**2. Evidence for the role of oxidative injury in autism and the rational use of antioxidant therapy in autism**

There is compelling evidence that cumulative damage by oxidative species play a role in many diseases including autism [17]. Reactive oxygen species (ROS) are unstable and aggressive molecules, which have the tendency to give their unpaired electron to other cellular molecules or snatch electrons from other molecules to attain stability [18]. ROS can be neutralized by antioxidant defence systems, including antioxidant enzymes and antioxidant compounds. Dismutation of the superoxide species, which is catalyzed by superoxide dismutases (SOD), leads to the formation of hydrogen peroxide. This can in turn be metabolized to water by catalases or peroxidases. Superoxide and hydrogen peroxide also undergo a series of ironcatalyzed reactions to yield hydroxyl free radicals (OH-). These are highly toxic themselves and can also generate more free radicals by reaction with other biomolecules, such as proteins or membrane fatty acids.

Antioxidants are compounds that reduce the production of free radicals and ameliorate the oxidative injury. In contrast to the rapid production of toxic oxygen species, the capacity of antioxidant systems, such as the enzyme SOD, catalase, glutathione (gamma-glutamylcystei‐ nylglycine, GSH) peroxidase, and vitamins C and E, is limited, and there is a lag time in their adaptation. Dietary antioxidants and micronutrients in the diet, such as zinc, influence the development and function of immune cells, the activity of stress-related proteins and antiox‐ idant enzymes, and help to maintain genomic integrity and stability [19, 20]. All these physiological functions occur through the action of proteins involved in the regulation of zinc homeostasis such as metallothioneins which bind zinc with high affinity but, at the same time, release free zinc ions in response to oxidative/nitrosative stress that modulates the expression of zinc-dependent genes, activates antioxidant enzymes and has impact on immune response [21]. Zinc may induce the synthesis of metallothionein that act as a scavengers of metals and free radicals [22, 23]. The release of zinc from metallothioneins represents an intracellular response to stress. Biochemical modification of stress-related proteins might represent a useful target to influence zinc homeostasis and related mechanisms in autism.

early diagnosis and treatment could be possible. However, the early diagnostic uncertainties and the nature of deficits unfortunately often delay the diagnosis. Besides limited social and communication skills, behavioural and emotional symptoms, abnormal sensory responses and activity levels often seen as attention deficits are common in ASD individuals [12-14]. Intel‐ lectual disability associates often with autism (30-60%) and epilepsy is seen in about 25% of individuals diagnosed with ASD [1, 8, 15]. Many autistic children exhibit behavioural and

sleep problems and aggression towards others or self as symptoms of their condition.

factors to development of autism [16].

98 Pharmacology and Nutritional Intervention in the Treatment of Disease

**of antioxidant therapy in autism**

or membrane fatty acids.

Unfortunately, there is no cure for autism and symptomatic treatment optimal for autistic individuals without major side effects is lacking. Improvement in patient care – both treatment and rehabilitation – directly influence the prospects of individuals with neurological disorders, including their abilities to integrate to the society and need for life-time support. Behavioural problems in ASD increase stress of people who take care of autistic children and dealing with these problems can be extremely challenging. The child's symptoms might result from an overload of demands (allergens, infectious agents, toxins, psychosocial stresses, inflammation, oxidative stress) in combination with weakness or susceptibilities, which impaired ability to respond to the demands (impaired energy production, inherited enzyme weakness, nutritional deficiencies, osteopathic disorders, sleep deficits, hormone imbalances, etc.) and increased vulnerability. There is evidence that many interrelated environmental factors may act as risk

**2. Evidence for the role of oxidative injury in autism and the rational use**

There is compelling evidence that cumulative damage by oxidative species play a role in many diseases including autism [17]. Reactive oxygen species (ROS) are unstable and aggressive molecules, which have the tendency to give their unpaired electron to other cellular molecules or snatch electrons from other molecules to attain stability [18]. ROS can be neutralized by antioxidant defence systems, including antioxidant enzymes and antioxidant compounds. Dismutation of the superoxide species, which is catalyzed by superoxide dismutases (SOD), leads to the formation of hydrogen peroxide. This can in turn be metabolized to water by catalases or peroxidases. Superoxide and hydrogen peroxide also undergo a series of ironcatalyzed reactions to yield hydroxyl free radicals (OH-). These are highly toxic themselves and can also generate more free radicals by reaction with other biomolecules, such as proteins

Antioxidants are compounds that reduce the production of free radicals and ameliorate the oxidative injury. In contrast to the rapid production of toxic oxygen species, the capacity of antioxidant systems, such as the enzyme SOD, catalase, glutathione (gamma-glutamylcystei‐ nylglycine, GSH) peroxidase, and vitamins C and E, is limited, and there is a lag time in their adaptation. Dietary antioxidants and micronutrients in the diet, such as zinc, influence the development and function of immune cells, the activity of stress-related proteins and antiox‐ idant enzymes, and help to maintain genomic integrity and stability [19, 20]. All these

A number of studies have implicated disturbed zinc metabolism to the neurobiology of autism. Many children with ASD are shown to suffer from zinc deficiency and excess copper levels [24, 25]. Low levels of zinc during development can adversely affect learning, memory, and attention [26]. Zinc deficiency has also been shown to associate with a behavioural syndrome characterized by reduced activity levels and slower response times [27]. Zinc is an important nutrient for the immune system, and supplementation with this mineral has been shown to reduce the duration of the common cold by suppressing the viral inflammation in the respi‐ ratory tract [28-30]. Zinc deficiency can result in a weakened intestinal immune system, which makes the digestive tract more prone to infection with certain parasites [31]. It is also reported in maldigestion and/or malabsorption that often associate with autism [32, 33]. There is evidence that zinc is required for intestinal wound healing and zinc is necessary to maintain the health and integrity of epithelial cells that line the intestines.

A variety of environmental factors that affect brain development during embryonic and perinatal periods may play a part in autism. These risk factors could be influenced by genetic mutations in genes involved in the inflammatory response such as TNF-alpha and interleukin 6 (IL-6) and in the maintenance of zinc homeostasis such as metallothioneins [34]. Il-6 has been associated with neurodegenerative disorders and autism [35, 36]. In genetic studies, measur‐ able differences associated with genes that encode enzymes and other proteins impacting the methylation cycle, the folate metabolism and the glutathione system are reported between children with autism and healthy controls [37]. In particular differences in allele frequency and/or significant gene-gene interactions for genes encoding the reduced folate carrier (RFC), transcobalamin II (TCN2), catechol-O-methyltransferase (COMT), methylenetetrahydrofolate reductase (MTHFR), and one of the glutathione transferases (GST M1) are found. These genetic results, combined with the biochemical observations of dysfunction in the methylation cycle, strongly suggest that variations in genes associated with this cycle and its related biochemistry are involved in the genetic predisposition to developing autism.

How genetic mutations contribute to autism is not clearly understood. A hypothesis for treatment of a genetic form of autism with intellectual disability and epilepsy caused by BCKDH (Branched Chain Ketoacid Dehydrogenase Kinase) mutations by dietary amino acid supplementation was recently put forward [38]. Mutations inactivating a protein called BCKDkinase prevent the breakdown of branched-chain amino acids. Normally, the amino acids are transported across the blood-brain barrier by special transporters. Since plasma amino acids compete with each other for transportation into the brain, the brain amino acid concentration will be substantially changed by low levels of branched-chain amino acids that affect the expression of transporters. The amino acids serve as precursors for neurotransmitters like dopamine and serotonin, which play a role in mood and pleasure-seeking, and whose activities are likely associated with autism.

Autistic children could have a vitamin A deficiency because of gastrointestinal inflammation caused by leaky gut syndrome, allergies or viral infections. Lower levels of vitamin E are

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*Vitamin B6and B12* Some forms of epilepsy are linked with deficiency of B vitamins. Lowered concentrations of B vitamins have been linked with cognitive decline and dementia in older adults. No statistically significant differences were found in plasma B12 levels between ASD cases and controls in meta-analysis [51]. Beneficial effects from high dose supplementation of vitamin B6 with magnesium are shown in a subgroup of ASD individuals. Magnesium is combined to the treatment to prevent hyperactivity that can be caused by vitamin B6 taken alone [52]. Peripheral neuropathy is a rare side effect of high dose vitamin B6 treatment which

*Folic acid* Folic acid, the synthetic form of folate or vitamin B9, during the first month of pregnancy may reduce child´s risk of autism [53]. Folate, vitamin B6 and vitamin B12 are important coenzymes of the homocysteine-degrading remethylation and transsulfuration pathways [54] and their deficiencies can lead to an elevated serum concentration of homocys‐ teine (hyperhomocysteinemia). In addition, B vitamins play a crucial role in the reduction of oxidative stress and in the methylation of different proteins [55]. Serum and plasma levels of folic acid are not affected in children with ASD when compared with control subjects and

*Vitamin C* Higher or not abnormal plasma levels of vitamin C have been reported in individuals with ASD when compared with controls [49, 50]. There is evidence that vitamin C brings about significant improvement in people with autism [56]. Vitamin C softens stools and can help in

*Magnesium (Mg)* Plasma magnesium levels are shown to be lower in autistic than control children [57]. Magnesium is usually combined with vitamin B6 supplement in ASD [52]. The efficacy of this treatment in ASD remains to be verified. Magnesium has been helpful for many autistic children who suffer from constipation. Magnesium is a smooth muscle relaxant, and it helps to pass stools by promoting rhythmic contractions of the intestinal smooth muscle. High magnesium supplementation can cause diarrhea as a side effect and the dose of magne‐

*Phenol sulfotransferase (PST)* Phenol sulfotransferase is an enzyme involved in liver detoxifica‐ tion. Researchers have proposed that PST is compromised in autistic children. A study [58] demonstrated that the PST enzyme system was functioning at sub-optimal levels in more than half of the autistic children tested. Since the deficiency of sulfur in the bloodstream and impairment of the PST system interferes with the body's ability to process and eliminate phenols, this may explain why many children with autism are so sensitive to phenols ingested via certain foods. Low levels of plasma sulphate are reported in autistic children when

*Coenzyme Q10* Classical mitochondrial diseases associate with a subset of autism cases. Both nuclear and mitochondrial genes can underlie mitochondrial dysfunction that is associated with autism [60]. Coenzyme Q10 administration in rats increases mitochondrial concentra‐

sium should be increased gradually until the desired effects are achieved.

reported in ASD patients than in healthy controls [49, 50].

generally disappears when supplementation is finished.

homocysteine show no association with ASD [51].

constipation by making the stools easier to pass.

compared with age-matched control children [59].

Recent studies have associated mitochondrial dysfunction with autism [39, 40]. Defects and malfunction observed in the mitochondria of autistic children suggest that oxidative stress in mitochondria could influence the onset of autism and explain the immunological anomalies present in autistic children. While many inherited genetic mitochondrial disorders occur in the mitochondria of all cells in the body, some are limited to specific cell sites, such as the brain cells which rely largely on mitochondria for energy [41, 42].
