the respective child. The dotted line represents the +2 S.D. threshold level of the reference range of hair molybdenum concentrations.
A significant inverse association of the molybdenum concentration with age (r = −0.463, p < 0.00001) in the autistic children is shown.
The association of molybdenum overload rate with age (1-15 years old) in autistic subjects (N = 2,550) is shown. The height of each bar represents the rate of molybdenum overload (%) in the respective age group of male (blue bar) and female (red bar).
The inverse association between hair molybdenum and zinc concentration in autistic subjects (N = 2,550) is shown. Each spot represents the corresponding logarithmic molybdenum and zinc concentration of the respective child.
A significant inverse relationship of the hair concentration of molybdenum with zinc (r = −0.509, p < 0.0001) in the autistic children is shown.
| Zn | Mo | Pb | Al | Cd | As | Hg | Cu | Mg | |
|---|---|---|---|---|---|---|---|---|---|
| Zn | 1 | −0.509 | −0.339 | −0.247 | −0.197 | −0.143 | 0.069 | 0.081 | 0.526 |
| Mo | **** | 1 | 0.428 | 0.411 | 0.286 | 0.195 | −0.065 | 0.058 | −0.388 |
| Pb | **** | 1 | 0.451 | 0.609 | 0.063 | −0.064 | 0.286 | −0.142 | |
| Al | **** | 1 | 0.376 | −0.015 | −0.082 | 0.091 | −0.159 | ||
| Cd | *** | 1 | 0.051 | −0.105 | 0.109 | −0.048 | |||
| As | ** | 1 | 0.104 | −0.188 | −0.273 | ||||
| Hg | 1 | 0.031 | 0.064 | ||||||
| Cu | 1 | 0.197 |
**: p < 0.01 ***: p < 0.001 ****: p < 0.0001
A) 7-year-old boy with most severe molybdenum overload (1,341 ppb); B) Only one boy with molybdenum deficit (7.3 ppb). Each bar represents the relative concentration of the respective trace element in his scalp hair specimen. The dotted horizontal line at 1.0 represents the reference control level of each trace element.
A) A girl suffering from high molybdenum (220 ppb) and iodine level and low zinc and magnesium levels; B) A typical metallome profile exhibiting high burdens with lead, aluminium, nickel etc. Each bar represents the relative concentration of the respective trace element in her scalp hair specimen. The dotted horizontal line at 1.0 represents the reference control level of each trace element.
In human, molybdenum is an essential trace element that plays important roles in four molybdenum-cofactor dependent enzymes, aldehyde oxidase, xanthine oxidase, mitochondrial amidoxime reducing component and sulphite oxidase, and its deficiency causes a severe phenotype characterized by a progressive neurological damage that leads to death shortly after birth [25-27]. In contrast, there are few reports on molybdenum overload or toxicity in humans, which originate from the well-documented adverse effects of elevated molybdenum levels on ruminants, and this effect is strictly limited to the special conditions in the rumen. In human, there is few study on the influence of high overload of molybdenum or information on the physiological or pathological levels in human blood or urine samples.
In this metallomics study, we demonstrate that high molybdenum overload in the children with neurodevelopmental disorders closely associates with zinc deficiency. In fact, more than one third (36.1 %) of the individuals with autistic disorders exhibit high molybdenum concentrations over the + 2 S.D. threshold level, defined as molybdenum overload (Figure 1). In particular, in the infantile group aged 0–3 years, more than one half (male: 54.0 %; female: 62.6 %) were found to suffer from molybdenum overloads (Figure 3). Furthermore, more severe molybdenum burdens over its +3 S.D. level (116 ppb) were observed in about 20 % individuals (male: 18.7 %; female: 21.8 %) in the infantile group.
The incidence rate of molybdenum overload in the elder group aged 10–15 years was downed to 7.8 % in male, and 5.3 % in female autistic subjects, respectively. These findings indicate the presence of critical “infantile time window” in the molybdenum overload and little gender difference.
On the biological interaction of molybdenum with other trace mineral elements, competition between molybdenum and copper had been reported [28], and in vitro studies indicated that the antagonism by copper occurs during molybdenum-cofactor biosynthesis [29], which might be affected under the condition of copper overload. In this metallomics study for the children with autistic disorders, there was neither significant competition nor corelation observed between molybdenum and copper element (r = 0.058, p > 0.05), probably because of the low copper overload rate of 2.2% in the autistic children examined.
Surprisingly, a high significant inverse association was observed between molybdenum and zinc concentration (r = −0.510, p < 0.0001) (Figure 4), and this close relationship with zinc was more marked than that of every toxic metal including lead, aluminium, cadmium and arsenic, r = −0.339, −0.247, −0.197 and −0.143, respectively; P < 0.01 (Table 2). Thus, high molybdenum burden and its close relationship with zinc deficiency in autistic children indicate that the overloading of molybdenum is probably another principal factor leading to zinc deficiency.
It should be emphasized that zinc is only one heavy essential metal exhibiting inverse relationship with high-burdened metals of not only toxic but also some essential metals such as molybdenum, manganese, chromium and iron, suggesting that zinc element has a character of antagonizing against high-burdened heavy metals and mitigating the adverse effects of them [5-8, 14, 16-19]. These findings are consistent with the fact that zinc has been used as the therapeutic drug for a hereditary Wilson’s disease which is a characteristic copper overload disease induced by the genetic disorder of a copper-transporter gene, ATP7B [30]. These evidences indicate that zinc plays as a central, key element for the bio-regulation and homeostasis of heavy metals in human body and has a critical characteristic mitigating toxic heavy metal burdens and ameliorating body mineral imbalances [5-8, 14, 16-19, 31, 32].
Many proteins with zinc-binding domains named “zinc-finger proteins (ZFP)” are involved in epigenetic modifications, such as DNA methylation and histone acetylation, which regulate transcription in physiological and pathological conditions [20-24]. In facts, the changes in the expression or function of these proteins are induced by zinc deficiency or by mutational function-loss of their ZFPs, leading to aberrant epigenetic reprogramming, which seems worsen the risk of neurodevelopmental disorders [23, 24].
Recently, Takeuchi et al reported a primary association between hair zinc level and brain functional connectivity in regions of the default mode network (DMN) in young healthy adults [33], and Curtin et al also found out that the close relationship between zinc level and functional DMN connectivity observed in healthy baby brains is not detected in the emergency of autism [34]. These findings accord with the evidence that the infants with autistic disorders are suffering from zinc-deficiency [5-8], suggesting that the disappearance of zinc-corelated DMN connectivity may be a characteristic functional disorder leading to ASD.
It was well known that many of the patients with neurodevelopmental disorders are suffered from comorbidities of immune disorders such as atopic dermatitis, food allergy and asthma [35-38] and that they are inevitable to take soybean foods rich in molybdenum, as protein sources, in place of milk and eggs. In large population-based surveys 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 [39, 40]. These findings suggest that early assessment of the imbalances (or dis-homeostasis) of body minerals in the child population is helpful for early diagnosis and open a novel intervention/therapeutic pathway for many children suffering from not only neurodevelopmental disorders but also allergic comorbid diseases [8, 14, 17].
In addition, soybean foods are known a representative containing high contents of phytic acid, a potent inhibitor of zinc absorption in intestine, leading to zinc deficiency. Thus, in the children feeding on soy-based infant formulas and weaning foods, the dietary intake of molybdenum is higher compared to breast milk-fed infants.
Eklund and Oskarsson reported in 1999 that soy-based formulas contain approximately six times more cadmium than cow’s milk formulas, and cereal-based formulas have 4–21 times higher levels [41]. In fact, many infantile children aged 0–3 years have been reported suffering from not only cadmium but also lead and aluminium [42]. It has been regrettably proved that commercial baby foods contain dangerous levels of toxic heavy metals, including arsenic, lead, cadmium, and mercury, by the Staff Report (February 4, 2021) of the Subcommittee on Economic and Consumer Policy Committee on Oversight and Reform in U.S. House of Representatives [43].
These findings suggest that the early assessment of a variety of body mineral imbalances (or dysregulations) such as toxic metal burdens and essential mineral deficiency in the child population is useful and essential for early diagnosis and intervention/treatment, opening a novel evidence-based personal therapeutic pathway for children with neurodevelopmentalal disorders [6-8, 10-17].
A valuable controlled clinical study on zinc supplementation for autistic children was reported by Russo and Devito [44]. They demonstrated that high plasma copper level 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) significantly decreased in autistic individuals following zinc and B-6 therapy. After that, 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 [45].
Arnold et al. reported 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 [46, 47]. Therefrom, zinc treatment was reported significantly superior to a placebo in reducing symptoms of hyperactivity, impulsivity, and impaired socialization in ADHD patients [48, 49]. 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 favourable effects in the treatment of ADHD children, pointing to the possible association between zinc deficiency and ADHD pathophysiology [50].
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, next in red blood cells, and in blood serum specimen [51]. 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 [52]. 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 behaviours [53]. 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 [54].
We also have experienced considerable cases of autistic children improved by the nutritional intervention supplementing deficit minerals, based on the individual data of the metallomics analysis (unpublished observation).
The therapeutic efficacy of the evidence-based micronutrient supplementation for the children with neurodevelopmental disorders remains to be confirmed by large controlled double-blind clinical study.
Recently, “A Call to Action” for “Preventing and Controlling Zinc Deficiency across the Life Course” was published by Lowe et al [55], and zinc deficiency is becoming a crucial pathogenic event in not only Wilson’s disease and neurodevelopmental disorders but also various heritable diseases such as diabetes, chronic inflammatory diseases, immune disorders, infections, cancer, neurodegenerative diseases etc [31, 32, 56-63]. It is hoped that an avenue to the precise preventive medicine, based on the individual data of clinical metallomics analysis, will be opened and realized in near future.
In this metallomics study for 2,550 autistic children, we demonstrate that many of them, particularly in the infantile group of 0–4 years, are suffering from severe high-burdens of molybdenum. It is notable that the molybdenum overload is associated inversely with the restricted two essential metals (zinc and magnesium), suggesting that molybdenum is a key trace element inducing zinc-deficiency and plays a principal role in the pathogenesis of the neurodevelopmental disorders. These findings suggest that the supplementation of the two deficit-nutrients, zinc and magnesium, is effective for ameliorating the adverse effects of high-burdened metals, not only toxic but also some essential heavy metals such as molybdenum, manganese and iron or copper. We hope that the early assessment of mineral imbalances of individual child opens a pathway to the evidence-based personalized precision medicine for the neurodevelopmental disorders and also for the comorbid such as allergic immune disorders, in near future.
The authors appreciate the child-subjects and their parents for their cooperation. They thank the laboratory team for their technical contributions to the trace element analysis.
The authors have no conflicts of interests.
None
The datasets underlining this article cannot be shared publicity due to for the privacy of individuals that participated in this study.
