Biomedicine & Pharmacotherapy 109 (2019) 174–180

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Association between catatonia and levels of hair and serum trace elements T
and minerals in autism spectrum disorder
⁎
Alexey A. Tinkova,b, , Margarita G. Skalnayab, Natalia V. Simashkovac, Tatiana P. Klyushnikc,
Anastasia A. Skalnayad, Geir Bjørklunde, Svetlana V. Notovaf, Elena V. Kiyaevag,
Anatoly V. Skalnya,b,h
a
Yaroslavl State University, Yaroslavl, Russia
b
Peoples’ Friendship University of Russia (RUDN University), Moscow, Russia
c
Scientific Center for Mental Health, Russian Academy of Medical Sciences, Moscow, Russia
d
Lomonosov Moscow State University, Moscow, Russia
e
Council for Nutritional and Environmental Medicine (CONEM), Mo i Rana, Norway
f
Orenburg State University, Orenburg, Russia
g
Federal Research Centre of Biological Systems and Agro-technologies of the Russian Academy of Sciences, Orenburg, Russia
h
All-Russian Research Institute of Medicinal and Aromatic Plants (VILAR), Moscow, Russia

A R T I C LE I N FO A B S T R A C T

Keywords: The objective of the study was to investigate the association between catatonia in autism spectrum disorder
Autism spectrum disorder (ASD) and the levels of hair and serum trace elements and minerals in children with ASD. The levels of hair and
Catatonia serum trace elements and minerals of boys suffering from ASD with (n = 30) and without (n = 30) catatonia, as
Mercury well as 30 age- and sex-matched neurotypical controls were assessed using ICP-MS. Hair calcium (Ca) and
Neurotoxicity
selenium (Se) levels were lower in ASD patients as compared to the controls. Hair mercury (Hg) levels in ASD
Children
patients were more than 3-fold and 2-fold higher as compared to the controls and children with catatonia in ASD.
Hair iodine (I) and manganese (Mn) were the lowest and the highest in ASD + Catatonia, respectively. Serum
aluminium (Al) and cadmium (Cd) levels in healthy controls were significantly higher in comparison to the
patients of both groups. Serum chromium (Cr), copper (Cu) levels were significantly increased in patients with
ASD and catatonia, whereas vanadium (V) levels were elevated in patients both with and without catatonia.
Multiple regression analysis demonstrated that hair Hg and serum Al and Cd levels were negatively associated
with catatonia in ASD in crude and adjusted models. Although the etiology of catatonia in ASD is unclear, the
obtained data demonstrate that catatonic symptoms in ASD may be at least partially mediated by altered trace
element levels. Further studies are required to elucidate the role of trace elements in the potential signaling
mechanisms of catatonia.

1. Introduction of catatonia in ASD are not known [6].

Autism spectrum disorder (ASD) is a complex neurobehavioral dis-
order characterized by social-interaction difficulties, communication
challenges, and restricted, repetitive patterns of behavior, interests
and/or activities (APA 2013). The worldwide prevalence of ASD is es-
timated to be 52 million cases [1]. The prevalence rate of ASD is region-
specific and vary from 0.9 cases of 1000 children in India [2] to 1 case
in 45 US children [3].
Catatonia is a syndrome characterized by motor dysregulation [4,5],
which is observed in certain neuropsychiatric disorders including ASD
at the rate of 4–17% [6]. At the same time, the causes and mechanisms
Although multiple mechanisms have been shown to play a role in
ASD (neuroinflammation, oxidative stress, excitotoxicity, etc.), the
particular causes of ASD are currently unclear [7–9]. Multiple factors
including genetics and environment, as well as their interaction, have
been proposed to be involved in ASD development [10,11]. Environ-
mental pollution has been shown to be associated with the incidence of
ASD [12]. It has been demonstrated that heavy metal pollution and
body accumulation may be associated with ASD. The most significant
indications were obtained for the association between mercury (Hg)
and ASD as demonstrated by recent meta-analyses [13,14]. Other me-
tals and metalloids including lead (Pb), arsenic (As), cadmium (Cd), and

⁎
Corresponding author at: Yaroslavl State University, Yaroslavl, Russia.
E-mail addresses: tinkov.a.a@gmail.com, tinkov.a.a@microelements.ru (A.A. Tinkov).

https://doi.org/10.1016/j.biopha.2018.10.051
Received 27 August 2018; Received in revised form 5 October 2018; Accepted 9 October 2018
0753-3322/ © 2018 Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
A.A. Tinkov et al.
aluminum (Al) [15] may play a significant role in ASD due to induction
of oxidative stress, neuroinflammation, excitotoxicity, as well as al-
teration of essential ion homeostasis. Essential elements including iron
(Fe) and manganese (Mn) may also play a role in ASD due to their
neurotoxicity [16], although the data from previous studies involving
children with ASD are contradictory.
Essential trace elements also play a significant role in ASD. In par-
ticular, ASD children were characterized by insufficient trace element
and mineral intake [17,18], resulting in decreased levels of these ele-
ments in the organism [19]. Deficiency of zinc (Zn) [20] and selenium
(Se) [21] was shown to be pathogenetically associated with ASD due to
their role in antioxidant system activity. Also, Zn deficiency was shown
to alter synaptic functions [22]. Certain essential trace elements, like
copper (Cu) and Fe, are redox-active metals and may contribute to
oxidative stress and play a potential role in the pathogenesis of ASD. In
this view, antagonism between Zn and Cu was proposed to contribute to
ASD through dysregulation of the metallothionein synthesis [20]. At the
same time, the relationship between essential and toxic trace elements
also has a significant impact on neurological health [23]. At the same
time, certain contradictions regarding trace element levels in ASD exist.
Particularly, De Palma et al. (2012) failed to detect any significant in-
terrelation between hair metal levels and ASD [24].
Although no studies on the levels of minerals and trace elements in
catatonia in ASD have been performed earlier, individual studies have
demonstrated the interplay between catatonia. Particularly, Mn [25]
and Pb [26] toxicity were shown to result in catatonia. Experimental
studies have also demonstrated an association between Hg exposure
and catatonia [27]. A significant elevation of serum calcium (Ca) was
also observed in patients with catatonic schizophrenia [28].
Therefore, the objective of the present study was to assess the effect
of catatonia on hair trace element and mineral levels in children with
ASD. To our knowledge, this is the first study of the interaction between
catatonia and trace element metabolism.
2. Materials and methods
The local ethics committee at the Scientific Center for Mental Health
(Russian Academy of Medical Sciences) approved the protocol of the
present study. All procedures performed were in accordance with the
principles of the 1964 Declaration of Helsinki and its later amendments
(2013). The parents of all children included in the study signed in-
formed consent forms. Psychiatric examination and collection of hair
and blood samples were performed in the presence of parents (or legal
representatives).
Ninety children aged from one to nine years living in Moscow were
enrolled in the current study. Psychiatric diagnosis of 30 boys with
childhood autism (ICD-10: F84.0) without catatonia and 30 boys with
ASD and catatonia was verified in the Scientific Center for Mental
Health, Russian Academy of Medical Sciences (Moscow, Russia). No
significant group difference in the scores on the Childhood Autism
Rating Scale (CARS) and the Clinical Global Impression-Severity scale
(CGI-S), that are used to identify autism severity [29,30], were found
between ASD children with or without catatonia (Table 1). Although
the group difference in CARS scores was nearly significant statistically,
the clinical value of such differences (less than 2 points) does not allow
detection of different autism severity. A significant group difference
was observed only for speech delay, which was more prevalent in
children with both ASD and catatonia. None of the patients with ASD
were characterized by obsessive-compulsive disorder (OCD) (Table 1).
The control group was presented by 30 age- and sex-matched neuro-
typical controls. The mean age of the neurotypical controls and the ASD
patients (without and with catatonia) was 4.8 ± 2.2, 4.7 ± 1.8 and
4.7 ± 2.1 years old, respectively. No significant group difference in
age values was observed.
None of the children examined in the present study lived near in-
dustrial sources of pollution. To prevent the influence of other side
175
Biomedicine & Pharmacotherapy 109 (2019) 174–180
Table 1
Psychiatric status of the patients with ASD in relation to the presence of cata-
tonia.
Parameter ASD ASD + Catatonia p-value
CARS, score 43.3 ± 6.2 45.1 ± 4.1 0.052
CGI-S, score 5.5 ± 0.7 5.3 ± 0.5 0.146
Speech delay, n (%) 0 (0%) 5 (17%) 0.041*
Cognitive deficit, n (%) 3 (10%) 8 (27%) 0.218
Infantile psychosis, n (%) 10 (33%) 14 (47%) 0.751
Obsessive-compulsive syndrome, n 0 (0%) 0 (0%) –
(%)
Hyperkinetic syndrome, n (%) 2 (7%) 0 (0%) 0.115
* -significant group difference at p < 0.05.
factors on hair trace element content, the following exclusion criteria
were used: the presence of other neuropsychiatric disorders including
cerebral palsy (G80-G83) and abnormal eating behaviors (ICD-10: F50);
vegetarianism; endocrine disorders (obesity, diabetes); metallic im-
plants (including dental amalgam fillings); acute traumas and in-
flammatory diseases; the use of mineral-enriched shampoos and hair
care products.
2.1. Sampling and trace element analysis
On the day of sampling, all children in the present study washed
their hair using their regular shampoos. Earlier studies demonstrated
that the use of commercially available shampoos does not cause a sig-
nificant difference in hair trace element content [31]. Only proximal
parts of occipital scalp hair strands were collected using ethanol-pre-
cleaned stainless steel scissors in a quantity of 0.05-0.1 g. The samples
were washed with acetone, rinsed three times with double distilled
water and dried on air at 60 °C to a stable weight.
0.05 g of cleaned hair samples were placed into Teflon tubes con-
taining 5 ml of concentrated (65%) nitric acid (HNO3) (Sigma-Aldrich
Co., St. Louis, MO, USA) and loaded into Berghof SW-4 DAP-40 (mi-
crowave frequency, 2.46 GHz; power, 1450 W) (Berghof Products &
Instruments, Eningen, Germany) system. Microwave digestion was
performed for 20 min at the maximal temperature of 170–180 °C. After
cooling the obtained solutions were added with distilled deionized
water (18 MΩ cm) prepared using DVS-M/1HА-1(2)-L (Mediana-Filter,
Podolsk, Russia) electric distiller to a total volume of 15 ml.
Blood samples were collected in the morning from the cubital vein
using 9-ml “Vacuette” tubes (Greiner Bio-One International AG,
Austria) with subsequent centrifugation (1600 g - 10 min) to obtain
serum. Only serum samples without signs of hemolysis were used for
analysis. The collected serum samples were diluted (1:15; v/v) with an
acidified (pH = 2.0) diluent consisting (v/v) of 1% 1-Butanol (Merck
KGaA, Darmstadt, Germany), 0.1% Triton X-100 (Sigma-Aldrich, Co.,
St. Louis, USA), and 0.07% HNO3 (Sigma-Aldrich, Co., St. Louis, USA)
in distilled deionized water.
The resulting samples were used for analysis of toxic (Al, As, Cd, Hg,
and nickel (Ni)) and essential trace elements and minerals (Ca, cobalt
(Co), chromium (Cr), Cu, Fe, iodium (I), magnesium (Mg), Mn, Se,
vanadium (V), and Zn) with inductively-coupled plasma mass spectro-
metry at NexION 300D (PerkinElmer Inc., Shelton, CT 06484, USA)
equipped with ESI SC-2 DX4 autosampler (Elemental Scientific Inc.,
Omaha, NE 68122, USA). Dynamic reaction cell (DRC) mode was en-
abled for analysis of certain elements, allowing reducing major atomic
interferences [24].
System’s calibration was performed using standard solutions (0.5, 5,
10 and 50 μg/L) of trace elements prepared from commercially avail-
able Universal Data Acquisition Standards Kit (PerkinElmer Inc.,
Shelton, CT 06484, USA). Internal online standardization was per-
formed using Yttrium (Y) and Rhodium (Rh) solutions (10 μg/L) pre-
pared from Pure Single-Element Standard (PerkinElmer Inc., Shelton,
A.A. Tinkov et al. Biomedicine & Pharmacotherapy 109 (2019) 174–180

CT 06484, USA). Laboratory quality control was regularly performed (p = 0.004) lower as compared to the controls, whereas no significant
before and after each set of analyses using analysis of the certified re- group difference was observed between the neurotypical controls and
ference materials (CRM) of human hair GBW09101 (Shanghai Institute the children with ASD + catatonia. At the same time, hair Se levels in
of Nuclear Research, Shanghai, China) and serum ClinCheck Plasma ASD + catatonia patients exceeded the values from those with ASD by
Control (lot 129, levels 1 and 2; RECIPE Chemicals + Instruments 12% (p = 0.018). Hair Hg levels in ASD patients were more than 3-fold
GmbH, Germany). The mean recovery rates for all analyzed trace ele- (p = 0.012) and 2-fold (p = 0.005) higher as compared to the controls
ments exceeded were within the range of 90%–110% during the whole and children with ASD + catatonia. Hair I levels in patients with
period of investigation. ASD + catatonia were nearly 2-fold (p = 0.020) lower as compared to
the patients without this syndrome. Surprisingly, the highest levels of
hair Mg were observed in patients with ASD + catatonia, being higher
2.2. Statistical analysis
as compared to the controls and ASD patients without catatonia by 42%
(p = 0.053) and 43% (p = 0.029), respectively. Despite a 25%
The obtained data were processed using Statistica 10.0 (StatSoft,
(p = 0.095) and 10% (p = 0.197) elevation in hair Al levels in ASD
Tulsa, OK, USA) software. Normality of data distribution was assessed
patients without and with catatonia the difference was not significant.
using the Shapiro-Wilk test. As the distribution of data was not
Similarly, hair Zn content in the studied groups was lower as compared
Gaussian, descriptive statistics of hair elements content included
to the control values by 20% (p = 0.367) and 17% (p = 0.510), al-
median and the respective 25 and 75 percentile boundaries. Non-
though being non-significant.
parametric Mann-Whitney U test was used for group comparisons.
Serum trace element and mineral content was characterized by
Correlation analysis was performed using Pearson’s correlation coeffi-
different patterns as compared to hair (Table 3). Particularly, serum Al
cient (r).
in healthy controls was 30% (p = 0.009) and 25% (p = 0.008) higher
Multiple regression analysis of the association between trace ele-
in comparison to ASD and ASD + catatonia. Similarly, serum Cd levels
ment levels and catatonia was performed. Particularly, Crude model
in both groups of patients were 2-fold (p = 0.049 and p = 0.001) lower
(Model 1) included only trace elements characterized by significant
as compared to the control values. Serum Cr concentration in children
group difference as independent predictors. Adjusted model (Model 2)
with ASD and ASD + catatonia catatonia was 99% (p = 0.003) and
was adjusted for the age of examinees, as well as clinical variables
48% (p = 0.057) higher than that in the healthy controls. At the same
(speech delay, cognitive deficit, infantile psychosis, and hyperkinetic
time, a significant 9% (p = 0.032) increase in serum Cu levels was
syndrome). Obsessive-compulsive syndrome was not included as a
observed only in patients with ASD + catatonia, but not ASD
covariate as no variability was observed (0% prevalence in all groups).
(p = 0.080). Serum V levels in both groups of patients exceeded the
Two types of dependent variables were used including the presence of
control values by 50% (p = 0.003 and p = 0.027), whereas a sig-
catatonia (“0” – no catatonia, “1” – catatonia, with “0” including both
nificant increase in Se concentration was observed only in ASD patients
controls and patients with ASD without catatonia) and catatonia in ASD
without catatonia (p = 0.040). Surprisingly, serum Mg levels were
patients (“0” – controls, “1” – ASD children without catatonia, “2” –
found to be elevated in the ASD group, exceeding the control values by
children suffering from ASD with catatonia).
6% (p = 0.033).
The level of significance was set as p < 0.05 for all analyses.
Correlation analysis (Table 4) demonstrated that in the total cohort
of children hair and serum levels of Co correlated positively, whereas
3. Results the association was negative for V and Zn. At the same time, the as-
sociation between hair and serum trace element levels was significantly
The obtained data demonstrate that catatonia significantly mod- affected by ASD and catatonia. Particularly, significant correlations
ulates the association between hair trace element and mineral levels were observed only in the groups of patients, but not neurotypical
and ASD (Table 2) with the majority of differences observed between controls. In ASD patients without catatonia, a significant positive and
the control and ASD group (without catatonia). Particularly, hair Ca negative relationship between hair and serum levels of Al and Zn was
and Se levels in patients with ASD were 30% (p = 0.002) and 23% observed, respectively. In turn, in patients with ASD + catatonia hair
and serum Co levels directly correlated, whereas an inverse association
Table 2 was found for V levels. At the same time, hair and serum Se levels were
Hair toxic and essential element content (μg/g) in children with ASD and cat- characterized by a significant negative correlation in children with both
atonia.
ASD and ASD + catatonia.
Element Control ASD ASD + Catatonia Multiple regression analysis (Table 5) demonstrated a significant
association between hair and serum trace elements and catatonia. In a
Al 6.860 (3.494-9.242) 8.566 (5.804-11.69) 7.529 (5.380-11.01)
As 0.031 (0.019-0.047) 0.033 (0.023-0.039) 0.033 (0.022-0.047)
crude model containing only the levels of the studied elements hair Hg,
Ca 297.06 (232.84- 209.07 (151.02- 248.21 (163.94-341.0) as well as serum Al and Cd levels were found to be inversely associated
410.0) 240.4) 1 with the presence of catatonia, with the model accounting only for 12%
Cd 0.021 (0.010-0.036) 0.022 (0.015-0.030) 0.024 (0.015-0.043) of parameter variability. After further adjustment for clinical variables,
Co 0.007 (0.005-0.011) 0.008 (0.006-0.011) 0.008 (0.006-0.012)
hair Hg levels remained significantly inversely associated with the
Cr 0.116 (0.099-0.196) 0.110 (0.085-0.150) 0.119 (0.094-0.181)
Cu 9.812 (7.954-11.12) 9.604 (8.454-11.02) 10.35 (8.786-11.87) presence of catatonia, although the predictive ability of the total model
Fe 12.25 (9.818-19.85) 12.37 (9.744-14.85) 11.66 (9.868-20.33) increased to 31%.
Hg 0.077 (0.039-0.163) 0.229 (0.072-0.393) 1 0.082 (0.040-0.157) 2 In regression models containing both ASD status and the presence of
I 0.359 (0.212-1.080) 0.700 (0.220-1.025) 0.353 (0.163-0.537) 2 catatonia as dependent variables serum Al and Cd levels were also
Mg 20.37 (15.29-31.00) 20.25 (14.22-25.19) 28.87 (19.51-42.57) 1,2
Mn 0.208 (0.155-0.318) 0.254 (0.194-0.325) 0.209 (0.171-0.382)
negatively associated with ASD children with catatonia. In turn, a po-
Ni 0.150 (0.112-0.200) 0.162 (0.113-0.208) 0.161 (0.121-0.235) sitive association with ASD and catatonia characterized the serum V
Se 0.443 (0.336-0.506) 0.341 (0.261-0.388) 1 0.382 (0.344-0.434) 2 concentration. All associations were significant after adjustment for age
V 0.017 (0.011-0.029) 0.019 (0.014-0.023) 0.020 (0.015-0.038) and clinical variables. Crude and adjusted models accounted for 18%
Zn 152.77 (99.02- 121.59 (77.17-156.1) 127.45 (92.53-164.5)
and 40% of the dependent parameter variability, respectively.
179.6)

Data expressed as Median (25–75 percentiles); 1 – significant group difference 4. Discussion

in comparison to control at p < 0.05; 1 – significant group difference in
comparison to ASD at p < 0.05. The obtained data demonstrate that catatonia and ASD are

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A.A. Tinkov et al. Biomedicine & Pharmacotherapy 109 (2019) 174–180

Table 3
Serum toxic and essential element levels (μg/ml) in ASD children and healthy controls.
Element Control ASD ASD + Catatonia

1
Al 0.020 (0.014-0.023) 0.014 (0.012-0.017) 0.015 (0.011-0.017) 1
As 0.0018 (0.0016 - 0.0020) 0.0017 (0.0016-0.0019) 0.0016(0.0015-0.0017)
Ca 106.71 (103.82-112.3) 109.16 (103.55-113.5) 110.29 (104.45-115.8)
1 1
Cd 0.00002 (0.00002-0.00002) 0.00001 (0.00001-0.00004) 0.00001 (0.00001-0.00003)
Co 0.590 (0.530-0.808) 0.680 (0.615-0.820) 0.690 (0.592-0.788)
Cr 1.020 (0.642-1.735) 2.030 (1.455-2.735) 1 1.510 (0.970-2.520) 1
Cu 1.170 (1.044-1.274) 1.256 (1.146-1.379) 1.275 (1.115-1.423) 1
Fe 1.350 (1.176-1.727) 1.502 (1.163-1.929) 1.412 (1.159-1.643)
Hg 0.0002 (0.0001-0.0002) 0.0002 (0.0002-0.0002) 0.0002 (0.0002-0.0002)
I 0.061 (0.056-0.067) 0.063 (0.055-0.068) 0.063 (0.057-0.076)
Mg 22.68 (21.99-23.21) 23.97 (22.46-25.60) 1 23.04 (22.23-24.45)
Mn 2.085 (1.683-2.730) 1.820 (1.550-2.355) 1.935 (1.653-2.375)
Ni 0.002 (0.002-0.002) 0.002 (0.002-0.003) 0.002 (0.002-0.003)
Se 0.087 (0.074-0.095) 0.076 (0.067-0.085) 1 0.082 (0.071-0.091)
V 0.006 (0.005-0.008) 0.009 (0.006-0.010) 1 0.009 (0.006-0.010) 1
Zn 0.993 (0.912-1.047) 0.974 (0.909-1.039) 0.999 (0.890-1.139)

Data expressed as Median (25–75 percentiles); 1 – significant group difference in comparison to control at p < 0.05; 1 – significant group difference in comparison to
ASD at p < 0.05.

Table 4 dysfunction in children [41], as well as in occupationally exposed

Correlation between hair and serum trace element levels in relation to ASD and adults [42]. Experimental studies also demonstrate that Hg overload
catatonia. may be associated with motor deficits [43]. Also, it has been noted that
Total Control ASD ASD + Catatonia Hg exposure results in oxidative stress and neuronal death in the motor
cortex of rats [44]. Taking into account alterations in the motor cortex
r p r p r p r p in catatonia pathophysiology [45], this association may provide a link
between Hg, ASD, and catatonia. At the same time, Blaurock-Busch
Al −0.127 0.232 −0.248 0.187 0.384 0.048*
−0.133 0.454
As 0.011 0.917 0.059 0.758 −0.167 0.405 0.105 0.553 et al. (2012) observed a slight but significant association between hair
Ca 0.009 0.933 −0.126 0.506 0.314 0.111 0.009 0.961 Hg levels and object use but not body use [36].
Cd −0.004 0.973 −0.023 0.905 0.030 0.883 0.111 0.534 A growing body of data indicates the role of Al in ASD [15]. Par-
Co 0.337 0.001* 0.176 0.352 0.352 0.072 0.480 0.004*
ticularly, it has been demonstrated that patients with ASD are char-
Cr −0.089 0.401 −0.035 0.854 0.305 0.122 −0.171 0.333
Cu 0.054 0.614 0.032 0.868 −0.074 0.713 0.118 0.505
acterized by Al accumulation in the brain [46]. Increased body Al
Fe −0.056 0.597 −0.025 0.898 0.011 0.957 −0.100 0.576 burden was also shown to be associated with behavioral disorders in
Hg 0.124 0.229 −0.057 0.779 0.245 0.137 0.077 0.689 ASD [47]. Experimental studies have also demonstrated the ability of Al
I 0.023 0.828 0.012 0.951 0.086 0.670 0.294 0.091 compounds to impair social behavior [48]. Although no indications of
Mg 0.053 0.618 −0.045 0.814 0.277 0.162 0.028 0.875
the association between Al exposure and catatonia exist, certain studies
Mn −0.010 0.924 −0.067 0.723 0.035 0.864 0.017 0.926
Ni 0.159 0.133 0.284 0.128 0.159 0.429 −0.059 0.740 provide evidence of the adverse effect of Al on motor function [49,50].
Se −0.044 0.678 −0.140 0.461 0.424 0.028* 0.504 0.002* Significantly higher plasma Cd levels were observed in a patient
V −0.288 0.006* −0.211 0.263 −0.172 0.392 −0.437 0.010* with ASD [51]. We have also revealed a significant association between
Zn −0.276 0.008* −0.287 0.125 −0.488 0.010* −0.161 0.362
serum Cd levels and markers of neuroinflammation in ASD [52]. At the
same time, no significant group difference in blood Cd concentration in
Data expressed as correlation coefficient (r) and the respective p values.
* - correlation is significant at p < 0.05. healthy and ASD children was observed in Jamaica [53]. Adams et al.
(2013) also demonstrated significantly reduced whole blood Cd levels
associated with altered hair trace element levels. Generally, the data of in ASD children as compared to the controls [54], is in agreement with
the present study are in agreement with the previous studies of hair the obtained data. Urinary Cd excretion was also found to be sig-
trace element content in children with ASD. However, this is the first nificantly decreased in children with ASD [55]. At the same time, an-
study performed on the association between trace element metabolism other study revealed significantly increased hair and urinary levels of
and catatonia. Cd in patient with ASD [56]. In turn, no data on the potential asso-
The most significant finding of the present study is an inverse as- ciation between Cd and catatonia are available, although indications of
sociation between toxic metal levels including hair Hg and serum Al the inverse association between Cd exposure and motor functions in
and Cd with catatonia in ASD. Despite the lack of direct indications of children exist [57].
the role of these metals in catatonia, multiple studies have demon- The vast majority of studies that examine hair toxic metal levels in
strated their role in the pathogenesis of ASD. ASD find a significant difference between children with ASD and typi-
Mercury was shown to play a significant role in ASD pathogenesis cally developing children (Table 6). Previously, scientists have pro-
due to its neurotoxic effects associated with oxidative stress, neuroin- posed that children suffering from ASD are characterized by more
flammation, excitotoxicity, and mitochondrial dysfunction [32]. Mul- profound alteration of metal handling and excretion in comparison to
tiple studies have demonstrated significantly increased levels of Hg in typically developing children [38,65], and this current study supports
the organism of children with ASD [14; 33]. Similarly, hair Hg levels that assumption. Particularly, the observed reduction in heavy metal
were found to be elevated in ASD and associated with autism severity levels in patients with ASD (with or without catatonia) may be asso-
[34–37]. However, individual studies detected a significant decrease in ciated with their sequestration in the organism due to the impaired
hair Hg levels in children with ASD [38,39]. Moreover, no significant metal detoxication in ASD [39]. Once sequestered in brain heavy metals
association between ASD and Hg-associated metal regulatory genes was may cause neurotoxicity through mechanisms being involved in ASD
observed [40]. pathogenesis [15] However, the mechanisms mediating group differ-
Mercury toxicity was also shown to be associated with motor ences in hair and/or serum metal levels between patients with only ASD
and individuals with both ASD and catatonia are unclear.

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Table 5
Multiple regression analysis of the association between trace element levels, clinical parameters and catatonia status in children with ASD.
Parameter Catatonia (0/1) ASD + Catatonia (0/1/2)

Model 1 Model 2 Model 1 Model 2

β p β p β p β p

H-Ca −0.115 0.371 −0.049 0.668 −0.219 0.074 −0.132 0.216

H-Hg −0.297 0.009 * −0.278 0.007 * −0.132 0.215 −0.100 0.292
H-I −0.201 0.154 −0.088 0.483 −0.102 0.445 0.015 0.899
H-Mg 0.201 0.120 0.055 0.644 0.224 0.069 0.083 0.458
H-Se 0.038 0.784 0.031 0.798 −0.088 0.503 −0.101 0.373
S-Al −0.306 0.021 * −0.235 0.052 −0.369 0.004 * −0.210 0.037 *
S-Cd −0.255 0.026 * −0.196 0.068 −0.312 0.005 * −0.281 0.013 *
S-Cr −0.064 0.627 −0.039 0.740 −0.069 0.585 −0.048 0.664
S-Cu 0.070 0.545 0.012 0.908 0.145 0.192 0.079 0.409
S-Mg 0.021 0.847 −0.133 0.221 0.096 0.357 −0.019 0.851
S-Se 0.182 0.126 0.151 0.161 0.063 0.576 0.034 0.734
S-V 0.226 0.113 0.162 0.210 0.340 0.013 0.243 0.045 *
Speech delay 0.334 0.001 * 0.295 0.002 *
Cognitive deficit 0.337 0.002 * 0.345 0.001 *
Infantile psychosis 0.260 0.019 * 0.382 < 0.001 *
Hyperkinetic syndrome −0.203 0.045 * −0.085 0.361
Age −0.130 0.235 −0.060 0.558
Multiple R 0.498 0.663 0.538 0.716
Multiple R2 0.248 0.440 0.289 0.513
Adjusted R2 0.121 0.309 0.180 0.400
p 0.036 * < 0.001 * 0.005 * < 0.001 *

Hypothetically, ASD patients with catatonia may be characterized by
various metabolic patterns including the activity of heavy metal-
handling enzymes as compared to children only with ASD. At the same
time, different modes of toxic and essential trace element levels in
patients with ASD and catatonia indirectly support this hypothesis.
The most significant associations between essential trace elements
and catatonia in ASD were observed for Cu, Cr, and V. In particular, the
data from the present study demonstrate a significant increase in serum
Cu levels and a trend to elevated hair Cu content in ASD patients with
catatonia. Earlier studies have shown a significant association between
Cu metabolism and ASD. Particularly, children with ASD are char-
acterized by significantly increased serum Cu levels [58]. Moreover,
increased Cu-mediated oxidation of membrane phospholipids in ASD
has been demonstrated [59]. At the same time, the existing data show
an association between altered Cu metabolism and catatonia.
Particularly, Cu overload in Wilson’s disease is associated with cata-
tonia [60]. Research has found serum ceruloplasmin to be increased in
patients with schizophrenia and especially in those with catatonia [61].
Although multiple studies have demonstrated increased hair Cr le-
vels in ASD, data on blood metal levels are insufficient and contra-
dictory [62,63]. At the same time, Yorbik et al. observed a significant
reduction in urinary Cr excretion in patients with ASD [55].
Increased V levels were observed in patients both with and without
catatonia. However, data on the levels of vanadium in ASD are scarce.
Certain studies demonstrated a significant increase in red blood cell V
content in patients with ASD [62, 65]. In turn, we have revealed sig-
nificantly lower levels of hair V in ASD children [64]. A significant
association between urinary [66] but not hair V and CARS, motor
functions and other clinical variables were demonstrated [36].
In turn, the significant group difference in comparison to the

Table 6
A summary of the reviewed studies on hair trace element levels in ASD in comparison to the controls.
Study N Elevated in ASD vs Controls Decreased in ASD vs Conclusion
Controls

Holmes et al. [38] ASD = 94 n.a. Hg Hair excretion patterns among autistic infants were significantly reduced
Controls = 45 relative to controls and correlated with autism severity
Fido and Al-Saad, [33] ASD = 40 Pb, Hg, U n.a. The children with autism had significantly higher hair levels of Pb, Hg and
Controls = 40 Y.
Kern et al. [39] ASD = 45 n.a. As, Cd, Pb Children with autism may have trouble excreting these metals, resulting in a
Controls = 45 higher body burden that may contribute to symptoms of autism.
El-baz et al. [34] ASD = 32 Hg n.a. Increased hair Hg level is indicative of the potential benefit of chelation
Controls = 15 therapy
Priya, Geetha, [35] ASD = 45 Cu, Pb, Hg Mg, Se Excess of toxic metals and deficiency of essential elements correlates with
Controls = 50 disease severity
De Palma et al. [24] ASD = 44 Mo, Li, Se n.a. Relationship between ASD and hair metal levels is weak and inconclusive
Controls = 61
Blaurock-Busch et al. ASD = 44 Al, As, Cd, Hg, Sb, Ni, Pb, V Ca, Fe, I, Mg, Mn, Mo, Heavy metal overload may play a role in ASD pathogenesis
[36] Controls = NS Zn, Se
Al-Farsi et al. [64] ASD = 27 Pb, Al, Si, Mo, V, Cr, Cd, Co, S, Na, Mg, Ca, Cu The body burden of heavy metals in autism is significantly increased
Controls = 27 Ni, B, Ba, K, Zn, Fe
Mohamed et al. [37] ASD = 100 Hg, Al, Pb n.a. Environmental exposure to Hg, Al, Pb may result in ASD
Controls = 100
Skalny et al. [65] ASD = 74 Se Cr, I, V, Be, Sn Children with ASD are characterized by reduced levels of toxic and essential
Controls = 74 elements except selenium

n.a. – data not available.

178
A.A. Tinkov et al.
controls was observed for Se (hair and serum) only in ASD but not in the
group with both ASD and catatonia, corresponding to the earlier pro-
posed role of Se and selenoprotein metabolism in ASD [67]. Blood Se
levels were found to be significantly reduced in patients with ASD [62,
68], although data on hair content are more contradictory [35, 69]. Our
recent study demonstrated a simultaneous elevation and decrease of
hair and serum Se levels, that may be indicative of increased Se loss and
formation of Se deficiency in ASD [70].
At the same time, the present study has certain limitations. First,
dietary intake of trace elements was not monitored during the study,
that may be beneficial to investigate whether the observed changes are
associated with dietary patterns. Similarly, data on urinary trace ele-
ment levels would be useful to clarify the role of increased or reduced
loss of essential and toxic trace elements in patients with ASD in rela-
tion to the presence of catatonia. Finally, although significant group
difference in trace element levels in the studied groups were revealed,
the potential mechanisms of these associations were not assessed.
Therefore, further studies involving assessment of the neuroin-
flammatory reaction, oxidative stress, excitotoxicity, and other parti-
cular mechanisms would be beneficial to highlight the role of trace
elements in catatonia in ASD.
Although the etiology of catatonia in ASD, as well as its pathoge-
netic association with trace element metabolism, is unclear, the ob-
tained data in the present study demonstrate a significant interaction
between catatonia and trace element and mineral metabolism in ASD.
Psychiatric status of the patients including the presence of catatonia
and other syndromes, as well as ASD subtypes [71] is essential to be
controlled in studies on ASD and trace elements, as they have a sig-
nificant impact on the outcome of the investigation. Particularly, a
grouping of ASD children without adjustment for the forms and clinical
features of the disease may result in incorrect results. At the same time,
further studies are required to elucidate the role of trace elements in
potential signaling mechanisms of catatonia and other syndromes in
ASD.
Conflict of interest
The authors declare no conflict of interest
Acknowledgement
The current investigation is supported by the Russian Foundation
for Basic Research within project № 18-315-00103.
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