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Middle East Journal of Medical Genetics:
doi: 10.1097/01.MXE.0000422775.82611.a3
Original articles

Assessment of metal content and oxidative stress in autistic Egyptian patients

El-Bassyouni, Hala T.a; Ismail, Samira I.a; El-Saeed, Gamila S.M.b; Alghroury, Eman A.c; Abd El Dayem, Soha M.d

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Author Information

aDepartment of Clinical Genetics, Human Genetics and Genome Research Division

Departments of bMedical Biochemistry

cClinical Pathology

dPediatrics, National Research Center, Giza, Cairo, Egypt

Correspondence to Hala T. El-Bassyouni, MD, PhD, Department of Clinical Genetics, Human Genetics and Genome Research Division, National Research Centre, El-Tahrir Street, Dokki, Giza, Cairo, Egypt Tel:+202 0122 3618884; fax:+002 02 702 5376;e-mail: halabassyouni@yahoo.com

Received April 28, 2012

Accepted September 15, 2012

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Abstract

Background: Many factors have been implicated in the onset of autism, including excess or deficiency in toxic or essential metals and impaired antioxidant systems. Exposure to metals is a putative risk factor for autism. Many metals can be implicated in autism as they typically disrupt enzyme functions and cell signaling processes and generate reactive oxygen species (ROS).

Patients and methods: Thirty-two autistic patients ranging in age from 2.6 to 12.7 years (mean age 5.6 years) were examined clinically. Twenty healthy children matched for age and sex were included as a control group. Serum mercury, lead, and nitric oxide were estimated in all patients.

Results: Eighteen patients presented with seizures and an epileptogenic focus in the electroencephalogram. The intelligent quotient assessment showed mental retardation in all patients: 18 had moderate retardation, two had severe retardation, and 12 had profound retardation. Assessment of the severity of autistic symptoms using the childhood autism rating scale indicated that six patients had mild, 12 had moderate, and 14 had severe autistic symptoms. The estimation of the level of mercury and lead in blood showed a significant increase compared with the control group (P=0.0001). Furthermore, nitric oxide was significantly increased compared with the control group (P=0.0001). These findings indicated a significant increase in both metal content and an imbalance in the oxidative status in the blood of autistic children.

Conclusion: Our findings suggest the hypothesis that exposure to metals and oxidative stress may be biomarkers of toxicity in autism. We recommend the consideration of supplementing autistic patients with antioxidants. We also recommend the study of introducing chelating agents for mercury and lead in patients with high toxic levels and their effect on clinical symptoms.

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Introduction

Autism (MIM 209850) is a neurodevelopmental disorder, with an estimated heritability greater than 90%. Nonetheless, its specific genetic etiology remains largely unknown (Combi et al., 2010). However, despite the strong influence of genetic factors, autism linkage studies and association studies of common SNPs have not identified any genes of major effect (Anney et al., 2011).

Autism is characterized by an impairment in the normal development of social and emotional interactions and related forms of communication. Autism [Autistic spectrum disorders (ASDs)] has three major symptoms: (a) limited or absent communication; (b) lack of reciprocal social interaction or responsiveness; and (c) restricted patterns of interest (Levy and Perry, 2011; Mintz et al., 2012). The prevalence of autism in the population is ∼60 in 10 000 children (Anney et al., 2011).

A close relationship has been reported between autism and epilepsy. In particular, the prevalence of epilepsy among autistics is much higher than that in the normal population and there is also an increased prevalence of abnormal potentially epileptogenic activity in children with ASD (Combi et al., 2010).

Exposure to metals may considerably contribute toward the symptoms associated with autism (Ghanizadeh, 2011; Thomas Curtis et al., 2011). Children with autism may have particular trouble in excreting heavy metals. Accumulation or altered mercury (Hg) and lead (Pb) clearance as well as concomitant oxidative stress, arising from redox-active metal and Pb toxicity, offers an intriguing component or a possible mechanism for oxidative stress-mediated neurodegeneration in autistic patients (McGinnis, 2004; Geier et al., 2010).

Taken together, these factors may be more important to the etiology of this symptomatically diverse disease spectrum and may offer insights into new treatment approaches and avenues of exploration for this growing disease. This could be helpful in the early diagnosis of young autistic patients and suggesting the possibility of antioxidant supplementation in an early intervention with autistic children (Al-Gadani et al., 2009).

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Patients and methods

The study included 32 autistic patients, and 20 healthy children matched for age and sex were included as a control group. They were recruited during 2010–2012 from the Clinical Genetics Department, National Research Center (Egypt). They ranged in age from 2.6 to 12.7 years (mean age 5.6±2.1 years). The recruitment and laboratory protocols of the study were carried out in compliance with the Declaration of Helsinki and approved by the NRC Ethical Research Committee. Patients were clinically examined to detect any malformation and anomalies. A three-generation pedigree was constructed with a special focus on positive consanguinity and similarly affected family members. In addition, the intelligent quotient or developmental age using (Stanford-Binet International Scale, 1986) the childhood autism rating scale (CARS) was determined using the method of Schopler (1986); anthropometric measurements including height, weight and head circumference, and electroencephalogram (EEG) recording were performed.

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Blood sample collection

A peripheral blood sample was obtained and then transferred to a plastic tube after coagulation of blood. Immediate centrifugation was performed for 10 min at 5000 rpm. Serum was stored at 4°C until the time of analysis.

Determination of Hg was performed using an Atomic Absorption Spectrometer-Hydride generation Graphit system (Solaar MQZ Environmental and Thermo Electron Corporation, Malvern, Pennsylvania, USA). 1000 mg/l Hg certified standard solution (Merck, Darmstadt, Germany), was used as a stock solution for instrument calibration.

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Sample preparation

One hundred microliter serum sample+400 µl water (dil. 1 : 5) (Kunert et al., 1979; Sardans et al., 2010).

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Determination of serum concentration of lead

Trace Pb determination was carried out using a graphite furnace Atomic Absorption Spectrometer equipped with a graphite furnace autosampler (Solaar MQZ environmental and Thermo Electron Corporation, USA).

Before measurement of Pb, samples were diluted five times with a mixture of Triton-X 100 (0.2%) and Dow Coming Antifoaming-B (0.2%). Then, a portion of the sample was introduced into the graphite atomization tube.

The standard addition technique was applied to construct the analytical calibration curve, in which an increasing volume of Pb solution was added to the sample to induce similar atomization behavior in both the samples and the standard. A volume of the standard solution (100 µg/l) was added to obtain the final concentrations of added Pb of 10, 20, 30, and 40 µg/l. Palladium solution (1000 µg/l) was used as a matrix modifier from which a volume of 10 µl was added before every sample or standard measurement of Pb. A portion of the sample (20 µl) was added to the atomization tube and then the heating program was started. The calibration curve was found to be linear over the calibration range used. The temperature program used for atomization included a maximum drying temperature of 120°C, ashing temperatures of 480, 1100°C, and atomization temperatures of 2000 and 2200°C for Pb (LeGendre and Alfrey, 1976).

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Measurement of serum nitric oxide

The serum nitric oxide (NO) level was measured by a colorimetric assay using a NO assay kit supplied by R&D system Inc. (Minneapolis, Minnesota, USA; catalog number KGE 100) (Tsikas, 2005).

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Statistical methods

Statistical package for social science program version 9.0 (SPSS, Chicago, Illinois, USA) was used for analysis of data. Data were summarized as mean and SD. The nonparametric test (the Mann-Whitney U-test) was used for the analysis of two quantitative data as data were not distributed symmetrically.

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Results

A total of 32 autistic patients were studied: 28 males and four females. Eighteen patients presented with seizures and an epileptogenic focus in EEG. The intelligent quotient assessment showed mental retardation in all patients: 18 had moderate retardation, two had severe retardation, and 12 had profound retardation. Assessment of the severity of autistic symptoms using the CARS showed that six patients had mild, 12 had moderate, and 14 had severe autistic symptoms (Table 1). Table 2 presents a comparison between Hg, Pb, and NO of autistic patients and controls. The estimation of the level of Hg and Pb in blood showed a significant increase compared with the control group (P≥0.0001) (Figs 1 and 2). Furthermore, NO was significantly increased compared with the control group (P≥0.0001) (Fig. 3).

Table 1
Table 1
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Table 2
Table 2
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Figure 1
Figure 1
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Figure 2
Figure 2
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Figure 3
Figure 3
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Discussion

Our results pointed to a higher risk of autism in boys than in girls, with a ratio of 7 : 1. This finding was consistent with that reported by Ben Itzchak and Zachor (2009), who found that 461 children (81%) of the 564 participants were male autistic patients. Autism is more than twice as common in boys as in girls, and this ratio increases to 5 : 1 at the high ability end of the autism spectrum (El-Baz et al., 2011). Muhle et al. (2004) reported a male to female ratio of 3 : 1 and hypothesized that male-to-male transmission in a number of families rules out X-linkage as the prevailing mode of inheritance. A previous study has highlighted that in high-risk families, males are more likely to inherit the condition than their female relatives, despite their shared genetic background. There is also evidence that the X chromosome has a higher proportion of genes involved in brain development and cognition than of autosomal genes (Gillis and Rouleau, 2011).

Our patients ranged in age from 2.6 to 12.7 years (mean age 5.6±2.1 years). Sousa et al. (2010) studied patients with autism with an age of onset before 3 years and affecting males predominantly (with an average sex ratio of 4 : 1).

Positive consanguinity was present in 10 of the 32 patients (31.25%) and similarly affected family members were present for eight of the 32 patients (25%). Gillis and Rouleau (2011) documented that familial aggregation studies have shown that the relative risk of developing autism in first-degree relatives of an autistic patient is 3–7%, which is 10 fold higher than the prevalence in the general population.

Delayed milestones were found in 28 of the 32 (87.5%) of our patients. Mays and Heflin (2011) noted that developmental delay was evident as early as 2 years of age. Moreover, 18 of the 32 patients (56.25%) developed seizures and EEG abnormalities. Gillis and Rouleau (2011) reported that comorbidity with epilepsy was estimated in 30% of autistic patients. Furthermore, Combi et al. (2010) reported that about one in four autistic children develop seizures. Hyperactivity was observed in 30 of the 32 patients (93.75%). Similarly, Guerini et al., 2011 proposed that autism is associated with hyperactivity.

According to CARS scores, 14 of 32 (43.75%) patients had a severe degree, 12 of 32 (37.5%) had a moderate degree, and six of 32 (18.75%) had a mild degree of autism. These findings were consistent with the findings of El-Baz et al. (2011) and Matson et al. (2011).

The Hg level was significantly higher in our patients with autism compared with the control participants. Geier et al. (2010)and Thomas Curtis et al. (2011) reported similar results and emphasized that Hg may significantly impact the pathogenesis of ASDs. Thomas Curtis et al. (2011) supported the role for heavy metal exposure in neuropathologies of autism. Geier et al. (2009) hypothesized that autism results from a combination of genetic and biochemical susceptibilities in the form of a reduced ability to excrete Hg and/or increased environmental exposure at key times during development. It was hypothesized that children with autism have a decreased detoxification capacity for Hg because of genetic polymorphism (Mutter et al., 2005).

Pb level was significantly higher in our patients with autism compared with the control participants. This finding is in agreement with the findings of El-Ansary et al. (2011), but was not in agreement with that of Tian et al. (2011), who concluded that there were no significant differences in blood Pb levels among children with autism and controls. Obrenovich et al. (2011) emphasized the importance of chelating agents as preventive measures for autism.

With respect to oxidative stress, NO, a marker of oxidative stress, was significantly higher in autistic patients compared with the controls. Ghanizadeh (2011) suggested that oxidative stress plays a significant role in autism. Similarly, James et al. (2004) and Priyaa and Geetha (2011) found increased levels of NO in individuals with autism, which would suggest excessive formation of reactive oxygen species.

Studies have shown supplementation of antioxidants along with a chelating agent to be a better treatment regimen than monotherapy with chelating agents. Treatment with chelating agents and the possible beneficial role of antioxidant supplementation was suggested to achieve the optimum effects (McGinnis, 2004; Flora et al., 2008; Villagonzalo et al., 2010).

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Conclusion

Our study suggests that heavy metals including Pb and Hg as well as oxidative stress may be biomarkers of toxicity in autism. Thus, we suggest the consideration of supplementing autistic patients with antioxidants. We also recommend the study of introducing chelating agents for Hg and Pb in patients with high toxic levels and their effect on clinical symptoms.

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Acknowledgements
Conflicts of interest

There are no conflicts of interest.

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Keywords:

antioxidants; autism; lead; mercury

© 2013 Middle East Journal of Medical Genetics

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