Abstract
Introduction
Autism is a complex, behaviorally defined neurodevelopmental disorder characterized by social deficits, language impairments, and repetitive behaviors. There has been a dramatic increase in the diagnosis of autism over the past decade.1,2
The etiology of this complex disease is highly heritable, but likely involves environmental factors. 3 Twin studies demonstrate concordance rates of 82%-92% in monozygotic twins and 1%-10% concordance rate in dizygotic twins. 1 Sibling recurrence risk (6%-8%) is 35 times the population prevalence.1,4
Genetic analysis suggests that as many as 15 genes might be involved in autism spectrum disorders (ASD), including variants on chromosomes 2q, 7q, 15q, and 17q.5–8
Children with ASD frequently have accompanying gastrointestinal, immunological, or nonspecific neurological symptoms.9–15
Zinc has a unique and extensive role in biological processes. Since the discovery of this element as an essential nutrient for living organisms,16–18 many diverse biochemical roles for it have been identified. These include roles in enzyme function, 19 nucleic acid metabolism,20,21 cell signaling 22 and apoptosis. 23 Zinc is essential for physiological processes including growth and development, 24 lipid metabolism, 25 brain and immune function.24,26
Dietary factors that reduce the availability of zinc are the most common cause of zinc deficiency. However, inherited defects can also result in reduced zinc. Both nutritional and inherited zinc deficiency produce similar symptoms, such as dermatitis, diarrhea, alopecia and loss of appetite. 27 With more prolonged deficiency causing growth impairment and neuropsychological changes such as emotional instability, irritability and depression.28–31
Deficiency of zinc in man has now been recognized to occur not only as a result of nutritional factors, but also in various disease states, including malabsorption syndromes, acrodermatitis enteropathica, Crohn's disease, alcoholism and cirrhosis of the liver.59,60
Low intracellular zinc has been found to be associated with DNA damage, oxidative stress, antioxidant defenses, and DNA repair,32,33 and zinc may serve as an important anti-oxidant. 34
Copper (Cu), a trace metal, is also an essential element for living cells. It plays an important role in redox reactions because of its easy conversion from Cu+ to Cu++. Copper is transported mainly by ceruloplasmin, a copper-binding antioxidant protein that is synthesized in several tissues, including brain.35,36
Copper levels are low in Menke's kinky hair syndrome 37 malnutrition 38 and Malabsorption. 39 Elevated copper levels are associated with infections, 40 inflammation, 41 trauma, 42 Wilson's disease, 43 excessive dietary intake 44 systemic lupus erythematosus, 45 as well as autism. 46
Because of the potential association between Zn and Cu levels and autism, we tested patients with autism for plasma concentration of these elements and then compared those levels with severity of disease symptoms.
Materials and Methods
Subjects
Experimental and Control
Plasma from consecutive individuals with diagnosed autism and neurotypical controls was obtained from patients presenting consecutively at the Health Research Institute/Pfeiffer Treatment Center. These individuals meet the DSM-IV criteria and many were diagnosed using The Autism Diagnostic Interview-Revised—ADI-R before presenting for treatment at the Pfeiffer Treatment Center, Warrenville, Il. *
The Pfeiffer Treatment Center is a comprehensive treatment and research center, specializing in the care of with neurological disorders, including autism.
Patient consent was obtained from all patients involved in this study and this study was approved by the IRB of the Health Research Institute/Pfeiffer Treatment Center.
Severity of disease
An autism questionnaire was used to evaluate symptoms. The questionnaire (Pfeiffer Questionnaire) asked parents or caregivers to assess the severity of the following symptoms: Awareness, Expressive Language, Receptive Language, (Conversational) Pragmatic Language, Focus, Attention, Hyperactivity, Impulsivity, Perseveration, Fine Motor Skills, Gross Motor Skills, Hypotonia (low muscle tone), Tip Toeing, Rocking/Pacing, Stimming, Obsessions/Fixations, Eye Contact, Sound Sensitivity, Light Sensitivity, Tactile Sensitivity, Pica/eats dirt, metal, Tics and Seizures. The symptoms were rated on a scale of 0-5 (5 being the highest severity) for each of these behaviors.
Serum/Plasma
All experimental and control plasmas were treated in an identical fashion—refrigerated (4C) immediately after collection and cell/serum separation, then used within 4 hours for inductively-coupled plasma-mass spectrometry.
Statistics
Inferential statistics were derived from
Results
Autistic individuals had elevated plasma levels of copper (108.9 μg/dL) compared to controls (86.5 μg/dL) (
Plasma copper concentration was significantly higher in autistic individuals compared to controls.
In 452 random individuals presenting to the Health Research Institute, we found no significant differences between the copper (
We found a correlation between Cu/Zn and expressive language [r = 0.3,n = 45,
Severity of hyperactivity correlated significantly with Cu/Zn [r = 0.3,n = 79,
Severity of hyperactivity correlated significantly with decreased Zn [r = −0.3,n = 79,
Discussion
There is much support for the role of GABA in the etiology of autism. Alterations in levels of GABA and GABA receptors in autistic patients indicate that the GABAergic system, which is responsible for synaptic inhibition in the adult brain, may be involved in autism.47–49
Zinc has been found to be associated with GABA and glutamate regulation, particularly through anxiolytic activity, modulating GABAergic inhibition and seizure susceptibility.50–52 Zinc deficiency has also been found to be associated with GABAergic impairment. 53
Copper, on the other hand, has been found to be a potent inhibitor of GABA-evoked responses, particularly in Purkinje cells. Copper toxicity, notably in Wilson's disease, could result, to some extent, from chronic GABAA receptor blockade. 54 Data strongly suggest that Cu and Zn might interact with each other with GABAA receptor complex and participate in modulation of synaptic transmission. 55
Dopamine-β-hydroxylase (DBH) is a neurotransmitter, synthesizing enzyme, which catalyzes the formation of norepinephrine from dopamine. Copper is a co-factor required for this enzyme's activity.57,58 Increased norepinephrine levels have been found in autistic individuals, 56 which, at least in part, could be explained by excess copper.
Our lab has also found that Cu/Zn SOD is decreased in autistic children, as well as in individuals with ADHD,61,62 and that in ADHD, lower Cu/Zn SOD is associated with increased copper. 62 This suggests that increased copper in autism may also be associated with increased oxidative stress.
Our study shows that autistic individuals have lower levels of zinc and significantly higher levels of copper when compared to neurotypical controls, and copper/zinc correlates with selected symptom severity in autistic children.
It is tempting to suggest that plasma copper concentration and/or copper/zinc could be used as a biomarker for diagnosis of autism, but copper and zinc levels are altered in many other disease states and nutritional deficiencies.
We suggest that low zinc and high copper may modulate GABA receptors, ultimately changing transmitter concentration. High copper may also be associated with high norepinephrine found in autistic children, and high epinephrine may, in turn, manifest as excitability and hyperactivity associated autistic symptoms. To evaluate this relationship, future studies will assess more patients with autism and evaluate GABA and norepinephrine levels, as they are associated with Cu and Zn levels.
Author Contributions
All authors shared responsibility for data collection/entry/analysis and assistance with manuscript preparation. AJR was responsible for the study design and preparation of the manuscript. All authors read and approved the final manuscript.
Funding
This research was funded by the Autism Research Institute.
Disclosures and Ethics
As a requirement of publication author(s) have provided to the publisher signed confirmation of compliance with legal and ethical obligations including but not limited to the following: authorship and contributorship, conflicts of interest, privacy and confidentiality and (where applicable) protection of human and animal research subjects. The authors have read and confirmed their agreement with the ICMJE authorship and conflict of interest criteria. The authors have also confirmed that this article is unique and not under consideration or published in any other publication, and that they have permission from rights holders to reproduce any copyrighted material. Any disclosures are made in this section. The external blind peer reviewers report no conflicts of interest.