Zinc Status in Autistic Children

Ozgur Yorbik,1 Cemal Akay,2 Ahmet Sayal,2 Adnan Cansever,3

Teoman Sohmen,1 and Ayhan O. Cavdar4*

1Child and Adolescent Psychiatry Department, GATA (Gulhane Military Medical Academy),Etlik, Ankara, Turkey12Toxicology Department, GATA, Etlik, Ankara, Turkey3Psychiatry Department, GATA, Etlik, Ankara, Turkey4Trace Element Research Group of TUBA (Turkish Academy of Sciences) andUNESCO Satellite Center in Ankara, Turkey2

The transition metal zinc is an essential element for normal development and functionof biological systems including the central nervous system (CNS). Zinc may be animportant trace element for the etiology and symptoms of subgroups of autisticchildren because it is an essential catalytic or structural element of many proteins andimportant for neural activity and the control of neuronal death. The aim of this studywas to evaluate zinc status in children with autistic disorder. Plasma, erythrocyte, andhair zinc levels were measured by atomic absorption spectrophotometry in the 45 and39 autistic children, respectively. Forty-one healthy children3 in the same age groupwere selected as controls. Plasma and erythrocyte zinc levels in the autistic childrenwere found to be significantly lower than normal (P < 0.05). The mean hair zinc levelof the patients was also low when compared with that obtained from normal Turkishchildren. These results may indicate chronic zinc deficiency or metabolic abnormalityof zinc in autistic children. Further studies are required to clarify underlying me-chanisms. J. Trace. Elem. Exp. Med. 17:101–107, 2004. � 2004 Wiley-Liss, Inc.

Key words: autistic disorder; zinc; trace elements; Turkey

INTRODUCTION

Zinc plays significant roles in the metabolism of proteins, charbon hydrates,and lipids [1]. It is incorporated into the active sites of many metalloenzymes [2]and probably is used in the central nervous system (CNS) as a neurotransmitteror neuromodulator [3]. It is stored within vesicles in presynaptic boutons [4], isreleased together with glutamate by membrane depolarization in a Ca2+-dependent manner [5], and modulates the activity of glutamate and gammaaminobutyric acid receptors [2]. Vesicular zinc ions have been suggested tocontribute directly to the death of neurons in the so-called ‘‘excitotoxic’’ injury toneurons after seizure activity [6], ischemia [7], and traumatic brain injury [8].Furthermore, zinc deficiency may alter brain function through changes in neu-rotransmitter content and receptor activity [9–11]. Zinc deficiency during pre-

*Correspondence to: Ayhan O. Cavdar, TUBA, Tunali Hilmi Cad. 111/9, 06100 Kavaklidere,Ankara, Turkey. E-mail: acavdar@tuba.gov.tr

Received 14 December 2001; Accepted 24 November 2003

� 2004 Wiley-Liss, Inc.

The Journal of Trace Elements in Experimental Medicine 17:101–107 (2004)DOI: 10.1002/jtra.20002

natal period may produce gross CNS malformations, including anencephaly inexperimental studies as well as in humans [12–15]. Zinc deficiency was suspectedto play a role in emotionally disturbed children or certain mental disorder suchas schizophrenia in the past [16].

Zinc may be an important essential trace element in the etiology and symp-tomatology of subgroup of autistic children because it is an essential catalytic orstructural element of many proteins and important for neural activity andcontrol of neuronal death. The aim of this study was to evaluate zinc status inautistic children by measuring plasma, red blood cell, and hair zinc levels becausezinc studies of this disorder are limited in the literature.

MATERIALS AND METHODS

Subjects for Blood Analysis

Forty-five Caucasian children (39 male, 6 female) aged between 4 and 12years who were referred by the Child and Adolescent Psychiatry Departmentof three main training hospitals of Ankara (Gulhane Military Medical School,Ankara University Medical School, and Hacettepe University MedicalSchool), and diagnosed as having autistic disorder according to the DSM-IVcriteria were included in this study. Forty-one age-, race-, sex-, and socioe-conomic status-matched healthy controls were selected as a normal controlgroup from the children who were evaluated in the Department of Pediatricsin Gulhane Military Medical School for their routine vaccinations (Table I). Acareful family history was taken from parents for seizure disorders, severehead injury, the history of psychotic disorder, and any other acute or chronicphysical or mental illnesses. Those patients with negative history of above-mentioned conditions were included in the study, whereas those children withany of these conditions or a history of any drug use during the last monthwere excluded.

Subjects for Hair Analysis

The hair samples were also obtained4 from 39 children (33 male, 6 female) outof 45 autistic patients aged between 4 and 12 years. The results of hair zincanalysis in 87 healthy Turkish children (age range between 3 and 16 years)obtained previously also was included in the study [17].

TABLE I. Demographic Information on Autistic and Normal Children

Autistic children (n: 45) Normal children (n: 41) Statistics P value

Age (mean ± SD) 6.5 ± 2.2 6.7 ± 2.5 t = )0.428 NSGender (male %) 86.7% 85.4% x2 = 0.30 NS

NS, not significant (P > 0.05).

102 Yorbik et al.

Procedures and Measures

Blood analysis. The blood samples were collected at 9 to 10 a.m. from thepatients after an overnight fasting. Ten milliliters of blood was taken to the glasstubes containing tripotassium ethylenediamine tetraacetic acid. The sampleswere centrifuged immediately at 4000 rpm for 15 min at 4�C, and plasma sampleswere transferred into polypropylene tubes. Remaining erythrocytes were washedthree times with 0.9% NaCl. Subsequently, 1 mL of the erythrocytes was placedinto polypropylene tubes and lysed with and additional 4 mL of cold dis-tilled water. Plasma and erythrocytes lysates were stored at )70�C in deep freezeuntil measurement.

Hair analysis. Hair samples were obtained from suboccipital region of thechildren’s scalp. Approximately 0.3 g of hair samples5 were washed, dried, andweighted by a balance (Mettler AT-201), then 3 mL of nitric acid (Merck) and0.5 mL of 30% hydrogen peroxide (H2O2) were added to each sample. After this,samples were placed in microwave digestion unit (MLS, 1200 Mega, Italy) foracid digestion of hairs, and diluted with 1% nitric acid during measurement [18].

Measurements of zinc in all samples (plasma, erythrocytes, and hair). Duringthe study deionized and distilled water was used for washing glassware andpreparation of standard solutions. 1000 lg/mL of certified zinc stock solution(Sigma) and ultrapure-grade nitric acid (Merck, Germany) were used for dilutionand standard preparation. Standard solutions of zinc (at 0.5, 1.0, and 1.5 lg/mL)were prepared daily. Plasma, erythrocytes, and digested hair samples were di-luted with 1% nitric acid during zinc measurement. Determinations of zinc wereperformed with flame Atomic Absorption Spectrophotometer (Varian 30/40model, Australia, A Varian DS-15 computer units connected with an EpsonLX-800 recorder). A Varian zinc hollow cathode lamp was operated at 213.9-nmwavelengths [19]. Results were given lg/g in hair.

Nutritional history of autistic children was not available in this study.Informed consent was obtained from all parents. Student t-test was performedfor the statistical analyses of the data.

RESULTS

Demographic data on autistic children are shown in Table I; there was nodifference in age and sex between normal and autistic children. The results ofzinc analyses of autistic children and controls are shown in Table II. The meanplasma zinc level of autistic children (13.00 ± 1.7 lmol/L vs. 16.82 ± 1.6 lmol/L) was significantly lower than that of controls (P < 0.05). Autistic children hadalso lower erythrocyte zinc levels (1.5 ± 1.6 lmol/L vs. 1.95 ) 1.9 lmol/L)

Zinc Status in Autistic Children 103

compared with normal children (P < 0.05). The mean hair zinc level of autisticchildren was found to be 131.7 ± 60 lg/g in the present study, whereas this valuefor normal Turkish children was 184.0 ± 19 lg/g, as has been reported recently[17].

DISCUSSION

Moynahan was one of the first to observe mood changes and aberrant visualbehavior in untreated children with the hereditary zinc deficiency disorder ac-rodermatitis enteropathica. These findings were similar to those seen in certainautistic children [20]. There are only a few studies in the literature that measuredzinc levels in plasma and hair of autistic children, and the results were contro-versial [21,22]. However, it has been shown that plasma, erythrocytes, and hairzinc levels were all low in autistic children as compared with the normal group inthe present study, suggesting chronic zinc deficiency [23]. The variable results inthe literature may be the result of the heterogeneity of autism or geographicareas. Our study included more patients than the other studies [21,22]. However,the reason of such a deficiency in these children is not known at present. Zincdeficiency, in general, may be the result of low dietary zinc intake, decreased zincabsorption, or increased zinc loss [23]. A possibility of a metabolic abnormalityinvolving zinc should also be taken into consideration.

Interestingly, immunological abnormalities have also been described in au-tistic disorder [24]. There are similarities between immunological changes (e.g.,reduced lymphocyte proliferative response to mitogens; abnormalities of T cell-mediated immunity; low total T lymphocyte’s count; decreased activity ofnatural killer cells) found in autistic children [24] and those observed in zincdeficiency [25]. It is possible that zinc deficiency may also contribute to immu-nological abnormalities in autistic children.

The transition metal zinc is an essential trace element for normal developmentand function of biological systems, including the CNS [1]. Zinc deficiency duringearly brain development may cause malformations [12–15], while during laterbrain development impairs neuronal growth, synaptogenesis [26,27], and causesbehavioral sequel [28]. Zinc is an important trace element for biogenic aminemetabolism [29–33]. Dopamine and serotonin are present in brain during earlydevelopment. It has been postulated that these biological amines effect neuraldifferentiation [33–35], and serotonin has been reported to effect neuroblast

TABLE II. Plasma, Erythrocyte, and Hair Zinc Levels in Autistic and Normal Children

Plasma zinc(lmol/L)

Erythrocyte zinc(lmol/L)

Hair zinc(lg/g)

n �XX ± SD n �XX ± SD n �XX ± SD

Autistic children 45 13.00 ± 1.7 45 1.51 ± 1.6 39 131.7 ± 60Normal children 41 16.82 ± 1.6 41 1.95 ± 1.9 97* 184.0 ± 19P <0.05 <0.05

*From [17].

104 Yorbik et al.

division [37], cell migration [38], and synapse formation [39], which may beimportant in etiology of autism. Developmental deviation may partly result fromabnormal biogenic amine metabolism mediated by zinc. However, the suscepti-bility of these processes to zinc deficiency is not clear.

Epileptic seizures occur more often in the children with autistic disorder thannormal children. Zinc status can affect seizure susceptibility and seizurethreshold [40]. Administration of zinc ions substantially reduces the frequency ofnoise-induced tonic and clonic seizures and death in mice as previously dem-onstrated [41]. These studies suggest that an optimal level of vesicular zinc ionsprotects the brain against seizure induction. Thus, zinc deficiency may contributeto epileptic seizures in autistic children.

In addition, oxidative stress could damage structure and function of neuronalcell membranes, and may also be important in the etiology of autistic disorder. Inour recent study on plasma and erythrocyte antioxidant enzyme activities,glutathione peroxidase and superoxide dismutase were found to have lower ac-tivity in autistic children than in normal children [42]. Superoxide dismutase is animportant antioxidant enzyme requiring zinc for its function. It has been shownthat zinc itself can also reduce oxidative stress by binding to thiol groups [43].

Zinc is now accepted as a critical nutrient for CNS development during pre-and postnatal life. Zinc finger proteins are important for neurotransmission inbrain, and zinc-dependent enzymes play a role in cell replication processes re-quired for brain growth. It is possible that maternal and early infant zinc defi-ciencies may adversely affect fetal and infant behavioral and neurologicaldevelopment as indicated by Cauldfield et al. [44].

CONCLUSIONS

There are few studies in the literature about zinc status in autistic disorder. Thepresent study shows that autistic disorder is associated with low zinc levels ofplasma, erythrocyte, and hair, but the etiological importance of these findings inautistic children is unclear at present. Further studies are needed to explainpossible zinc deficiency and/or metabolic abnormality involving zinc in autisticdisorder. Prenatal and postnatal nutritional factors, environmental conditions(such as the existence of toxic elements such as lead and cadmium) and rela-tionship with copper and ceruloplasmin should be studied further in this disorder.

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