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Nigerian J Paediatrics 2018 vol 45 issue 3

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Neurotransmitter and amino acid levels in Nigerian children with autism spectrum disorders
Niger J Paediatr 2018; 45 (3):129 - 134
ORIGINAL
Fagbayi TA
CC – BY Neurotransmitter and amino acid
Taiwo A
Esezobor CI
levels in Nigerian children with
Okpuzor J
autism spectrum disorders
Lesi FEA
Bello-Mojeed M
Ogun O
DOI:http://dx.doi.org/10.4314/njp.v45i3.1
Accepted: 25th June 2018
Abstract : Autism Spectrum Dis-
were done using IBM SPSS ver-
order (ASD), often referred to as
sion 20. A total of 31 plasma sam-
Fagbayi TA (
)
Autism, is a clinically heterogene-
ples (18 Autism cases and 13 age-
Okpuzor J
ous neurodevelopmental disorder
sex matched controls) were ana-
Department of Cell Biology &
with core-defining features of
lyzed with mean ages 8.44±4.87
Genetics, University of Lagos
impaired socialization, impaired
for cases and 8.15±4.88 for con-
Nigeria
verbal and nonverbal communica-
trols. No significant intergroup
Email: tawakalt.08@gmail.com;
tion, and restricted and repetitive
difference was observed in
the
020802058@unilag.edu.ng
patterns of behaviour. The disor-
individual amino acid levels with
der is presently diagnosed behav-
the exception of glutamate (t =
Taiwo A
iourally, however the search for
5.472, df = 2.324, p = 0.000063),
Synapse Technologies, VI, Lagos
possible biomarkers that could aid
glutamine (t = 8.342, df = 14.780,
Nigeria
earlier diagnosis have been on the
p = 0.000001) , GABA (t = 6.601,
increase. The present study aims
df = 24.593, p = 0.000001), trypto-
Esezobor CI, Lesi FEA
to investigate plasma amino acid
phan (t = 3.568, df = 16.472, p =
Department of Peadiatrics,
levels as a potential biomarker in
0.002) and cysteine (t = 4.000, df =
University of Lagos Teaching
ASD screening. Plasma levels of
13.762, p = 0.001).The amino acid
Hospital (LUTH), Nigeria
20 amino acids (AA) (including
profile and the glutamate - GABA
neurotransmitters-GABA
and
levels can serve as biochemical
Bello-Mojeed M, Ogun O
glutamate) of autistic individuals
markers for ASD, and can thus be
Child & Adolescent Unit, Federal
and typical age and sex matched
utilized as screening for earlier
Neuropsychiatric Hospital, Oshodi
control, were determined using
diagnosis of the disorder.
Annex, Lagos, Nigeria
reversed-phase high performance
liquid
chromatography
(RP-
Keywords: Autism Spectrum Dis-
HPLC). All statistical analyses
order (ASD), Biomarkers, Neuro-
(independent
t-test,
spearman
transmitters, GABA, Glutamate,
correlation, cohen’s d effect size)
Diagnosis.
Introduction
there are comorbid conditions). Presently, diagnosis of
the disorder is typically behavioural through the admini-
Autism Spectrum Disorder (ASD), often referred to as
stration of standardized interviews and questionnaire,
Autism, is a clinically heterogeneous neurodevelopmen-
sometimes with direct observations (Autism Diagnostic
tal disorder with affected children exhibiting marked
Interview (ADI) and the Autism Diagnostic Observation
behavioural phenotypes deviant from the norm (i.e. of
Schedule (ADOS) which are designed based on the di-
their age-matched peers) .The core-defining features are
1
agnostic criteria of the Diagnostic and Statistical Manual
impaired socialization, impaired verbal and nonverbal
of Mental Disorders (DSM) (published in revisions by
communication, and restricted and repetitive patterns of
the American Psychiatric Association [APA]) and the
behaviour
2
and
more
recently,
impaired
social/
International Statistical Classification of Diseases &
communication deficits and restricted interests/repetitive
Related Health Problems (ICD), also published in revi-
behaviours . These behavioural phenotypes are often
3
sions by the World Health Organization (WHO). Cur-
detected between eighteen months to three years of age
rently there is no known cure for Autism.
and persist to adulthood. Different levels of severity in
Autism is believed to have a 1% prevalence World-
wide , with different rates of occurrences observed in
6
the cognitive ability of affected children are observed,
ranging from near mental retardation to geniuses . The
4
different countries. In a clinic based population study in
disorder is often detected before the age of three years
Enugu, south-eastern Nigeria, prevalence of Autism
more often in males than female, with a male-female
Spectrum Disorders had been noted to be 0.8% of the
ratio of occurrence of 4: 1 and affected children usually
5
total population of children that attended the clinic over
a one year period and 11.4% among children with intel-
7
have no visible physical abnormalities (except when
130
lectual disability . A study in Egypt and Tunisia docu-
8
plasma levels of 18 essential amino acid and two neuro-
mented the prevalence of children with ASD among
transmitters (GABA and glutamate) in individuals with
children with developmental disorders to be 33.6% and
Autism and their sex and age-matched controls.
11.5% respectively .
9
Despite great efforts to move forward and clarify the
precise mechanisms underlying the pathophysiology of
Materials and methods
Autism, its pathophysiological mechanisms still remains
Ethical Approval
largely unknown . However several hypotheses have
10
been postulated. One of such hypothesis is the neuro-
Ethical approval to carry out the research was obtained
transmitter pathophysiology of Autism.
Increasing
from Lagos University Teaching Hospital (LUTH)
number of literature have implicated elevated levels of
Health Research and Ethics Committee (ADM/DCST/
neurotransmitters and related amino acids in autistic
HREC/954), Idi-Araba, Lagos and the Ethics Review
individuals compared to age and sex-matched controls
Committee,
Federal
Neuro
Psychiatric
Hospital
11,12,13,14, 15
. This is often correlated with dysfunctional
(FNPHY/ERC/13/088), Yaba, Lagos.
neuronal connectivity (hyper and hypo connectivity) in
regions of the brain involved in cognitive and social
Experimental Model
Enrolment criteria for control
functions
16,17,18,19
.Glutamate and Gamma Aminobutyric
subjects
Acid (GABA) are the main excitatory and inhibitory
Diagnosis of Autism
age: ≥3 years
neurotransmitters in the human brain and both have im-
portant roles during early development of the nervous
age: ≥3 years
Absence of neurological disorder
system and during synapses . At the developing stage,
20
signed informed consent
GABA acts as an excitatory neurotransmitter, modulat-
ing neuronal migration and a wide range of functions
Recruitment and Sample Collection
that lead to the correct formation of neuronal circuits
while in the mature adult brain its function is mainly
Five millilitres (5ml) of whole blood was collected into
inhibitory, generating synchronous rhythms of cortical
EDTA bottles via venipuncture from affected individu-
assemblies (adequate synapses), allowing for time-
als after written informed consents were obtained from
precise communication between neurons and cerebral
their parents and/or guardians. Collected samples were
regions/centers
21,22
. This is of particular importance as
transported in cooling bags from site of collection to the
Autism has been shown to be a disorder of impaired
laboratory where they were centrifuged for plasma sepa-
functional integration (modulated by neuronal connec-
ration. The plasma samples were decanted, aliquoted
and stored at -24 C until analysis. Participants were re-
0
tivity), a factor responsible for characteristic features
observed
21
such as hyperactivity and epileptic seizures.
cruited from the Neuro-paediatric clinic of University of
Cumulative evidence indicates that dysfunctional excita-
Lagos Teaching Hospital (LUTH) and the Child and
tory and inhibitory synaptic activities underlie several of
Adolescent Unit of the Federal Neuro-Psychiatric Hos-
the characteristics of Autism .
23
pital, Oshodi Annex Lagos. While the control subjects
were recruited from the Medical Centre, University of
Moreover, the GABAergic and glutamatergic neuronal
Lagos and the Pathology Unit, Orthopaedic Hospital,
system, highly implicated in genetic studies of Autism
Igbobi, Lagos and the University of Lagos Health Cen-
as a result of mutations in genes coding for GABA and
tre, Akoka, Lagos. All caseshad been previously diag-
nosed with ASD using the DSM-1V .
2
glutamate receptors, have been postulated to be respon-
sible for the observed differential neurotransmitter levels
in autistic individuals . Also, given that the GABAergic
12
Plasma Quantification of Amino Acids (AA)
and glutamatergicneuronal pathway appear to be conver-
Plasma samples were deproteinized as described by
12,25
gent nodes of genetics, epigenetics and probably envi-
.
ronmental factors that may cause Autism, the GABA
A 300΅l aliquot of plasma was homogenized with 450΅l
and glutamate receptors may form important targets for
of 5% sulfosalicylic acid and centrifuged at 12000rpm in
4 C for 10 minutes. The resulting supernatant was de-
0
pharmacological interventions. Studies on amino acid
(AA) profile/levels of autistic individuals have become
canted, filter sterilized through 0.22΅m membrane filter
common
12,24
. Since AA exert various influences on one
(Millipore) into 1.5 ml amber vial (Agilent PN 5182-
another in the process of synthesis/degradation, any
0716). Amino acid concentrations were measured using
changes in plasma neurotransmitter levels could be at-
an automatic HPLC system (1200 series: Agilent Tech-
tributed to changes in other amino acids . This is be-
12
nology Inc., USA) with a reversed-phase (RP-HPLC)
cause most essential amino acids are mostly precursors
method and pre-column derivatization with Ortho-
Phthalaldehyde (OPA: Agilent-PN5061-3337)
26,27
for the formation/production of some neurotransmitters.
. Af-
For instance, glutamine which is an essential AA, is the
ter derivatization, 20΅l of derivatized mixture was in-
precursor for glutamate and tryptophanis the precursor
jected and run through a C-18 column (Agilent ZOR-
for serotonin. To the best of the authors’ knowledge
BAX Eclipse Plus C-18 4.6Χ250, 5΅m) for 40 minutes
there are no studies of the amino acid and neurotransmit-
at a flow rate of 1.5 ml/min. Eluted amino acids were
ter levels of individuals with Autism in Nigeria. The
detected and quantified via fluorescence detector (FLD:
present study is a comparative description/analysis of
Agilent G1321C). All statistical analysis were done
131
using IBM SPSS version 20. All results are presented as
GABA ( t = 6.601, df = 24.593, p )= 0.000001), trypto-
mean±SD. The sample (blood and plasma) preparation
phan (TRP: t = 3.568, df = 16.472, p = 0.002) and cys-
and storage was done in the Post Graduate Laboratotory
teine (CYS: S t = 4.000, df = 13.762, p = 0.001) (Table 1;
of th department of Cell Biology and Genetics, Univer-
Fig. 1) from a two-tailed independent t-test, unequal
sity of Lagos, while the HPLC analysis of the depro-
variances assumed. Further analysis with nonparametric
teinized plasma samples was done in the Central Re-
Mann-Whitney U test revealed a nonsignificant differ-
search Laboratory (CRL) of the University of Lagos.
ence of mean for glutamate ( t = 2.000, Exact p = 0.052)
but significant for the Kruskal-Wallis test ( t = 3.868, df
= 1, p = 0.049) using an asymptotic 2-sided test (Fig 1a).
As expected the plasma levels of glutamate, GABA and
Results
glutamine were significantly higher in autistic individu-
als than controls with mean differences, 9.838΅g/ml
A total of 31plasma samples (18 Autism cases and 13
(95% confidence interval [CI]: 6.008 to 13.667΅g/ml),
age-sex matched controls) were analyzed with mean
42.596΅g/ml (95% CI: 29.294 to 55.898΅g/ml) and
ages 8.44±4.87 for cases and 8.15±4.88 for controls. No
133.562΅g/ml (95% CI: 99.392 to 167.732΅g/ml) with
significant difference ( t = 0.164, df = 29, p = 0.871) was
Cohen’s d effect sizes 2.81, 2.66 and 4.33 respectively.
observed in ages of both groups. The result also indi-
A spearman ranked correlation analysis revealed strong
cated no significant intergroup difference in the mean
positive correlations between the concentrations of the
concentration levels of individual amino acid analyzed
amino acids with significant group difference- GLN-
with the exception of glutamate (GLU: t = 5.472, df =
GLU (Spearman’s rho 0.529 p <0.05), GLN-GABA
2.324, p = 0.000063), glutamine (GLN: t = 8.342, df =
(0.796 p <0.01), GLN-TRP (0.654 p <0.01), GLN-CYS
14.780, p = 0.000001) ,
(0.654 p <0.01), GABA-TRP (0.662 p <0.01) and be-
tween GABA-CYS (0.838 p <0.01
Table 1 : Plasma Levels of Neurotransmitters and Amino Acids in Autistic Individuals and control subjects
Plasma Amino Acid Concentration(΅g/ml)
Independent t-test
Amino Acids/
Neurotransmitters Autism Case Control
t
p-value
Aspartate
29.446±1.958e-2
20.793±2.854e-2
0.506
0.657
Glutamate
10.743±7.176e-3
0.905±1.626e-4
5.472
0.000063***
Serine
24.958±2.091e-2
28.805±1.754e-2
-0.382
0.718
Glutamine
141.332±5.461e-2
7.769±1.872e-2
8.342
0.000001***
Histidine
40.143±6.838e-2
0.000±0.000
1.017
0.416
Glycine
5.700±8.061e-3
0.000±0.000
1.000
0.500
Threonine
4.859±3.069e-3
30.174±4.084e-2
-1.38
0.238
-
a
Arginine
0.000±0.000
0.000±0.000
-
Alanine
3.699±6.485e-3
3.030±3.954e-3
0.233
0.825
GABA
62.205±2.152e-2
19.609±1.234e-2
6.601
0.000001***
Tyrosine
8.540±1.619e-2
3.996±3.479e-3
0.923
0.373
Cystine
23.205±1.009e-2
11.603±2.741e-3
4.000
0.001**
Valine
1.777±3.777e-3
6.493±7.599e-3
-1.184
0.308
Methionine
9.625±2.178e-3
9.144±4.049e-3
0.252
0.811
Tryptophan
29.190±1.919e-2
5.158±1.194e-2
3.568
0.002**
-
a
Phenylalanine
0.000
19.400
-
Isoleucine
10.670
2.840±1.697e-4
-
-
a
Leucine
15.950
0.000±0.000
-
-
Lysine
63.020
2.955±2.980e-3
-
-
Proline
3.385±9.661e-3
0.113±3.926e-4
1.435
0.169
Values are expressed as mean±SD; ** p <0.05 *** p <0.00001
a. t cannot be computed because the standard deviation of at least one of the groups is 0.
a)
Diagnosis
b)
Diagnosis
132
c)
Diagnosis
d)
Diagnosis
been implicated in Alzheimer’s disease, epilepsy and
15
most neurophysiological disorders
. Apart from the 3
main amino acids involved in the excitatory and inhibi-
tory connectivity in the brain, significant elevated levels
was also observed in the amino acid – Tryptophan
(TRP). This is of no surprise as TRP is the main precur-
sor for serotonin synthesis, the serotonergic neuronal
system have also been implicated in Autism pathogene-
sis .
31
e)
Diagnosis
Fig 1 : Mean plasma concentrations of Neurotransmitters
and Amino Acids with statistically significant levels
Conclusions and Recommendations
between autistic individuals and typical age and sex
matched controls. a) Glutamate, b) Glutamine, c)
The results reported from the present study supports the
GABA, d) Cystine and e) Tryptophan.
neurotransmitter pathophysiology of Autism, thus it is
proposed that the neurotransmitter/amino acids plasma
levels could serve as biomarkers in the aetiology of Au-
tism. However, it should be noted that several other con-
Discussion
ditions such as comorbidity (other developmental disor-
ders), pharmacological conditions (use of psychoactive
Observed differential plasma levels of the neurotrans-
medications) and gender could also influence plasma
mitters and related other amino acids amongst autistic
neurotransmitter and amino acid levels. T he African
individuals against controls conform with the neuro-
population is still greatly understudied and underrepre-
transmitter pathophysiology of Autism and other re-
sented in Autism research, thus it is recommended that
ported studies
12,13,14,15,24
. Glutamine (GLN), glutamate
more studies and awareness of Autism research be en-
(GLU) and γ -amino butyric acid (GABA) are essential
couraged in Nigeria and Africa.
amino
acids
for
brain
metabolism
and
func-
tion .Glutamine had the highest observed mean concen-
28
Conflict of interest: None
trations of all the analysed amino acids. This is similar
Funding: None
to the findings reported where glutamine concentration
12
was higher than 24 out of the 25 amino acids analyzed.
This trend is not uncommon as glutamine is believed to
have the highest plasma level of all essential amino ac-
Acknowledgment s
ids. The strong correlation observed between GLN-
GLU, GLN-GABA levels, could be due to the glutamine
First, we would like to acknowledge and appreciate the
-Glutamate (GABA) cycle . In order to maintain a
29
families that participated in this study, also the Univer-
rhythm of neuronal synapses/connectivity, the GABA-
sity of Lagos Central Research Committee (CRC) for
glutamate levels in the brain are strictly monitored via
funding this study (CRC NO.: 2014/01).
the glutamine-glutamate cycle through uptake, reuptake
mechanisms between postsynaptic, astrocytes and pre-
synaptic neuronal cells respectively
20,23
.
It is believed that glutamate does not cross the blood
brain barrier (BBB) , this might account for it having
30
the least mean concentrations (10.743±7.176e-3) among
the variables with significant group difference in this
study. Also, its presence extracellularly is deemed to be
neurotoxic and its extracellular elevated levels have
133
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