Open Access
Austin Journal of Autism & Related Disabilities
Rapid Communication
Identification of New Putative Biomarkers for Autism in
Urine by Mass Spectrometry
NI Silva; DOC Mariano; AA Cunha; I Lebrun*
Laboratory of Biochemistry and Biophysics Instituto
Butantan- city of São Paulo, São Paulo, Brazil
*Corresponding author: Ivo Lebrun
Laboratory of Biochemistry and Biophysics Instituto
Butantan- city of São Paulo, São Paulo, Brazil.
Email: ivo.lebrun@butantan.gov.br
Received: June 12, 2023
Accepted: July 10, 2023
Published: July 17, 2023
Introduction
Autism Spectrum Disorder (ASD) is a clinical syndrome cha-
racterized by neurodevelopmental changes that impair social
interaction and communication and result in repetitive or res-
tricted patterns of interest [1]. Studies have identified distinct
metabolic profiles in children with ASD, with an estimated 5%
to 30% suffering from some type of metabolic disorder [2].
Therefore, the aim of this work was to develop a pilot study
of a non-targeted metabolomics and proteomics approach to
identify potential candidate metabolic biomarkers in the urine
of children with ASD.
Material and Methods
In total, 44 children were recruited. Of these, 22 boys were
diagnosed with ASD and 22 boys with typical development. Be-
tween the two groups of subjects, there was no statistical diffe-
rence between the ages. The group of boys with ASD in the age
Austin Journal of Autism & Related Disabilities Citation: Silva NI, Mariano DOC, Cunha SS, Lebrun I. Identification of New Putative Bioma-
Volume 9, Issue 1 (2023) rkers for Autism in Urine by Mass Spectrometry. Austin J Autism & Relat Disabil. 2023; 9(1):
www.austinpublishinggroup.com 1064.
Silva NI © All rights are reserved
Abstract
Introduction: Autism Spectrum Disorder (ASD) is characteri-
zed by conditions affecting social interaction, communication and
behavior, and sensory sensitivity. Studies have reported an associa-
tion of ASD with various metabolic alterations. The aim of this study
was to develop a pilot study of a non-targeted metabolomics and
proteomics approach to identify candidate metabolic biomarkers in
urine from children with ASD.
Material and Methods: Prospective cross-sectional study with
case-control design. Cases were male children (n=22) with ASD,
aged 3-10 years, and controls were neurotypical children (n=22)
matched for sex and age. Metabolomic analysis was performed
by mass spectrometry with laser desorption ionization and time-
of-flight analysis (MALDI-TOF). Proteomic analysis was performed
by Liquid Chromatography coupled to Mass Spectrometry (LC-MS).
Results: Metabolomic analysis identified the 655 mass spectrum
with high frequency in the urine samples of both groups, classified
as coproporphyrins II and IV. The 1911 Da spectrum showed a hi-
gher frequency in urine samples from children with ASD and was
classified as GT2 ganglioside (d18:0/14:0). Proteomic analysis also
detected the compound with molecular weight of 1911 and its frag-
ments, but they were classified as uromodulins.
Conclusion: The non-targeted metabolomic and proteomic ap-
proach allowed the identification of promising candidate bioma-
rkers for ASD, although the results of the analyses differed with
respect to the nature of the identified compounds.
Keywords: Autism; Biomarkers; Urine; Coproporphyrins; Gan-
gliosides; Uromodulin
range of 3 to 10 years had a mean age of 6.6 years (±1.62). The
control group with equivalent age range had a mean age of 7.1
years (±1.93).
Ethical Aspects
All procedures were carried out in accordance with the Na-
tional Health Council Resolution No. 466 (2012). The present
project was presented to the Research Ethics Committee of
the Faculty of Medicine of the University of São Paulo, in the
meeting of 12/14/2011, and approved with protocol number
449/11. Written informed consent was obtained from the pa-
rent or legal guardian. The screening of children with ASD was
carried out at the Municipal Specialization Center for Autism, lo-
cated in the city of Limeira - SP, and at the Association of Parents
and Friends of Autism of Baixa Mogiana - Fonte Viva, located in
the city of Mogi - Guaçu - SP. The participating children were
Silva NI
Figure 1: Possible mechanism of action of the identified compoun-
ds in urine samples of indiduals with autismo: coproporphyrin II,
coproporphyrin IV, ganglioside GT2 (d18:0/14:0), uromodulin and
its fragments.
first diagnosed by psychiatrists and pediatricians and referred
to these institutions. There, the diagnosis was confirmed by a
multidisciplinary team according to the criteria of the ICD 10.
The control group was constituted by boys with typical de-
velopment. The enrollment of participants in the control group
was carried out in five schools (Children's Center - Lucinda Tank
Kühl, EMEIEF- Pastor Ismael Pereira do Lago, EMEIEF- Maria
Aparecida Machado Julianelli, EMEIEF- Prof. Noedir Tadeu San-
tini, EE Leontina Silva Busch) and one religious institution (Dis-
pensário Madre Teresa de Calcutá) in the city of Limeira - SP.
Inclusion criteria for children with ASD were: male sex, age
between 3 and 10 years, and being treated in the participating
centers. The exclusion criteria were: not participating in the tre-
atment at the selected institutions, being a supporter of any
nutritional intervention, having celiac disease and allergy or
intolerance to cow's milk, and having a diagnosis of a disease
that affects the metabolism and excretion of proteins and ami-
no acids, such as liver and kidney disease. The inclusion criteria
for neurotypical children were: typical development, be the sex
male and possess equivalent age of the child with ASD in the
survey. The exclusion criteria were: being adept at any nutritio-
nal intervention, having neuropsychiatric disease, having celiac
disease and cow's milk allergy or intolerance, and having a diag-
nosed disease that compromises the metabolism and excretion
of proteins such as liver and kidney disease.
Urine Collection and Preparation
The first urine of the day was collected and stored in a ste-
rile universal collector. The samples were stored at - 80°C. The
samples were slowly thawed at a temperature of 4°C, homo-
genized (Centrifuge Sorva ll Super Speed RC2-B, Thermo Fisher
Scientific, Waltham, MA, USA), filtered through a 45μm PVDF
filter (Merck Millipore S/A ®, Cotia, SP, Brazil) and fractionated
in volumes of 1.5mL. The post new fractionation, were stored at
a temperature of - 80°C until the moment to the experimente.
Metabolomic Analysis by Laser Desorption Ionization and
Time of Flight (MALDI-TOF) Mass Spectrometry
A 2mL aliquot of urine from the control groups and patients
with ASD was lyophilized. It was then resuspended in 500μL of
0.1% TFA solution, desalted, and concentrated using C18 Zip Tip
tips. At the end of this step, a final volume of 7μL was obtained
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for each sample. Immediately thereafter, 2μL of each sample
was co-crystallized with 2μL of α-Cyano-4-Hydroxycinnamic
acid (CHCA) (supersaturated solution diluted in 50% ACN/0.1%
TFA matrix) and applied to the sampler. Samples were analyzed
in positive linear mode over a range of 400-5000 mass/charge
(m/z) with the laser power fixed at 61%. Prior to data acquisi-
tion, the instrument was calibrated using the MALDI TOFMIX
TM kit (LaserBio Labs®). After determining the most relevant
spectra, a search was performed in the human metabolite data-
bases HMDB (The Human Metabolome Database) and METLIN
to identify the possible compounds of the most relevant spec-
tra.
Proteomics Analysis by Liquid Chromatography-Mass Spec-
trometry (LC-MS)
Samples were processed according to the protocol used for
mass spectrometry. Trypsin digestion materials were analyzed
by liquid chromatography coupled to mass spectrometry (LC-
MS) using a binary UFLC system coupled to an electrospray ion
trap time of flight (ESI-IT-TOF) mass spectrometer. For LC-MS
analyses, samples were resuspended in water/0.1% acetic acid
and analyzed on a C18 column (Discovery C18, 5μm, 50mmx
2.1mm) with (A) acetic acid/water (0.1% v/v, 99.9% v/v) and
(B) acetic acid/ACN/water (0.1% v/v, 90% v/v, 99.9% v/v) as sol-
vents. Using a constant flow rate of 0.2mL/min, the gradient was
varied from 0 to 40% solvent B for 35 min at 37°C and monito-
red at 214nm using a Shimadzu SPD-M20A PDA detector. After
the chromatographic step, mass spectrometric analyses were
performed according to the following parameters: the interfa-
ce voltage used was 4.5kV and the detector voltage was 1.8kV
at a temperature of 200°C; fragmentation was performed with
argon collision gas at 50% energy; MS spectra were acquired in
positive mode and collected in the range of 350-1400m/z; ions
between 390-1400 m/z were automatically selected for frag-
mentation; MS/MS spectra were collected in the range of 50
- 1950 m/z. The fragmentation pattern for each sample was pro-
cessed using the Peaks Studio V7 program [3], and for “de novo”
sequencing and proteomic and sequence comparison analyses,
searches were performed against the SwissProt database (taxo-
nomy: Homo sapiens - human) (taxon identifier: 9606).
Statistical Analysis
Descriptive statistical analysis was the method of choice for
the presentation of the results.
Table 1: Result of metabolomic analysis by laser desorption ionization
and time-of-flight mass spectrometry (MALDI-TOF). Compounds iden-
tified with significant frequency in databases from molecular weight
spectra.
Frequency
Molecular Mass Chil-
Compound HMDB ID Neuro-
formula (Da) dren
with
typical
children
ASD
Copropor-
HMDB0000611 C36H38N4O8 655
phyrin II
Copropor-
HMDB0000644 C36H38N4O8 655 68% 77%
phyrin IV
C86H-
Ganglioside HMDB0012023 1911 95% 14%
150N4O42
GT2
(d18:0/14:0)
Austin J Autism & Relat Disabil 9(1): id1064 (2023) - Page - 02

Table 2: Compounds that were identified from the proteomic analysis
of urine samples from the study participants.
Frequency
Compound Amino acid sequence Mass (Da) Children Neurotypical
with ASD children
Uromodulin VIDQSRVLNLGPITR 1679.96 18% 0%
Uromodulin SVIDQSRVLNLGPITR 1767 14% 0%
Uromodulin SGSVIDQSRVLNLGPITR 1911.05 77% 14%
Results
Metabolomic Analysis by Laser Desorption Ionization and
Time of Flight (MALDI-TOF) Mass Spectrometry
The analysis identified in the urine sample from both groups
observed significant frequency of the 655 Da mass spectrum.
The 1911 Da mass spectrum identified showed a higher fre-
quency in the urine samples from children with ASD. The com-
pounds were classified according to the Human Metabolome
Database (HMDB) [4]. A summary of the results is given in table
1.
Proteomics Analysis by Liquid Chromatography-Mass Spec-
trometry (LC-MS)
The proteomic analysis also detected the spectrum of 1911
Da. In this analysis it was classified as uromodulin glycoprotein.
Different fragments of uromodulin were also identified in urine
samples from children with ASD. The results are summarized in
Table 2.
Discussion
The spectrum of molecular weight 655 Da, present at high
frequency in the urine samples of both groups, was identified
as coproporphyrins II and IV. Coproporphyrins are end products
of the spontaneous oxidation of the methylene bridges of co-
proporphyrinogen resulting from heme synthesis and are excre-
ted in both feces and urine [5]. Heavy metals can inhibit the
enzymes uroporphyrin decarboxylase and coproporphyrinogen
oxidase [6,7]. Studies have associated elevated blood lead and
mercury concentrations with increased urinary excretion of co-
proporphyrins in children with ASD and neurotypical children
[8,9]. In this regard, Woods et al. (2010), Kern et al. (2011b),
and Nataf et al. (2006) observed higher concentrations of por-
phyrins in the urine of individuals with ASD compared to neu-
rotypical individuals [10-12]. Kern et al. (2011a) examined the
concentration of porphyrins in urine samples from neurotypical
children. The authors concluded that the increased excretion
of porphyrins may be related to the environment to which the
children are exposed. In environments where heavy metal ex-
posure is higher, the prevalence of elevated porphyrin levels in
children's urine is high [8].
The 1911 Da spectrum most commonly found in urine sam-
ples from children with autism was identified as ganglioside
GT2 (d18:0/14:0). Glycosphingolipids are a class of compoun-
ds that contain a hydrophobic ceramide or sphingoid lipid tail,
usually anchored to the outer leaflet of the plasma membra-
ne [4,13]. They are involved in neuronal cell maintenance and
repair, memory formation, and synaptic transmission [14,15].
Information on the ganglioside GT2 (d18:0/14:0) is particularly
scarce. Based on an animal model, Iber et al. (1992) proposed
that gangliosides of the GT2 series are formed in the Golgi com-
plex of the liver [16]. It is a component of the cellular plasma
membrane that modulates cellular signal transduction events.
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In a rodent study, high levels of GT2 gangliosides were observed
during early periods of brain development [17]. The mechanism
of action of the ganglioside family in the etiology of ASD is not
well understood. Evidence links ganglioside deficiency to defi-
cits in the myelination process. Another possible mechanism is
that gangliosides are also known to regulate the function of in-
sulin receptors, and dysregulation of these receptors has been
shown to be associated with an increased incidence of ASD [18].
According to the proteomic analysis, the 1911 Da spectrum
and the other compounds correspond to the uromodulin glyco-
protein and its fragments. This compound is known as Tamm-
Horsfall protein. It is a glycoprotein expressed exclusively in the
kidney by epithelial cells lining the Thick Ascending Limb (TAL)
of the loop of Henle. The biological function of uromodulin is
complex and only partially understood. Alterations in the ex-
cretion of this compound or its fragments are associated with
physiological conditions such as renal disease, cardiovascular
disease, and renal transplantation [19,20].
The higher frequency of different uromodulin fragments
(1911 Da spectrum) in urine samples from children with ASD
may be related to the association between pediatric kidney di-
sease and neurodevelopmental disorders [21]. Children with
ASD have high rates of bladder and bowel dysfunction, noctur-
nal enuresis, and daytime urinary incontinence. They often take
longer to achieve urinary continence and have urinary symp-
toms of urgency and delayed urination [22]. In a recent study,
Mioto et al. (2019) found a prevalence of chronic kidney dise-
ase of 25.4% in a population of adults with ASD and intellectu-
al disability (mean age 42.9 years) [23]. Rosen, Yoshida, Croen
(2007) observed in a case-control study a higher frequency of
diagnosis of genitourinary infections in the first two years of life
in children with ASD compared to neurotypical children [24].
Ramsey et al (2013) reported elevated uromodulin concentra-
tions in plasma samples from children with ASD compared to
plasma samples from neurotypical siblings [25]. Figure 1 illus-
trates the potential mechanisms of action of the identified com-
pounds in individuals with autism.
Conclusion
The non-targeted metabolomics and proteomics approach
allowed the identification of promising candidate biomarkers
for ASD. Although the results of the analyses differed with res-
pect to the nature of the compounds identified, the possibility
of the existence of all the compounds identified in the urine
samples cannot be excluded.
Endnotes
This article is part of the Doctoral Thesis entitled "Identifica-
tion of Candidates for Urinary Biomarkers for Autism Spectrum
Disorder", submitted to the Postgraduate Program in Sciences
of the Disease Control Coordination of the Ministry of Health of
the State of São Paulo, to obtain the title of Doctor of Science.
Author Dr. N. I. S. Advisor Dr. I. L.
Author Statements
Acknowledgments
We thank all the institutions that allowed us to recruit volun-
teers. We also thank the parents of children with ASD and neu-
rotypical children who agreed to their children's participation in
the to participate in the research project.
PhD. Carlos Alberto Labate and Dra. Thaís Regiani Cataldi,
Austin J Autism & Relat Disabil 9(1): id1064 (2023) - Page - 03

Laboratory of Plant Functional Genetics, Department of Genet-
ics, Escola Superior de Agricultura “Luiz de Queiroz”, University
of São Paulo. They participated in the discussion of data related
to the research results.
Author Contributions
Author contributions Coordinator: I. L.; Conception and de-
sign: N. I.S., D.O.C.M., A.A.C., I. L.; Collection and assembly of
data: N.I.S., D.O.C.M., A.A.C., I. L.; Data analysis and interpreta-
tion: N. I.S, D.O.C.M., A.A.C., I. L.; Manuscript writing: N.I.S., I.L.
Final approval of manuscript: All authors.
Funding
This work was supported by Fundação Butantan.
Data Availability
The datasets generated during and analysed during the cur-
rent study are available in the Mendeley Data and Digital Com-
mons Data repository, DOI:10.17632/65brs8vc97.2
Conflict of Interest
All authors declare that they have no conflict of interest.
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