INTRODUCTION

INTRODUCTION
Autism spectrum Disorder:

Autism spectrum Disorder is a complex neurodevelopmental disorder,

which refers to a rage of conditions characterized by some degree of

impaired social behavior, communication and language, and a narrow

range of interests and activities that are both unique to the individual and

are carried out repetitively. This disorder is characterized by some core

features in two areas—social communication and restricted, repetitive

sensory–motor behaviors (NZ Khan et al., 2012). It results from early

altered brain development and neural reorganization, so sign symptoms of

ASD begin in early childhood and tend to persist into whole life (WHO

2019, ML Bauman and TL kemper, 2012) .In most cases the conditions are

evident during the first 5 years of life (WHO 2018). Signs of autism differ

from individual to individual, people can experience large and different

varieties of symptoms thus why it’s called” Spectrum of disorder”. Not all

autistic children exhibit all the signs. Some children with ASD often have

problems with social, emotional, and communication skills. They might

show some repetitive behaviors and might not want change in their daily

life activities. Many children also have different ways of learning, paying

attention, or reacting to thing (CDC, 2019). There are two types of

developmental courses seen in ASD. One is delayed development, which is

gradual in onset & diagnosis is made within 3-4 years of age. Another

pattern is regressive development (G A Stefantos, 2008), in which

developmental milestones are normal or near normal then regression

occurs. Most common regression is loss of language (E Werner et al.,

2005). Regressive autism can be diagnosed slight lately even up to 9

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years of life (L Z waigenbaum, 2001) . Diagnosing ASD can be difficult

since there is no single medical test, like a blood test, or imaging as there

is no single causation of this disorder.

Different types of ASD

Initially autism was believed to be a form of childhood schizophrenia, but

by the 1980s according to Diagnostic and Statistical Manual of Mental

Disorders, DSM III it was officially known as autism (Volkmar and Klin,

2005). From 1994 to May 2013, In DSM IV autism was represented along

with other four neurological condition. They included Asperger's

syndrome, pervasive developmental disorder—not otherwise specified

(PDD-NOS), autistic disorder, childhood disintegrative syndrome, and Rett

syndrome. In 2013 fifth version of DSM-V was published. Rett syndrome is

excluded from ASD in DSM-5 .now it is considered a discrete neurological

disorder. Now rest four different conditions are combined into one

umbrella diagnosis that is ASD (Maenner et al., 2014; autism society,

2016).

Epidemiology

ASD can happens in all racial, ethnic, and socioeconomic groups all over

the world though in many low and middle income countries prevalence is

unknown. According to World Health Organization (WHO) one in 160

children has an ASD (WHO, 2019).The latest survey done by Centers for

Dieses control and prevention (CDC) in March 27, 2020. CDC report states

that the prevalence rate of ASD is increased which is around 1 in 54

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children (CDC,2020).The World Health Organization (WHO) estimates the

international prevalence of ASD at 0.76%; however, which is

approximately 16% of the global child population. The increased

prevalence can be attributed to methodological factors such as

broadening of the concept and diagnosis of autism, referral patterns,

availability of services and better ascertainment due to heightened

awareness among public and improved detection and early diagnosis (E.

Fombonne, 2020). Boys are four times more affected than girls (NR

Tartaglia et al, 2017). In a recent met analysis the male to female ratio is

decreasing that is 3:1 (R Loomes et al, 2017). The female autism

phenotype, gender biases and stereotypes of ASD as a male disorder play

role in female children being misdiagnosed or diagnosed later (S Bargiela

et al., 2016; E Giarelli et al.,2010).

Etiology of Autism

As a whole ASD is considered to be a multi-factorial disorder that results

from genetic and various non-genetic risk factors such as environmental,

biochemical, metabolic factors during critical developmental period (WHO,

2018). It is generally accepted that it is caused by abnormalities in brain

structure or function. Brain scan shows difference in the shape and

structure of the brain in children with autism compared to neurotypical

children. Researchers do not know the exact cause of autism but are

investigating a number of theories including the links among heredity,

genetic and medical problem (Autism society, 2018). There is no single

specific genes linked to ASD have been identified, but multiple genes are

involved in the pathogenesis of ASD. Autism is a presenting feature of

some rare genetic syndromes, including Fragile X syndrome, Williams

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syndrome and Angelman syndrome (KS Reddy, 2005). Several prenatal

and perinatal complications also have been reported as risk factors for

autism, Such as maternal gestational diabetes (AH Xiang et al.,2018),

parents age over 30 (MS Durkin et al.,2008), bleeding during and after

first trimester, use of antiepileptic medication (e.g. valproate) during

pregnancy (AD Rasalam et al.,2005), and Low vitamin D levels in early

development have been hypothesized as a risk factor for autism(H

Mazahery et al., 2016). Previously some study suggested a causal

association between MMR (Mumps, Measles and Rubella) vaccine with ASD

(A Hviid et al., 2019). Despite extensive research, no reliable study has

shown a link between autism spectrum disorder and any vaccines (Zain et

al., 2015; Rao et al., 2011;LE Taylor et al., 2014).

Correlation of neurotransmitters dysfunction to ASD:

As Autism is a complex neurodevelopmental disorder so Researchers has

also focused on the study of neurotransmitters, in search of any sensitive

and specific markers of ASD. It also used as a potential therapeutic

intervention. Scientists from the University of Missouri school of medicine

and MU Thompson center for Autism and Neurodevelopment disorders

identified a link between neurotransmitter imbalances with brain

connectivity between the regions of the brain that play a pivotal role in

social communication and language. It is evident that certain

neurotransmitter systems are impaired in the brains of autistic patients,

causing the symptoms observed in the disease. The role of several central

neurotransmitters (e.g., 5-HT, ACh, DA, GABA and Glu) in initial brain

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development is crucial. Certain imbalance of brain neurotransmissions in

the early developmental phase of the CNS may demonstrate early

pharmacological intervention that facilitates to cure and or even preclude

some of the severe behavioral symptoms of ASD (E. nermin et al., 2018).

Pharmacological and biochemical observations suggest that autism could

be associated with central dopaminergic imbalance (Ernst. M et al 1997).

Several researchers suggested autistic behavior arises from dysfunctions

in the midbrain dopaminergic system. There is a dysfunction of the

mesolimbic cortical circuit leads to social deficits, while a dysfunction of

the nigrostriatal circuit leads to stereotyped behaviors (Paval, 2017).

Patients with autism display signaling alterations in the mesolimbic

cortical dopaminergic pathway, e.g. reduced release of dopamine in the

prefrontal cortex and reduced response in the nucleus accumbens (Ernst

M et al., 1997). Dopaminergic fibers arising from the ventral tegmental

area project to the prefrontal cortex and to regions of the limbic system,

such as the nucleus accumbens, forming the mesocorticolimbic circuit.

This circuit was found to be involved in high order brain functions, such as

emotional social behavior, reward, motivation and cognition (Hellings et

al., 2017). Dopaminergic pathway has been related to behavioral skills

belonging to executive functioning, such as analyzing, planning and

prioritizing (Seeman, 2010). Children with ASD were found to show

deficits in tasks linked to executive functioning, including

response/selection, planning/working memory and flexibility (Hellings et

al., 2017). Various genetic studies have demonstrated that any mutations

of dopaminergic pathway-associated genes such as the DA transporter

(DAT) (Hamilton et al., 2013), DA receptors (Hettinger et al., 2008), and

enzymes of DA biosynthesis (Nguyen et al., 2014) are involved in ASD.

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Autism is strongly associated with a mutation in the DA transporter gene

SLC6A3. This gene encodes for a protein that contributes to regulation of

DA levels in the CNS. DAT is the crucial regulator of Dopamine

homeostasis and alteration of this homeostasis due to DAT dysfunction is

associated with ASD and other neuropsychiatric conditions (Hamilton et

al., 2013; Hellings et al., 2017; Paval, 2017). Many researchers reported

that urinary homovanillic acid (HVA; a main metabolite of dopamine)

levels are higher in autistic children (Cohen, 1974; Cohen and young,

1977; Gillberg et al., 1983; Lelord et al., 1978 and Garreau et al., 1984).

Biomarkers of ASD

Diagnosis of ASD currently done with patient’s behavioral criteria, but

some biomarkers that can assist or support early diagnosis or evidence

based intervention (Murphy et al., 2016). There are many biomarker have

been investigated such as neurotransmitter based e.g. elevated serotonin

level (cook et al., 1996), higher urinary HVA level (Cohen, 1974; Cohen

and young, 1977; Gillberg et al., 1983; Lelord et al., 1978 and Garreau et

al., 1984).mitochondrial dysfunction based (Cook et a., 1996), oxidative

stress marker (WR Mc Ginnis, 2004), metabolic marker, immune

dysregulation and many more.

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Guidelines and recommendation

Guidelines and protocols are reflections of collected wisdom and sets of

mandatory rules, principles or recommendations for practices in

particular fields. Some guidelines are developed for managing ASD,

such as NICE guidelines (NICE guideline, 2019), recommendations by

American Academy of Child & adolescent psychiatry (Volkmar et al.,

2014), Indian Clinical Practice guideline for Autism (subramanyam et

al., 2019) , Evidence based Recommendations by European Society of

Child and Adolescent Psychiatry (ESCAP) ([Link] et al., 2020), Clinical

guidelines of Spain, Australia (Whitehouse et al., 2018) and Guidelines

of Scotland/ United Kingdom( SIGN 145, 2016).Till now there is only

advances in early diagnosis and intervention, but no curable treatment

foe autism, all of these guidelines do not specify a specific protocol for

the management of autism. Researchers of all over the world have

conducted research on autism to minimize the symptom and maximize

their abilities to lead a near normal life for ASD patients. All the

recommended treatments mentioned in above guidelines are applied

behavioral analysis (ABA), speech therapy, sensory integration therapy,

pharmacotherapy, complementary alternative medicine (CAM) therapy

etc.

Current concept of management of Autism

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According to CDC, management of Autism spectrum disorder includes

applied behavior analysis, social skills training, occupational therapy,

physical therapy, sensory integration therapy, and the use of assistive

technology.

The types of treatments usually can be divided into the following

 Behavior and Communication Approaches

 Dietary Approaches

 Pharmacotherapy

 Complementary and Alternative Medicine (CDC, 2019).

Complementary and alternative medicines:

According to the National Center for Complementary and Alternative

Medicine (NCCAM) define complementary and alternative medicine (CAM)

is a ‘‘group of diverse medical and health care systems, practices, and

products that are not presently considered to be part of conventional

medicine’’.

The various approaches to complementary and alternative medicines

(CAMs) used for the treatment of autistic disorders (ADs) includes Vitamin

B6 and Magnesium (RE Nickel & IY Child, 1996), Omega-3-fatty acids (MP

Freeman et al., 2006), Vitamin C (MC Dolske et al., 1993), Folic Acid

(James et al., 2009), Melatonin (M Angley et al., 2007), Vitamin B12 (SJ

James et al., 2004: M Angley et al 2007), Probiotics (J Garvey, 2002) etc.

Other CAM therapies include the use of mind-body medicines (yoga, music

therapy), Acupuncture, animal assisted therapy, Hyperbaric oxygen

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therapy (HBOT) (DA Rossignol and LW Rossignol, 2006 : DA Rossignol,

2007) dietary supplements (amino acids, gluten free/casein free diets), GI

medications(secretin), immune therapies, etc (SE Levy &SL Hyman,

2015) .

Role of high dose of vitamin B6 with magnesium in Autism:

Vitamin B6 and magnesium are included in complementary alternative

medicine group that are safely use so long. Vitamin B6 a water soluble

vitamin is essential for over 100 enzymatic reactions, including the

synthesis of major neurotransmitters (serotonin, dopamine, and others)

and glutathione (needed for detoxification). Magnesium is a

macromolecule, used to counteract the possibility of hyperactivity, which

can occur from vitamin B6 toxicity. The reason behind improvement from

high-dose vitamin B6 is still unclear, but a possible explanation is that

many autistic children have both 1) a decreased ability to convert vitamin

B6 to its active form e.g. PLP form, and 2) defective enzymes for

producing key central neurotransmitters that needed an unusually high

amount of the active form of vitamin B6.

It is suggested that oral vitamin B 6 with magnesium supplementation is

beneficial for children with autism and in decreasing their sign symptoms

and normalize biochemical parameters. High dose of vitamin B 6 and

magnesium might be considered as a useful therapy in children of ASD.

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RATIONALE:

The prevalence of autism spectrum disorder is increasing throughout the

world including Bangladesh. A lot of researches have been conducted for

ASD but the treatment options are still unsatisfactory. With the

encouraging results of several studies, recently one study has been

conducted at pharmacology department of Bangabandhu Sheikh Mujib

medical university (BSMMU) and found some improvement on

neurobehavioral status of children with autism by supplementation of

vitamin B6 and magnesium. One of the recommendations of this previous

study is dose escalation of Vitamin B 6 to get more improvement on

neurobehavioral status as well as normalization of biochemical parameters

in autistic children. The synergistic effect of Vitamin B 6 and Magnesium is

so obvious in Autism Spectrum Disorder. Findings of this study may

provide research evidence that the administration of high doses of vitamin

B6 with magnesium could improve the behavioral sign symptoms and to

normalize the urinary homovanillic acid level in autistic children. And to

establish high doses of vitamin B6 with magnesium as an effective

treatment option for the patient of autism spectrum disorder. This result

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might open a new horizon of treatment to make the lives of many autistic

patients worthwhile.

RESEARCH QUESTION:

Is administration of high doses of vitamin B6 and magnesium is effective

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OBJECTIVES

General Objectives:

To assess the effect of high doses of vitamin B6 and magnesium on

urinary excretion of homovanillic acid and neurobehavioral sign symptoms

in patients of ASD.

Specific objectives:

i. To estimate urinary homovanillic acid conc. at baseline and after 12

weeks in both interventional group and placebo group.

ii. To estimate the severity of autistic sign symptoms in autistic

children at baselines and after 12 weeks in both interventional and

placebo group.

iii. To compare urinary homovanillic acid level after 12weeks between

interventional and placebo group.

iv. To compare the changes of neurobehavioral sign symptoms after 12

weeks between interventional and placebo group.

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v. To correlate homovanillic acid level with neurobehavioral sign

symptoms in autistic children after Vitamin B 6 & Magnesium

supplementation.

OPERATIONAL DEFINITIONS

Autism Spectrum disorder (ASD): Autism Spectrum Disorder (ASD)

refers to a range of conditions characterized by some degree of impaired

social behavior, communication and language, and a narrow range of

interests and activities that are both unique to the individual and are

carried out repetitively.

Pervasive Developmental Disorder (PDD): The diagnostic category of

pervasive developmental disorders (PDD) refers to a group of disorders

characterized by delays in the development of socialization and

communication skills (NIH, 2018).

Asperger’s Syndrome: Asperger’s syndrome is an autism spectrum

disorder that is characterized by impaired social interaction, by repetitive

patterns of behavior and restricted interests, by normal language and

cognitive development but poor conversational skills and difficulty with

nonverbal communication, and often by above average performance in a

narrow field against a general background of impaired functioning

(Merriam Webster, 2018).

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Dopamine: Dopamine is an important endogenous catecholamine which

exerts widespread effects both in neuronal (as a neurotransmitter) and

non-neuronal tissues (as an autocrine or paracrine agent). Within the

central nervous system, dopamine binds to specific membrane receptors

presented by neurons and it plays the key role in the control of

locomotion, learning, working memory, cognition, and emotion.

Homovanillic Acid: A major dopamine metabolite used to synthesize

major epinephrine and norepinephrine and excreted in urine; HVA is

usually measured with the major catecholamines and other

catecholamines metabolites.

Magnesium: A mineral involved in many processes in the body including

nerve signaling, the building of healthy bones, and normal muscle

contraction. About 350 enzymes are known to depend on magnesium

(Medicine Net, 2016).

Pyridoxine (Vitamin B6): A water soluble vitamin act as an essential co-

enzyme in the synthesis of myelin sheet, its deficiency leads to

development of peripheral neuropathy (Frye et al., 2014).

Randomized Controlled clinical Trial (RCT): A clinical trial comparing

the effects of intervention in an interventional group against a control

group following random allocation of patients to interventional control

groups (Hoque, 2009).

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Double blind: Participants and assessors are kept ignorant about the

treatment assignment (Hoque, 2009).

Neurobehavioral: Of or relating to the relationship between the action

of the nervous system and behavior disorders such as aphasia, alexia and

childhood learning disabilities (Merriam Webster, 2018).

Placebo controlled: Patients receive inactive intervention (eg, placebo)

to match the experience of participants with that of interventional group

(Hoque, 2009).

Placebo: A placebo is any component of therapy that is without specific

biological activity for the condition being treated (Schachter and Rubin,

2012).

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