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\begin{document}


\def\RunningAuthor{Ali B Roomi et al.}
\firstPage{3127}
\articleType{Original Article}
\receivedDate{14 Mar 2020}
\acceptedDate{14 May 2020}
\revisedDate{14 Apr 2020}
\journalVolume{2020, 11}
\journalIssue{3}
\journalDoi{ijrps.v11i3.2422}
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\title{Evaluation of lead, copper and zinc levels for autistic children in Thi-qar province/Iraq}
\author{Khansaa~A~Hussien\textsuperscript{1},  
            Ali~B~Roomi\textsuperscript{*}\textsuperscript{2,3}, 
            Shamam~K~Oudah\textsuperscript{4}~\\[5pt]{\textsuperscript{1}National University of Science and Technology, Thi-Qar, Iraq}~\\{\textsuperscript{2}Ministry of Education, Directorate of Education Thi-Qar, Iraq}~\\{\textsuperscript{3}College of Health and Medical Technology, University of Al-Ayen, Thi-Qar, Iraq}~\\{\textsuperscript{4}College of Pharmacy, National University of science and Technology, Thi-Qar, Iraq}}

\begin{abstract}
Autism (AU) is a developmental disability identified by a substantial deficiency in communication and social interaction. The cause of Au is not known yet. The purpose of this study is to evaluate if exposure to lead, zinc and copper can affect causin Autismsm. In this study, 54 children diagnosed with AU were involved. Ninety (90) \% of them were under the age of three years, and 10\% were over the age of three years in the Nasiriyah Autism Centre 2019-2020. The results revealed that lead and copper levels did not affect the female children as much as males. On the other hand, the zinc levels showed a significant statistical difference between female and male. The lead, zinc and copper level did not have any impact on the children with Autism Spectrum Disorder. Lead, zinc, and copper were recognised as one of the chief causes for Autism in Children in Thi-Qar Province. The children who were exposed to substantial metal metabolism and the environment in Thi-Qar province were affected by AU or ASD. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Autism Spectrum Disorder,\newline zinc,\newline copper,\newline lead,\newline heavy metals,\newline autistic symptoms
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Ali~B~Roomi~\\ Phone:\ +9647810036600~\\  Email:\ dr.ali\_bader@alayen.edu.iq
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Autism Spectrum Disorder (ASD) or AUtism (AU) is a neurodevelopmental state. It is described as impairments in social communication \unskip~\citep{781603:18611937,781603:18611989}, social reciprocity, restricted and repetitive patterns of behaviour \unskip~\citep{781603:18612198,781603:18611918}. 

The aetiology of AU may include a complex mixture of immunological, genetic factors, environmental and neurological \unskip~\citep{781603:18612198}. ASD is a developmental disability characterised by diagnostic criteria that involve impairments in social interaction, social communication repetitive patterns of behaviour and the presence of restricted activities \unskip~\citep{781603:18611990}. 

The initial investigations of the prevalence of AU were published in the (1960 and 1970) when AU was considered to be severe state, generally characterised by intellectual disability \unskip~\citep{781603:18611930}. Possible reasons for AU are toxic trance element exposure in combination with a poor nutritional state \unskip~\citep{781603:18611920}. 

Autistic children feel at least several months, even for a year or more of healthy growth, followed by regression and failure to progress or a loss of function, although AU may be apparent soon after birth \unskip~\citep{781603:18611917}.


\begin{table*}[!htbp]
\caption{\boldmath {Serum lead (Pb)concentration of AU.} }
\label{tw-afae1089c52e}
\centering 
\begin{threeparttable}

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\begin{tabulary}{\linewidth}{p{\dimexpr.3155\linewidth-2\tabcolsep}p{\dimexpr.3452\linewidth-2\tabcolsep}p{\dimexpr.3393\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{3}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Lead (mg/dl) }\\
\rowcolor{kwdboxcolor}Sex  & N & Mean \ensuremath{\pm} SD\\
\tblmidrule 
Female  &
    21 &
  12.63\ensuremath{\pm} 3.92 a\\
Male &
  33 &
  12.18\ensuremath{\pm} 4.41 a\\
L.S.D  &
  \multicolumn{2}{p{\dimexpr(.6845\linewidth-2\tabcolsep)}}{\cAlignHack 1.36 }\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{- All value represents(Mean\ensuremath{\pm}SD) values with non-identical superscript (a, b or c..etc.) were carefulsignificantly different (P \ensuremath{\leq } 0.05), n=no. of patients.}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Relation betweenlevel of serum Pb with age in AU patient} }
\label{tw-b1b02fc0b5ec}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
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\begin{tabulary}{\linewidth}{p{\dimexpr.3217\linewidth-2\tabcolsep}p{\dimexpr.3536\linewidth-2\tabcolsep}p{\dimexpr.3247\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{3}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Lead (mg/dl) }\\
\rowcolor{kwdboxcolor}Age. Year & N & Mean \ensuremath{\pm} SD\\
\tblmidrule 
1-3   &
    17 &
  13.35 \ensuremath{\pm} 4.84 a\\
4-5  &
  23 &
  11.7\ensuremath{\pm} 3.74 b \\
More than 5  &
  14  &
  12.22\ensuremath{\pm} 4.14 ab \\
L.S.D &
  \multicolumn{2}{p{\dimexpr(.6783\linewidth-2\tabcolsep)}}{\cAlignHack 1.36}\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{Legend as in table (1)}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Serum zinc (Zn)concentration of AU patient} }
\label{tw-c1894b769dc1}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
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\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{3}{p{\dimexpr(.9999999999999999\linewidth-2\tabcolsep)}}{\cAlignHack Zinc (mg/dl) }\\
\rowcolor{kwdboxcolor}Sex & N & Mean \ensuremath{\pm} SD\\
\tblmidrule 
Female  &
    21 &
  106.57\ensuremath{\pm} 35.04 a\\
Male &
  33 &
  78.68\ensuremath{\pm} 32.85 b\\
L.S.D &
  \multicolumn{2}{p{\dimexpr(.67\linewidth-2\tabcolsep)}}{\cAlignHack 10.87 }\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{Legend as in table (1)}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Relationship between the level of Serum Zn with age in AU patient} }
\label{tw-84cc9ce5d787}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
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\begin{tabulary}{\linewidth}{p{\dimexpr.33\linewidth-2\tabcolsep}p{\dimexpr.3594\linewidth-2\tabcolsep}p{\dimexpr.3106\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{3}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Zinc (mg/dl) }\\
\rowcolor{kwdboxcolor}Age. Year & N & Mean \ensuremath{\pm} SD\\
\tblmidrule 
1-3   &
    17 &
  82.38 \ensuremath{\pm} 28.78 b\\
4-5  &
  23 &
  82.95\ensuremath{\pm} 24.34 b\\
More than 5  &
  14  &
  109.00\ensuremath{\pm} 35.31 a \\
L.S.D &
  \multicolumn{2}{p{\dimexpr(.67\linewidth-2\tabcolsep)}}{\cAlignHack 11.24}\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{Legend as in table (1)}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Serum copper (Cu) concentration of AU patient} }
\label{tw-d5e35b3d4156}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.33\linewidth-2\tabcolsep}p{\dimexpr.3508\linewidth-2\tabcolsep}p{\dimexpr.3192\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{3}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Copper (mg/dl) }\\
\rowcolor{kwdboxcolor}Sex & N & Mean \ensuremath{\pm} SD\\
\tblmidrule 
Female  &
    21 &
  136.03\ensuremath{\pm} 38.12 a\\
Male &
  33 &
  134.45\ensuremath{\pm} 45.18 a\\
L.S.D &
  \multicolumn{2}{p{\dimexpr(.67\linewidth-2\tabcolsep)}}{\cAlignHack 13.74 }\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{Legend as in table (1)}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Relation between levelof serum Cu with age in AU patient} }
\label{tw-d40ac700cc13}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.33\linewidth-2\tabcolsep}p{\dimexpr.3542\linewidth-2\tabcolsep}p{\dimexpr.3158\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{3}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Copper (mg/dl) }\\
\rowcolor{kwdboxcolor}Age. Year & N & Mean \ensuremath{\pm} SD\\
\tblmidrule 
1-3   &
    17 &
  142.94\ensuremath{\pm} 45.97 a\\
4-5  &
  23 &
  134.70\ensuremath{\pm} 38.06 ab\\
More than 5  &
  14  &
  126.12\ensuremath{\pm} 45.19 b \\
L.S.D &
  \multicolumn{2}{p{\dimexpr(.67\linewidth-2\tabcolsep)}}{\cAlignHack 13.72}\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{Legend as in table (1)}
\end{tablenotes}
\end{threeparttable}

\end{table*}

    
\section{Materials and Methods}
\textbf{Biochemical Measurement}

Blood specimens were obtained and collected from the autistic children in Thi-Qar Province/Iraq (33 male 61.1\% and 21 female 38.8 \%) in EDTA tubes. A 0.5 ml from the blood specimens designated for element analysis was then stored at\ensuremath{-}20 \ensuremath{^\circ}C. All metal analysis for lead, zinc and copper determination was conducted using the Atomic absorption spectrophotometry equipped with a graphite furnace. \textbf{\space }


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\centering \makeatletter\IfFileExists{images/2545af0c-67b6-4cbe-96b5-c34a3fb2eaa3-upicture2.png}{\includegraphics{images/2545af0c-67b6-4cbe-96b5-c34a3fb2eaa3-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Relationship between Pd level and age in AU patients}}
\label{f-cd6292c08d61}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/2dd68a8a-46d0-41f2-9274-b4bc86c27e19-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/2dd68a8a-46d0-41f2-9274-b4bc86c27e19-upicture3.png}{\includegraphics{images/2dd68a8a-46d0-41f2-9274-b4bc86c27e19-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Serum Zn levels in AU patient}}
\label{f-f2bc0354a0cc}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/184ec4d0-339d-4556-95e1-a1ebe0215002-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/184ec4d0-339d-4556-95e1-a1ebe0215002-upicture4.png}{\includegraphics{images/184ec4d0-339d-4556-95e1-a1ebe0215002-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Relationship between Zn level and age in AU patients}}
\label{f-bd8147445237}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/dfa32a66-d186-4513-981e-7d131205d19d-upicture5.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/dfa32a66-d186-4513-981e-7d131205d19d-upicture5.png}{\includegraphics{images/dfa32a66-d186-4513-981e-7d131205d19d-upicture5.png}}{}
\makeatother 
\caption{\boldmath {Serum Cu levels in AU patient}}
\label{f-faa8f425f816}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/be222332-8ff9-4cd6-ae05-769430a62ce7-upicture6.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/be222332-8ff9-4cd6-ae05-769430a62ce7-upicture6.png}{\includegraphics{images/be222332-8ff9-4cd6-ae05-769430a62ce7-upicture6.png}}{}
\makeatother 
\caption{\boldmath {Relationship between Cu level and age in AU patients}}
\label{f-af66837a2ea7}
\end{figure}
\egroup
\textbf{Statistical Analysis} \textbf{\space }

Data analysis was accomplished using SPSS version 24.0. Values were reported as Mean \ensuremath{\pm} stander deviation SD. Independent-samples T-test and non-parametric tests were used to compare the various study groups in concentrations of lead, zinc and copper for 54 children witAutismsm aged (0{\textendash}12) years and revealed that most of the cases' P-value (P \ensuremath{\leq } 0.05) estimated was statistically significant.
    
\section{Results and Discussion}
This study was carried on with fifty-four (54) autistic disorder children. Ninety (90\%) of them were diagnosed before the age of three years. The remaining Ten (10\%) were tested and diagnosed after completing three years. These results are in agreement with another study \unskip~\citep{781603:18611929} who observed that mothers and fathers become concerned of autistic disorder at age 12-30 months. Also, finding that AU is a complex neurodevelopmental disability described by deficiencies in social reciprocity and communication skills and always is diagnosed before the age of 3 years \unskip~\citep{781603:18611992,781603:18611922}. Another study reported that the male/female ratio was (2.8:1) of patients diagnosed with ASDs \unskip~\citep{781603:18611992}, while other research said male to female ratio (4:1) \unskip~\citep{781603:18612106}.  Many studies demonstrated that males are at least four times more prone to AU disorder than females \unskip~\citep{781603:18611985}. Other studies showed that the ratio increased more than twice was usual in boys, and the case was so in girls in AU disorder \unskip~\citep{781603:18611992,781603:18611993}. Table~\ref{tw-afae1089c52e} and Figure~\ref{f-20daae3fe07b} demonstrated that of lead concentrations for 54 children with autistic disorders. This study found no significant differences in lead levels between autistic children where the concentrations of lead for autistic female was (12.63 \ensuremath{\pm} 3.92 mg/dl), and for male was (12.18 \ensuremath{\pm} 4.41 mg/dl) that reported in this study agrees with the results of \unskip~\citep{781603:18611990}.\textbf{\space }

Exposure to Pb disrupts neurological development of children and physiological processes \unskip~\citep{781603:18611932,781603:18612704}. In some babies with AU, raised levels of blood Pb may cause the generation of serum anti-ribosomal P antibodies \unskip~\citep{781603:18612704}. The mechanism of lead toxicity is through the exchange of calcium and zinc by lead \unskip~\citep{781603:18611935}. 

The lead can cause an improper release of neurotransmitters \unskip~\citep{781603:18611921,781603:18611925} and crashes brain function by a lower concentration of calcium \unskip~\citep{781603:18611938}. Several studies showed a significant increase in the Pd in children with AU \unskip~\citep{781603:18611936}. Table~\ref{tw-b1b02fc0b5ec} and Figure~\ref{f-cd6292c08d61} demonstrated no significant increase in the level of Pd in study groups. This finding in the study agreement with other studies which reported did not observe a substantial rise in blood Pb in children with ASD \unskip~\citep{781603:18611990,781603:18611921}. Moreover, Pb levels are significantly associated with gene expression and plasma neurotransmitters in children with ASD \unskip~\citep{781603:18612104}.

Many risk factors are included in the aetiology of AU; one is an essential element deficiency. Therefore, it is necessary to trace element concentrations in human bodies to observe and investigate their effect on health \unskip~\citep{781603:18612756,781603:18611940}. Zinc is an essential trace element, which is of great importance in the synthesis of nucleic acid/protein, replication of cells, and growth and repair of tissues. Some reasons are showing the role of GABA in AU aetiology \unskip~\citep{781603:18611916}. GABA levels as a neurotransmitter are reduced in autistic individuals. This amino acid is reduced, leading to restlessness, aggressiveness, irritability, and seizures. 

Zinc is associated with glutamate regulation and GABA, primarily during anxiolytic activity, seizure susceptibility and modulating GABAergic inhibition \unskip~\citep{781603:18612924}. Zinc supplementation not treated ASD or promoting behaviour and cognition \unskip~\citep{781603:18612924,781603:18611928}. There is some evidence that suggested children with ASD might already have a deficiency, and according to sensitivity analysis, which has been performed in the current study, zinc supplementation may be an essential novel therapy \unskip~\citep{781603:18612756,781603:18612105}. 

In this analysis, the plasma of Zn was examined and showed that there was a significant statistical difference between plasma Zn concentrations in autistic patients for autistic female was (106.57 \ensuremath{\pm} 35.04 mg/dl), and for male was (78.68 \ensuremath{\pm} 32.85 mg/dl) the results of Zn concentrations of autistic patients and controls are shown in Table~\ref{tw-c1894b769dc1} and Figure~\ref{f-f2bc0354a0cc}. Individually, some studies, included in this review, had shown a significant relationship between Zn deficiency and AU \unskip~\citep{781603:18611939}. Other studies have not supported such findings \unskip~\citep{781603:18612924}. Some studies have shown the lower levels of zinc in autistic patients, although their results were not significant \unskip~\citep{781603:18611919}. Oxidative stress could defect the function of neuronal cell membranes, damage the structure of a neuronal cell and autistic disorder. In another study on plasma and erythrocyte antioxidant enzyme activities, superoxide dismutase and glutathione peroxidase were found to have lower activity in autistic children than in healthy children \unskip~\citep{781603:18611926}.

Results listed in the Table~\ref{tw-84cc9ce5d787} and Figure~\ref{f-bd8147445237} are compatible with findings in another study. It reported zinc plays a vital role in cell proliferation, nucleic acid/protein formation, tissue growth and repair, mainly in pregnant ladies and infants. Zinc works as an active element in 300 types of enzymes \unskip~\citep{781603:18611927,781603:18613142}. \textbf{\space }

Copper plays a vital role in the oxidation and reduction reactions and is an essential trace element for living cells. Copper and zinc interact with intestinal mucosa, influencing the absorption of Cu and Zn. It is converted from Cu\ensuremath{^{+}} to Cu\ensuremath{^{++}} and back to Cu\ensuremath{^{+}}\unskip~\citep{781603:18611916}. Autistic babies development shows up at times to grow typically until age one to three years. At that point, the abrupt changes happen that shows the nearness of an ASD \unskip~\citep{781603:18611916}. Results listed in the Table~\ref{tw-d5e35b3d4156} and Figure~\ref{f-faa8f425f816} showed a no significant difference in copper levels,  female was (136.03\ensuremath{\pm} 38.12 mg/dl), and the male was (134.45\ensuremath{\pm} 45.18 mg/dl). The present study is in agreement with the results of  \unskip~\citep{781603:18611924}.

Several of the toxic impacts of zinc are, in fact, due to copper deficiency. When zinc medication is given for a long time without copper supplementation or when zinc is given in a large dose of copper, these conditions cause a decrease in the absorption of copper \unskip~\citep{781603:18613361}. Other studies found that babies with AU disorder have larger levels of toxic metals, and levels of toxic metals are connected strongly with the cruelty of AU disorder in children \unskip~\citep{781603:18613403}. Results listed in the Table~\ref{tw-d40ac700cc13} and Figure~\ref{f-af66837a2ea7} compatible with findings in another study reported that autistic people had raised plasma levels of copper \unskip~\citep{781603:18611934}. In the children with ASD (6{\textendash}10years), Se levels and hair Fe were 21\% and 16\% higher, while in serum, only copper levels were raised.
    
\section{Conclusions}
In the present work, we can conclude that the\textbf{\space }lead and copper levels were not significantly different in the female group compared to the male group. In contrast, the zinc levels showed a significant statistical difference between the female group and the male group. Whereas, the lead, zinc and copper level was of insignificant difference in children with autism spectrum disorder (ASD). Lead, zinc, and coppe recognised as one of the chief causes for autistic Children in Thi-Qar Province. A defect in heavy metal metabolism and environmental exposure in Thi-Qar province where is responsible for the high level of heavy metals in children. 

\textbf{Abbreviations}

ANOVA: Analysis Of Variance, ASD: Autism Spectrum Disorder AU: AUtism, LSD: Least Significant Difference, SD: Standard Deviation and SPSS: Statistical Package Social Sciences.
\section*{Acknowledgement}The study was supported by the staff of Al-Hussein teaching Hospital in the collection of samples are gratefully acknowledged.

\textbf{Authors Contributions}

Khansaa and Roomi designed the study, performed the experiments and wrote the manuscript. Roomi and Shamaanalyseded the data. All authors read and approved the final manuscript.

\textbf{Funding Support}

This work was not financially supported. 

\textbf{Conflict of Interest}

None
    

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