Neuroscience & Medicine, 2013, 4, 271-276
Published Online December 2013 (
Open Access NM
Biochemical and Neurobiological Study of
Moroccan Autists
Hakim Harchaoui1, Fatima-Zahra Azzaoui1, Ahmed Ahami1, Samira Bou lbar o u d 2, Mohamed El Hioui1
1Equip of Clinic and Cognitive Neurosciences and Health, Laboratory of Biology and Health, Department of Biology, Faculty of
Science, IBN TOFAIL University, Kenitra, Morocco; 2Equip of Neuroendocrine Physiology, Faculty of Science, IBN TOFAIL Uni-
versity, Kenitra, Morocco.
Received August 7th, 2013; revised September 5th, 2013; accepted October 3rd, 2013
Copyright © 2013 Hakim Harchaoui et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Autism spectrum disorder (ASD) is a range of complex neurodevelopment disorders, characterized by social impair-
ments, communication difficulties, and restricted, repetitive, and stereotyped patterns of behavior. Its diagnosis is very
difficult and consists of only clinical criteria. The aim of this study is to confirm autism disorder, among three children
(2 girls and 1 boy), recruited in a social association of children in a precarious situation, located in Rabat, Morocco, and
to evaluate some biological and neurobiological parameters among these children. The confirmation of autistic status is
realized using Questionnaire (E2) and the evaluation of biological parameters (organic acids, urinary peptides, toxic
metals and essential elements) is done in urine and hair of these children. Obtained children’s scores of E2 Ques-
tionnaire, is found in the favor of autism. Moreover, abnormal levels of urinary organic acids, urinary peptides, toxic
and essential metals are observed.
Keywords: Autism; Neurobiology; Organic Acids; Toxic Metals; Essential Elements
1. Introduction
Autism, as defined by “The Diagnostic and Statistical
Manual of Mental Disorders”, 4th edition (DSM IV)
(2000), is a disorder of neural development characterized
by impaired social interaction and verbal and non-verbal
communication, and by restricted, repetitive or stereo-
typed behavior. The diagnostic criteria require that sym-
ptoms become apparent before a child is three years old.
In Morocco, very few studies are realized about autism
[1] which cannot provide the prevalence in the Kingdom.
However, the prevalence of this neurodevelopmental
disorder is increasing; in the USA, from 1998 to 2007,
the number of 6- to 21-year-old children receiving ser-
vices for an ASD (autism spectrum disorders) in public
special education programs increased from 54,064 to
258,305 [2]. In the recent CDC report, it is found that the
overall estimated prevalence of ASD is one in 88 chil-
dren during [3].
The etiology of autism is still discussed and some stu-
dies report that autism affects approximately three times
as many males as females. Taken together with studies
looking at families and twins, this suggests that autism
has a genetic component. It is likely that a number of
genes are involved. However, the exact mechanism by
which genes are implicated in autism is unclear [4].
Other findings suggest that some neuroanatomical im-
pairments in brain areas and/or brain chemistry changing
could be involved in this neuropathology [5,6].
Moreover, some studies have reported many nutria-
tional deficiencies in autism [7], and accumulations of
organic acids in urine can indicate metabolic dysfunction,
nutrient insufficiencies, or even microbial overgrowth [8].
The urinary organic acids test measures selected metabo-
lites which serve as important diagnostic indicators of
abnormal metabolism in the case of autistic children. The
measurement of organic acids in urine evaluates four
critical areas of metabolism: gastrointestinal function,
cellular energy and mitochondrial metabolism, neuro-
transmitter metabolism, and amino acid/organic acid bal-
ance as influenced by vitamin/mineral cofactors [9]. In the
urine of many autistic children, abnormal metabolites
such as opioid peptides and [10] abnormal levels of different
organic acids [11,12] were observed. Some researches
Biochemical and Neurobiological Study of Moroccan Autists
about this pathology implicated insufficient enzymatic
activity and increased gastrointestinal permeability, and
the absorption of toxic byproducts of incompletely digest-
ed proteins from dairy (casein) and cereals (gluten), [13,14].
Furthermore, environmental pollution could be a pos-
sible factor contributing to this disease [15]; many metals
could be implicated in autism since they typically disrupt
enzyme functions and cell signaling processes, generate
oxygen free radicals (ROS) and elicit autoimmune reac-
tions [16]. Metals involved in physiopathological proc-
esses include essential elements such as zinc (Zn), copper
(Cu), iron (Fe), calcium (Ca), manganese (Mn), nickel
(Ni) and silicon (Si), or toxic metals such as cadmium
(Cd), cobalt (Co), aluminium (Al), lead (Pb), mercury
(Hg) and arsenic (As) [17,18]. The laboratory analysis of
different biological matrices (urine, blood, hair) is very
useful in identifying this deficiency or toxicity and allow
for health professionals establishing possible strategies of
treatment. The aim of this study, in one hand is to con-
tribute to this field, and still virgin in Morocco, and in the
other hand, to confirm autistic diagnosis of studying
children and to measure some biological and neurobio-
logical parameters in hair and urine of these children.
2. Patients and Methods
2.1. Ethical Clearance and Informed Consent
This study was approved by the Internal Ethics Commit-
tee of Faculty of Science, Kenitra, Morocco and by the
GDRI of Neurosciences France-Morocco Ethics Com-
mittee. The recruitment of patients is based on the fol-
lowing criteria:
- Written consent of the children’s parents,
- Children physically healthy and not previously under-
gone chelation therapy, free of any medication, with-
out any casein-free and gluten-free diet and any medi-
cal issues.
2.2. Patients
Three children (2 girls and 1 boy) are involved in the
present study. They are aged between 15 to 18 years old.
The children are diagnosed for autism and they are in-
serted into an associative structure in Rabat, Morocco,
that cares for children suffering from neurobehavioral
Patient (A): Aya, female child aged 4 years is diag-
nosed with autism. Aya is the second of three sisters and
she is physically normal. She had frequent infections
breathing during the first months of life. She learned to
walk alone between 24 and 36 months. Between 2cd and
4th year, she had an irresistible urge to eat something. At
the age of 3 or 4 years, she is “going into a shell”; she
became so distant and lost in thought. Moreover, she was
indifferent to any affection and seems to be happier when
we do not take care of her. Before 5 years, she was able
to speak but not to answer. Her abnormal behavior has
been discovered between 7 and 12 months.
Patient (B): Salwa, female child of 18 years is diag-
nosed with autism. Salwa is the younger child of her
family, she is physically normal. Salwa had a difficulty
of sucking and diarrhea. She learned to walk alone be-
tween 8 and 12 months. Between 2cd and 4th year, she
suffers from PICA syndrome. At the age of 3 or 4 years,
she is “going into a shell”; she became so distant and lost
in thought. Moreover, she was indifferent to any affect-
tion and seems to be happier when we do not take care of
her. Before 5 years, she was able to speak but not to an-
swer. Her abnormal behavior has been discovered be-
tween 13 and 24 months.
Patient (C): Ismail 17 years old, male diagnosed with
autism. Ismail is the only child in his family, he is physi-
cally normal with an excellent health. He learned to walk
alone between 8 and 12 months. Between 2 and 4 years,
he sucks often metallic objects. A child at the age of 3 or
4 years was “locked in his shell” or so distant and lost in
thought. He is indifferent to any brand of affection and
seems being happier when you do not take care of him.
Before 5 years, he was able to speak but not to answer.
The abnormal behavior of the child has been discovered
between 2 and 3 years.
2.3. Methods
An E2 Questionnaire, including 79 amnestic items for the
diagnosis of autism, completed by parents, is used. It
covers the period from birth to age of 5 years. All items
are summed to obtain a total score. If an item is applica-
ble to the assessed individual, it is noted (+1), if not, it is
noted (1). The total score is the difference between the
presence of autism (+) and its absence (). A total score
of “20” is in the favor of autism [19].
The urine and hair samples were collected from the
patients for the following biological tests: Tests of uri-
nary organic acids including neurotransmitters, fatty ac-
ids, amino acids, bacterium and fungal metabolites. Test
of urinary peptides including casamorphin and gluten-
morphin. Test of toxic metals (Ur, Al, As, Pb, Hg, Ag)
and essential elements (Ca, Mg, Na, k, Cu, Cr, Mo, Se,
Be, Ru, Zn, Va, Li) in the hair samples. All the analysis
is realized by Great plain laboratory, Inc (USA).
The urinary organic acids and urinary peptides results
are expressed as ratios to the urinary creatinin concentra-
tion in mmol/mmolcreatinine. Certified hair standards
and in-house standards were used as part of the labora-
tory quality control and for the validation of results. Re-
sults are expressed as micrograms per gram, equivalent
to parts per million.
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Biochemical and Neurobiological Study of Moroccan Autists
Open Access NM
3. Results
3.1. Evaluation of Children’s Autistic Status by
E2 Questionnaire
The scores of patients A, B and C, in the E2 Question-
naire are respectively 36, 28 and 26 (Table 1).
3.2. Organic Acids and Peptides in
Children’s Urine
The patient A has a deficit in pyroglutamic acid (17.69
mmol/mmolcreatinine) and in ascorbic acid (1.09 mmol/
mmolcreatinine). However, she has an excess of vanyl-
mandelic acid (7.80 mmol/mmolcreatinine), 3-oxoglu-
taric acid (0.67 mmol/mmolcreatinine), arabinose (147.57
mmol/mmolcreatinine), Cytramalyc acid (2.69 mmol/
mmolcreatinine), 4-hydroxyphenylacetic acid (150.73
mmol/mmolcreatinine) and in Oxalic acid (150.73 mmol/
mmolcreatinine), (Table 2).
The patient B has a deficit in pyroglutamic acid (15.48
mmol/mmolcreatinine) and an absence of ascorbic acid
(0.00 mmol/mmolcreatinine). However, she has an ex-
cess of Suberic acid (3.89 mmol/mmolcreatinine), 3-
oxoglutaric acid (0.84 mmol/mmolcreatinine), arabinose
(123.86 mmol/mmolcreatinine), Citric acid (855.71
mmol/mmolcreatinine) and in 2-Oxo-glutaric acid (3.08
mmol/mmolcreatinine), (Table 2).
The patient C has a deficit in pyroglutamic acid (19.37
mmol/mmolcreatinine) and an absence of ascorbic acid
(0.00 mmol/mmolcreatinine). However, he has an excess
in Methylmalonic acid (13.55 mmol/mmolcreatinine),
arabinose (696.55 mmol/mmolcreatinine), 4-hydroxy-
phenylacetic acid (54.07 mmol/mmolcreatinine) and in
2-Oxo-glutaric acid (144.57 mmol/mmolcreatinine), (Ta-
ble 2).
Moreover, an abnormal level in both Casomorphin
1.28 (P/C ratio) and Gliadorphin 1.09 (P/C ratio) is reg-
istered among patient A, and in only Gliadorphin 0.78
(P/C ratio) in patient B (Table 3).
3.3. Levels of Toxic Metals and Oligoelements in
Autistic Children’s Hair
Levels of some Toxics exceed widely the reference range;
Patient A has a high level of Al (6.6 µg/g) and low ex-
ceeding in As (0.082 µg/g) and Pb levels (1.1 µg/g). Pa-
tient B has the same level of Al than patient A, but he has
a higher level of Hg (1.2 µg/g) and Ag (0.2 µg/g). Patient
C has a very high level of Al (14 µg/g) and Ur (0.12
µg/g), (Table 4).
Levels of some oligoelements are less or more than the
normal range; Patient A suffer from a deficiency in Ca
(349 µg/g), Mg (18 µg/g), Cu (7.6 µg/g). However, the
levels of Na (410 µg/g), K (1200 µg/g), Cr (0.53 µg/g), B
(8.5 µg/g) and Ru (0.80 µg/g) exceed the normal ranges.
Patient B has no deficiency in these oligoelements, but
an excess in Na (290 µg/g), K (270 µg/g), B (2.8 µg/g),
Ru (0.16 µg/g), Zn (240 µg/g), Va (0.19 µg/g) and Li
(0.19 µg/g).
Table 1. The children’s scores in questionnaire E2.
Patient APatient BPatient C Normal Score
Total36 28 26 <20
Table 2. Levels of organic acids in the urine of autistic children (mmol/mmolcreatinine).
Organic acids Patient A Patient B Patient C Reference range
Amino acid metabolites Pyroglutamic acid 17.69 15.48 19.37 20 - 115
Fatty acid metabolites Subericacid 1.81 3.89 1.88 0 - 2
Methylmalonic acid 1.81 1.64 13.55 0 - 5
Various organic acids
Ascorbic acid 1.09 0.00 0.00 10-200
Homovanilic acid 2.62 2.14 4.98 0-10
Vanylmandelic acid 7.80 3,69 5.82 1 - 6.6
3-oxoglutaric acid 0.67 0.84 0 0-0.5
Arabinose 147.57 123.86 696.55 0 - 47 Yeast/fungal
Cytramalic acid 2.69 1.53 1.53 0 - 2
Bacterium 4-hydroxyphenylacetic acid 150.73 4.89 54.07 0 - 50
Citric acid - 855.71 446.77 180 - 560
Krebs Cycle
2-Oxo-glutaric acid 42.12 3.08 3.08 15 - 200
Various Oxalicacid 150.73 30.27 144.57 0 - 100
Biochemical and Neurobiological Study of Moroccan Autists
Table 3. Levels of urinary peptides in autistic children (Re-
lative (P/C)* Ratio report.
Peptides Patient A Patient B Patient C
Casomorphin (Milk) 1.28 0.54 0.28 <0.56
Gliadorphin (Wheat) 1.09 0.78 0.09 <0.58
*P/C ratio: peptides (ng/ml)/Creatinin (mg/dl).
Table 4. Concentrations of toxic metals (µg/g).
Toxic metals Patient A Patient B Patient C Reference range
Uranium (Ur) 0.003 0.052 0.12 <0.06
Aluminum (Al) 6.6 6.6 14 < 0.8
Arsenic (As) 0.082 0.043 0.03 <0.060
Lead (Pb) 1.1 0.92 0.89 <1
Mercury (Hg) 0.13 1.2 0.18 <0.40
Silver (Ag) 0.04 0.20 0.06 <0.16
Patient C is deficient in Mo (0.024 µg/g) and Se (0.89
µg/g) only and he has an excess in Ca (2260 µg/g), Mg
(250µg/g), K (51 µg/g), Cu (40 µg/g), Zn (300 µg/g), Va
(0.13µg/g), Li (0.56 µg/g), (Table 5).
4. Discussion
The pathogenesis of autism remains elusive, autism is
considered a multifactorial disorder that is influenced by
genetic, environmental and immunological factors. Rim-
land questionnaire (E2 Questionnaire) is used in this
study to confirm the autistic status of patients. The ob-
tained scores in this questionnaire show that the patients
are autistic. [20] had used this questionnaire, also, to dis-
tinguish the autistic from non-autistic studied children.
The analysis of organic acids in human body fluids
such as plasma, urine and cerebro-spinal fluid is very
important. This is necessary for the diagnosis of inborn
errors of metabolism of amino and organic acids and for
individual patient’s diagnosis of nutrient requirements
[8]. Organic acids are important metabolites in major
metabolic pathways such as the Krebs cycle or the pen-
tose phosphate pathway. The accumulation of organic
acids in biological fluids, especially in urine, can also pro-
vide useful information for an early diagnosis of meta-
bolic disorders and neurological diseases [8,11,12,21].
The current finding shows a high to very high levels in
some organic acids (suberic, methylmalonic, 3-oxoglu-
taric, Cytramalic, 4-hydroxyphenylacetic, Oxalic acids
and arabinose) among at least one of the children. [22]
found a marked increase in the analogs of Krebs cycle
metabolites in the urine of two brothers with autistic fea-
tures. These metabolites included citramalic, tartaric (3-
Table 5. Concentrations of essential elements (µg/g).
elements Patient APatient B Patient C Reference
Calcium (Ca) 349 919 2260 350 - 1000
Magnesium (Mg)18 120 250 35 - 120
Sodium (Na) 410 290 70 12 - 90
Potassium (K) 1200 270 51 7 - 36
Copper (Cu) 7.6 15 40 12 - 35
Chrome (Cr) 0.53 0.35 0.35 0.20 - 0.40
Molybdenum (Mo)0.043 0.033 0.024 0.029 - 0.06
Selenium (Se) 1.0 1.0 0.89 0.95 - 1.7
Bore (B) 8.5 2.8 1.4 0.30 - 1.5
Rubidium (Ru) 0.80 0.16 0.038 0.01 - 0.06
Zinc (Zn) 130 240 300 130 - 220
Vanadium (Va) 0.053 0.19 0.13 0.02 - 0.07
Lithium (Li) 0.030 0.19 0.56 0.01 - 0.02
OH-malic), and 3-oxoglutaric acids and compounds ten-
tatively identified as a citric acid analog and partially
identified as a phenylcarboxylic acid and in arabinose.
Moreover, [23] detected abnormal amino acid metabo-
lism, increased oxidative stress, and altered gut micro-
biome in autistic children.
Quantification of urinary homovanillic acid (HVA)
and vanillylmandelic acid (VMA) can be a very impor-
tant tool in the study of disorders of dopamine and sero-
tonin metabolism among autistic children. This study
demonstrates a higher level of VMA among one patient.
[11,12] found, recently, that The levels of HVA and
VMA were higher in the urine of autistic children com-
pared to control ones.
Furthermore, the current results show a high level of
casein and gluten in patients. Several research suggests
gluten-free and casein-free diets for autistic children [24-
26]. A recent investigations, prove the efficiency of free-
casein and free-gluten diet on improvement of autistic
children’s health status [27,28].
Besides of metabolic factors’ involvement in autism
syndrome, impairments in metallic trace element could
have an effect also; the increase in autism incidence in
the last two decades has led scientists to propose the en-
vironmental pollution as a possible factor contributing to
this disease [15]. Heavy metals are environmental con-
taminants whose levels have been dramatically increased
by the modern industrialization. Many metals could be
implicated in autism since they typically disrupt enzyme
functions and cell signaling processes, generate oxygen
free radicals (ROS) and elicit autoimmune reactions [16].
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Biochemical and Neurobiological Study of Moroccan Autists 275
A number of studies have implicated exposure to
various metals in the development of autism [17,18,29]
but, to date, the mechanisms by which metals exposure
may cause autism have been difficult to ascertain.
All these findings are consistent with our results that
find very high levels of Ur, Al, As, Pb, Hg and Ag are
registered in at least one of the patients.
In the present study, impairment in the level of the es-
sential element is observed; high levels of Na, K, Cr, B,
Ru, Zn,Va, Li and low level of Mo and Se are found in at
least of two patients. However, concerning Ca and Cu,
the results show high levels in some patients and low
among others. Previous studies found higher levels of Zn
in both hair and teeth [18] of autistic subjects. Others [30]
found a significant elevation in the concentration of Cu,
Pb, and Hg and significant decrease in the concentration
of Mg and Se in the hair and nail samples of autistic sub-
jects. These variations could be well correlated with de-
gree of autism severity.
5. Conclusions
The studied children are autistic and suffer from impair-
ments in organic acids, oligoelements and high levels of
toxic metals.
Rimland questionnaire and biological study, using
analysis of biological matrix, can be effective for con-
firming autistic status of patients and detecting abnormal
metabolic and nutritional conditions in autistic children.
In future investigations, these analyses could help to es-
tablish a medical protocol for treatment or at least im-
provement of health status of these children.
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