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CHROMB-18753; No. of Pages 7 ARTICLE IN PRESS

Journal of Chromatography B, xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Identification of organic acids as potential biomarkers in the urine of

autistic children using gas chromatography/mass spectrometry夽
Joanna Kałużna-Czaplińska a,∗ , Ewa Żurawicz a , Wiktoria Struck b , Michał Markuszewski b
a
Institute of General and Ecological Chemistry, Department of Chemistry, Lodz University of Technology, Żeromskiego 116, 90-924 Łódź, Poland
b
Department of Biopharmacy and Pharmacodynamics, Medical University of Gdańsk, Hallera 107, 80-416 Gdańsk, Poland

a r t i c l e i n f o a b s t r a c t

Article history: There is a need to identify metabolic phenotypes in autism as they might each require unique approaches
Received 11 October 2013 to prevention. Biological markers can help define autism subtypes and reveal potential therapeutic tar-
Received in revised form 21 January 2014 gets. The aim of the study was to identify alterations of small molecular weight compounds and to
Accepted 23 January 2014
find potential biomarkers. Gas chromatography/mass spectrometry was employed to evaluate major
Available online xxx
metabolic changes in low molecular weight urine metabolites of 14 children with autism spectrum dis-
orders vs. 10 non-autistic subjects. The results prove the usefulness of an identified set of 21 endogenous
Keywords:
compounds (including 14 organic acids), whose levels are changed in diseased children. Gas chromatog-
Organic acids
Gas chromatography–mass spectrometry
raphy/mass spectrometry method combined with multivariate statistical analysis techniques provide an
Biomarkers efficient way of depicting metabolic perturbations of diseases, and may potentially be applicable as a
Autism novel strategy for the noninvasive diagnosis and treatment of autism.
Principal component analysis © 2014 Elsevier B.V. All rights reserved.

1. Introduction
Autism spectrum disorders (ASD) is a complex developmen-
tal disability which impairs social interaction and communication.
It is characterized by Asperger syndrome, childhood disintegra-
tive disorder, and pervasive developmental disorder not otherwise
specified (PDDNOS) [1,2]. ASD typically appears during the first 3
years of life. Autism is a multifactorial disorder, which means that a
lot of factors must be considered when determining the case. These
factors include complex genetic interactions, nutritional deficien-
cies or overloads, pre- and postnatal exposure to chemicals or
Abbreviations: 3PGPH, 3-phosphoglycerate dehydrogenase; CE–MS, capillary
electrophoresis–mass spectrometry; CNVs, copy number variations; CRC, col-
orectal cancer; CSF, cerebrospinal fluid; GAII, glutaric aciduria type II; GC–MS,
gas chromatography–mass spectrometry; LC–MS, liquid chromatography–mass
spectrometry; MADD, multiple acyl-CoA-dehydrogenase deficiency; MSTFA, N-
methyl-N-trimethylsilyl-trifluoroacetamide; NMR, nuclear magnetic resonance;
PCA, principal component analysis; PKU, phenylketonuria; PLSCV, partial least
square cross-validation; PPA, propionic acid; PSPH, phosphoserine phosphatase;
SLC, solute carrier; SLOS, Smith–Lemli–Opitz syndrome; SNPs/VNTRs, sin-
gle nucleotide polymorphisms/variable number of tandem repeats; TMCS,
trimethylchlorosilane.
夽 This paper is part of the special issue “Metabolomics II” by G. Theodoridis and
D. Tsikas.
∗ Corresponding author. Tel.: +48 426313091; fax: +48 4263131 28.
E-mail addresses: joanna.kaluzna-czaplinska@p.lodz.pl, jkaluzna@p.lodz.pl
(J. Kałużna-Czaplińska).
viruses, errors during the embryonic neural tube closure process,
dysfunctional immune systems and allergies [3–6].
Many causes of autism have been suggested, but the information
is still incomplete. There is indisputable evidence of the influence
of genes on the pathogenesis of autism. Studies of identical twins
show that if one twin is autistic, the probability that the other will
be autistic is as high as 58% for boys and 60% for girls, while in the
case of fraternal twins the probability falls to 21% for boys and 27%
for girls [7]. Thus, it becomes clear that in order to explain the com-
plexity of autism, many factors need to be considered, including
the impact of genetic heterogeneity, reduced penetrance, epige-
netic factors, the involvement of multiple genes, gene–gene and
gene–environment interactions [8].
The recent research has focused on a number of known neu-
rometabolic disorders identified as having an autistic phenotype,
as well as on the theories related to other metabolic abnormal-
ities thought to contribute to the development of autism [9].
These metabolic disorders include: phenylketonuria (PKU), dis-
orders of purine metabolism, biotinidase deficiency, disorders of
cerebrospinal fluid (CSF) neurotransmitters such as deficiencies
of folic acid, Smith–Lemli–Opitz syndrome (SLOS), methylmalonic
acidemia, dicarboxylic aciduria and other organic acids acidemias
[9–14]. The coexistence of numerous psychiatric, behavior, neu-
rodevelopmental as well as metabolic disorders in autism [15]
requires the study of the relationship between these patholog-
ical states. It is suspected that defects in the gut–blood barrier
make it possible for the central nervous system to have access to
compounds which can be neurotoxic, among them organic acids,

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causing the severity of symptoms in autism spectrum disorders
(ASD) [16,17]. Organic acidurias may interfere with the regula-
tion of glutamatergic and gamma-aminobutyric acid (GABA)ergic
neurotransmission, neuroenergetics, handling of metabolic water,
regulation of transport of hydrophilic compounds from and to
the brain compartment via the blood–brain barrier, as well as
the process of myelination [18]. Although it is difficult to extrap-
olate the mechanisms of organic acid blood-to-brain influx and
brain-to-blood efflux transport, some general rules have been
provided [19,20]. One of the main functions of the blood–brain
barrier is to control the passage of products of metabolism
and xenobiotics. The blood–brain barrier includes brain capillary
endothelial cells and the blood–CSF barrier, which consists of
epithelial cells. Molecules cross the blood–brain barrier via mech-
anisms which include diffusion of lipid-soluble small molecules
as well as catalyzed transport by one of three families of trans-
porters: carrier-mediated transport, receptor-mediated transport,
active efflux transporters [21,22]. In the transport of organic
acids across the blood–brain barrier, the following families of
the solute carrier (SLC) group of membrane transport proteins
are involved: SLC13 (Na+ -sulfate/carboxylate cotransporter) [23],
SLC16 (monocarboxylate transporter) [24], SLC21 (organic anion
transporter) [25], SLC22 (organic cation/anion/zwitterion trans-
porter) [26], SLC27 (fatty acid transport proteins) [27]. It is believed
that disruption of the blood–brain barrier in autistic children
can be the result of acute stress, leaky gut and food intolerance
[28].
Because of a number of disorders occurring in autism, there is
a need to identify metabolic phenotypes in autism as they might
each require unique approaches to prevention or treatment. There
are treatment strategies consisting in the correction of biochem-
ical imbalance in autistic patients such as: eradication of fungal
(Candida) overgrowth and restoration of gut integrity, removal
of potential sources of opioid peptides from the diet (gluten-
free/casein-free diets) [29,30]. Pharmacotherapy of children with
autism in the context of a number of metabolic disorders seems to
be a very promising form of treatment, but we need to emphasize
that there is no clear evidence for the efficacy of this approach in
the study of large groups of children.
The study of biological markers may be an important step to
solve this problem [31,32]. They can help to define autism sub-
types and reveal potential therapeutic targets. A perfect marker
should be detectable with good sensitivity and specificity in biolog-
ical samples from the patient [33]. Metabolomics, which analyzes
low molecular weight compounds such an as organic acid and
amino acid, was used as an effective tool for the diagnosis of dis-
ease and phenotype prediction. Biological fluids such as urine and
plasma contain metabolites which can provide valuable bioinfor-
mation on the metabolism of organism [34]. Compared with the
blood sample, the analysis of urine is non-invasive and can give
similar results.
Metabonomic studies in conjunction with multivariate anal-
ysis are proved to be a useful information-rich strategy for
investigating the metabolic state of biological fluids based on
modern analytical techniques such as nuclear magnetic reso-
nance (NMR), liquid chromatography/mass spectrometry (LC–MS),
capillary electrophoresis–mass spectrometry (CE–MS) and gas
chromatography–mass spectrometry (GC–MS) [35–37]. Due to its
relatively low cost, high separation efficiency and commercial mass
spectra libraries, gas chromatography/mass spectrometry is very
often used in metabonomic research of biological samples in dif-
ferent analyses [38].
The aim of this study was to identify alterations of small molec-
ular weight compounds and find potential biomarkers in the urine
of autistic children using gas chromatography coupled with mass
spectrometry.
Please cite this article in press as: J. Kałużna-Czaplińska, et al., J. Chromatogr. B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.01.041
2. Materials and methods
2.1. Participants
The study was restricted to children with a diagnosis of autism
in compliance with the criteria detailed in the Diagnostic and Sta-
tistical Manual of Mental Disorders [39]. All autistic children were
assessed and diagnosed by clinicians specializing in the diagnosis
and management of autistic children from the Navicula.
2.2. Materials
2.2.1. Sample collection
All overnight urine samples were collected from 14 autistic chil-
dren (4–10 years) who underwent rehabilitation in the Navicula
– Center in Lodz and 10 non-autistic children as healthy controls
(4–10 years). All procedures were carried out, having the written
consent of a parent. The study was performed in agreement with
the Standards and Ethics in Biological Rhythm Research [40]. Urine
was stored at −20 ◦ C until analysis.
2.2.2. Chemicals and reagents
Heptadecanoic acid (98%) used as an internal standard
and N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA),
trimethylchlorosilane (TMCS), urease, methoxyamine, pyridine
were obtained from Sigma–Aldrich (Poznań, Poland). High perfor-
mance liquid chromatography-grade ethyl acetate and methanol
were obtained from POCh (Gliwice, Poland).
2.3. Sample preparation
For sample preparation, we used a simple modification of
method described by Zhang et al. [41]. 200 ␮L of urine with 40
units of urease were incubated at 35 ◦ C for 20 min to decompose the
urea present in the sample. Next, 800 ␮L of methanol and 100 ␮L of
heptadecanoic acid (1 mg/mL in ethyl acetate) used as IS (internal
standard) were added. The solution was extracted and centrifuged
at 12,000 × g, 5 ◦ C for 15 min. The supernatant was transferred to a
vial, and then evaporated to dryness under nitrogen at room tem-
perature. 30 ␮L of methoxyamine in pyridine was added to a vial
and the solution was vortexed for 5 min. The reaction of methox-
imation lasted 24 h at room temperature. Next, the sample was
trimethylsilylated for another hour by adding 60 ␮L of MSTFA with
1% TMCS. Before the GC–MS analysis, 50 ␮L of hexane was added
to the vial. All samples were prepared according to the same pro-
cedure.
2.4. GC–MS analysis
1 ␮L of derivatized sample was injected splitless into an Agi-
lent 6890N Network GC system and 5973 Network Mass Selective
equipped with a capillary column (J&W Ultra Inert HP-5ms; Agi-
lent Technology; 30 m × 0.25 mm internal diameter; film thickness,
0.25 ␮m). The injector temperature was set at 250 ◦ C. Helium was
used as a carrier gas at a constant flow rate of 0.9 mL/min through
the column. The column temperature was initially kept at 50 ◦ C
for 1 min and then increased to 240 ◦ C at 15 ◦ C min−1 and further
increased at 30 ◦ C min−1 to 300 ◦ C. The MS quadrupole tempera-
ture was set at 150 ◦ C and the ion source temperature at 200 ◦ C.
Masses were acquired from m/z 50–800. GC–MSD ChemStation
Software (Agilent) was used for autoacquisition of GC total ion
chromatograms (TIC) and fragmentation patterns. Each compound
had a unique fragmentation pattern composed of a series of split
molecular ions, whose mass charge ratios and abundance could
be compared with the standard mass chromatogram in the NIST
(National Institute of Standards and Technology) mass spectra
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library by the ChemStation Software. For each peak, the software
will generate a list of similarities comparing them with every sub-
stance within the NIST library. Peaks with the similarity index more
than 80% were assigned compound names [42–44], while those
having less than 80% similarity were listed as unknown metabolites.
The chromatograms were subjected to noise reduction and peaks
with the intensity higher than threefold of the signal-to-noise (S/N)
ratio were recorded prior to the peak area integration. The relative
intensity of each peak was normalized against the internal standard
in GC–MS run. All known artifact peaks such as peaks due to the
column bleed and MSTFA artifact peaks did not enter the final data
analyses. Integrated peak areas of multiple derivative peaks belong-
ing to the same compound were summed and considered a single
compound. Each sample was characterized by the same number of
variables and each of these variables was represented throughout
the observations by the same sequence. Thus, a data matrix was
generated by intensities of the commensal peaks from all the sam-
ples to characterize the biochemical pattern of each sample. The
obtained matrix was then employed for the correlation analysis
and pattern recognition. For each case three urine aliquots were
processed throughout the whole experimental process to test the
reproducibility of sample preparation and GC–MS analysis. Analyti-
cal accuracy and precision were assessed by a consecutive injection
of the three derivatized samples into the GC system. To assess the
stability of the analytical system, quality control (QC) samples were
measured after every fourth run. Prior to QC sample measurement,
aliquots of the same volume of all urine samples from this study
were pooled. They were prepared once and measured in tripli-
cate. All urine samples were analyzed with GC–MS according to
the method described above. The stability of the response per com-
pound was expressed in relative standard deviation (RSD) of peak
areas.
2.5. Data processing and pattern recognition
After the GC–MS analysis each sample was represented by
GC–MS TIC, and the peak areas of compounds were integrated. The
peak area ratio of each compound to the corresponding internal
standard was calculated as the response. The majority of the peaks
in the chromatograms were identified as endogenous metabolites
by NIST mass spectra library, including amino acids, organic acids,
Fig. 1. Examples of four (from 8 to 15 min) typical GC–MS total ion current chromatograms (TIC) of urine samples obtained from autistic children.
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3
carbohydrates and fatty acids. 83 signals were obtained, but only
43 signals which could be autoidentified by the NIST library by the
comparison of the fragmentation patterns composed of all frag-
ment ions were analyzed. 10 Organic acids in the “83 metabolites”
were further identified through the correlation of retention times
and fragmentation patterns, when compared with the commer-
cially available corresponding standards.
Data were statistically evaluated using statistical analysis pack-
age Statistica, version 9.0 (Statistica 9.0, StatSoft, Poland). Firstly,
individual differences in the profiles of metabolite levels between
the two groups were evaluated by the comparison of statistical
parameters and after performing the t-test and U-Mann–Whitney
test. The difference in the p-value of <0.05 was considered statisti-
cally significant. The Principal component analysis (PCA) and partial
least square cross-validation (PLSCV) were performed within the
Matlab environment (Matlab 7.0, Mathworks, Natick, MA, USA).
Before the analysis the dataset (24 profiles × 43 metabolite levels)
was autoscaled. The PCA was applied to check the dataset struc-
ture and assess the variability of the profiles belonging to groups
of autistic vs. non-autistic children.
3. Results
In order to assess the stability of the analytical system, quality
control sample was performed during GC–MS analyses. The relative
standard deviations values of the obtained peak areas of each 43
metabolites used for PCA analysis were lower than 15%.
Fig. 1 shows an example of the overlap of four typical TIC pro-
files of injected autistic urine samples in the same aliquot. The
data showed stable retention time with no drift of the peaks,
which reflects the stability of GC–MS analysis and reliability of
the metabolomic data. Before the start of the principal component
analysis, the levels of metabolites were autoscaled to remove the
dependence of the rank of the metabolites on the average concen-
tration and the magnitude of the changes. A score plot of PC1 vs.
PC2, for 24 profiles of all the measured metabolites showed sep-
aration between the groups of autistic and non-autistic children
(Fig. 2).
Therefore, it might be concluded that the PCA analysis dis-
tinguished between autistic and non-autistic children using the
metabolomic profiles, collected by the established GC–MS system.
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Fig. 2. PC1 vs. PC2 score plot derived from total ion chromatograms (TIC) obtained
from urine of 14 autistic children and 10 non-autistic children for all measured
metabolites. A, autistic children; C, control group. All data used were autoscaled.
Individual differences in the profiles of metabolite levels
between the two groups were found after performing the t-test and
U-Mann–Whitney test. Considering the difference in the p-value of
<0.05 to be statistically significant, 21 metabolites were identified
as potential biomarkers. The potential marker metabolites which
were responsible for the separation of the autistic group from the
non-autistic group of children are presented in Table 1. Next, the
two groups were analyzed again by means of PCA. The results of PCA
support the observations provided by general data analysis. There
is a clear separation between the profiles belonging to different
groups, which is generally reflected by the first three principal com-
ponents with nearly 56% of data variance explained. This is a slightly
better result than that when all the determined metabolites were
Fig. 3. PCA score plot discriminating autistic specimens from normal specimens
based on GC–MS marker metabolites for 21 selected low molecular weight com-
pounds. A, autistic children; C, control group.
used in PCA analysis (Fig. 3). Secondary PCA model was constructed
using marker metabolite intensities as variables. ROC analysis using
the cross-validated predicted Y values was performed to validate
the robustness of PCA model. The sensitivity and specificity trade-
offs were summarized for each variable using the area under the
ROC curve denoted as AUC and calculated using the trapezoidal rule
(Fig. 4). The area under the ROC curve is a direct indication of the
efficiency and clinical applicability of the diagnostic method. It is
widely accepted that diagnostic methods are considered to be clini-
cally applicable when the value for the area under ROC curve is not
less than 0.6 and have a very good diagnostic measure when the
value is greater than 0.8 [45]. In this paper the diagnostic system
based on the metabolites can be attributed to the good because it
has values circa 0.8.

4. Discussion
Table 1
Potential marker metabolites found in GC–MS chromatograms of urine samples of
autistic and control groups. At PC1/PC2/PC3 scores plot, the distribution of metabolite pro-
files from the autistic and non-autistic children is consistent with
No Metabolite Retention Differentiation p-Value*
the diversity of these profiles observed in the analysis of individual
time (min) for autistic
samples
metabolite ranges. The group of samples from non-autistic con-
trol children is more homogeneous than the group from autistic
1 Propionic acid 5.28 ↓ 0.019
children. There is a clear distinction between those two groups of
2 ␣-Hydroxybutyric acid 5.32 ↑ 0.002
3 Oxalic acid 5.98 ↑ <0.001 samples. These results prove the usefulness of the identified set of
4 ␤-Hydroxybutyric acid 6.28 ↑ <0.001 21 endogenous compounds, whose levels are changed in diseased
5 Homocysteine 6.78 ↑ 0.036 children and confirm their usefulness in predicting their health
6 Phosphoric acid 7.44 ↓ 0.001
status and potential biomarkers.
7 Butanoic acid 7.59 ↓ 0.022
8 Succinic acid (butanedioic 7.90 ↑ 0.009
acid)
9 l-Serine 8.21 ↓ 0.007
10 Sebacic acid (decanedioic 8.72 ↓ 0.001
acid)
11 l-Threonic acid 9.83 ↑ 0.026
12 ␣-Hydroxyglutaric acid 9.96 ↑ 0.002
13 p-Hydroxyphenylacetic 10.53 ↑ 0.036
acid
14 Tartaric acid 10.58 ↑ 0.031
(2,3-dihydroxybutanedioic
acid)
15 Ribonic acid 10.71 ↑ <0.001
16 d-Arabinitol 11.28 ↑ 0.009
17 Citric acid 11.92 ↑ 0.006
18 l-Tyrosine 12.72 ↑ 0.036
19 Tryptophan 13.89 ↓ 0.036
20 d-Mannitol 14.05 ↑ 0.004
21 m-Hydroxybenzoic acid 14.37 ↑ <0.001

(↑) Means increased level of metabolite in urine of autistic children, (↓): decreased.
*
Statistical p-value calculated using the Mann–Whitney test (significance at Fig. 4. ROC curve determined using cross-validated predicted Y-values of GC–MS
p < 0.05). PCA model.

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Moreover, it is very important that the motivation for this analy-
sis was not to replace the established clinical evaluations of autism,
but to identify the metabolites which hold the potential to augment
the metabolic biomarkers gleaned from urine, as routine screening
or surveillance.
The levels of ␣-hydroxybutyric acid, oxalic acid,
␤-hydroxybutyric acid, homocysteine, succinic acid
(butanedioic acid), l-threonic acid, ␣-hydroxyglutaric acid, p-
hydroxyphenylacetic acid, tartaric acid (2,3-dihydroxybutanedioic
acid), ribonic acid, d-arabinitol, citric acid, tyrosine, d-mannitol,
m-hydroxybenzoic acid in urine were higher in the group of autis-
tic children than those in non-autistic children. Metabolites whose
levels were reduced in the urine of autistic children compared with
non-autistic children are propionic acid, phosphoric acid, butanoic
acid, l-serine, sebacic acid (decanedioic acid) and tryptophan. The
metabolites are known to be involved in multiple biochemical
processes, especially in energy and lipid metabolism [46].
4.1. Metabolites excreted in elevated amounts
One of the metabolites whose level was significantly higher in
the group of autistic children than that in non-autistic children
was citric acid. An increased level of citric acid observed in current
investigations can suggest an amino acid deficiency or problems
with protein metabolism in autistic children. Succinic acid (butane-
dioic acid) cannot play its role in the production of cellular energy
via the citric acid cycle when coenzyme Q10 (CoQ10) is inadequate.
Elevated succinic acid excretion is considered a potential marker for
deficiency of CoQ10 and riboflavin in children with autism [9,47].
Another metabolite whose level in the urine of autistics was
increased in comparison with the control group was l-threonic
acid, which is a product of oxidative degradation of ascorbic acid
[48]. Ascorbic acid in plasma and cells acts as a powerful scav-
enger or reducing antioxidant, capable of donating its electrons
to reactive oxygen species and eliminating them. The mechanism
of oxidative degradation of ascorbic acid by hydrogen peroxide to
threonic acid involves the loss of two electrons, the intermediate
free radical can be further oxidized to produce dehydroascorbic
acid. Dehydroascorbic acid, which is not stable, can be decomposed
to 2,3-diketogulonic acid and then to oxalic and l-threonic acids
[49,50]. The higher level of urinary threonic acid may indicate an
increased degradation of ascorbic acid which in turn may indirectly
indicate oxidative stress [44].
Tartaric acid (2,3-dihydroxybutanedioic acid) naturally occurs
in fruit, vegetables and in fermented products [51,52]. Tartaric acid
is a metabolite of yeasts [53]. This acid, an analog malic acid, acts
as an inhibitor of the citric acid cycle enzyme fumarase [54], which
is a catalyst in the interconversion of malate and fumarate. It was
noted that tartaric acid is a highly toxic substance, 12 g of this acid
can prove to be a fatal dose with death occurring from 12 h to 9
days after ingestion [55]. The symptoms of gastrointestinal caused
by the response to this acid includes vomiting, diarrhea, abdominal
pain, thirst. Tartaric acid may also lead to cardiovascular collapse
and/or acute renal failure. It can damage the muscles and kidney
[56,57], and may even lead to fatal human nephropathy (kidney
damage) [58,59]. What is particularly important, both muscle and
kidney damage was observed by Shaw et al. [60] in two brothers
with autism. Shaw et al. described elevated levels of tartaric acid in
the urine of autistic children [60,61]. In the case of a 2-year old child
with autistic symptoms, nystatin therapy was found to help reduce
the level of tartaric acid, improve eye contact, decrease hyperac-
tivity, and increase vocalization. Lonsdale et al. [62] presented the
case report of autistic sibling. The urine analysis of both children
showed elevated levels of arabinose and one of the children had
an elevated level of tartaric acid. In the next study Evans et al.
[63] reported no significant differences in the measured excretions
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5
of tartaric acid in autistic children. Based on this information we
can conclude that the differences in the excretion of tartaric acid
in the urine of non-autistic and autistic children may result from
dysbiosis.
␣-Hydroxybutyrate is a by-product of the conversion of cys-
tathione to cysteine [64]. According to our best knowledge, elevated
levels of this acid in the urine of autistic children have not been
reported so far. Nevertheless, the literature includes descriptions
of disorders associated with the high levels of ␣-hydroxybutyrate
in urine. Elevated excretion of this acid is linked to the increased
destruction of lymphocytes in infectious diseases [65], increased
cytoplasmic NADH2 /NAD ratio [66], birth asphyxia, lactic acido-
sis, glutaric aciduria type II, dihydrolipoyl dehydrogenase (E3)
deficiency, propionic acidemia [67]. It is also suggested that the
increased level of the acid in urine may result from smoking, poor
diet and lack of exercise [68]. According to this report, increased
levels of ␣-hydroxybutyric acid in the urine of children with autism
can be associated with poor diet in autism and increased excretion
of propionic acid.
␤-Hydroxybutyric acid is synthesized in the liver from acetoac-
etate (formed from acetyl-CoA) and when the concentration of
glucose in blood is low, it can be used by the body as a source
of energy [69]. The increased level of ␤-hydroxybutyrate in body
fluids is known as a “ketone body”, which is characteristic of
metabolic acidosis. Another reason for the increased levels of ␤-
hydroxybutyraic acid is deficiency in cytochrome oxidase enzymes
of the electron transport system [70]. Cases of the elevated levels
of butyric acid in the urine of autistics reported in literature relate
to children [60,71,72].
The higher level of m-hydroxybenzoic acid can be a marker of
intestinal dysbiosis, which often occurs in the case of autistic chil-
dren. Moreover, the results may also show elevated values as a
result of ingestion of food [64]. The increase in ␣-hydroxyglutaric
acid (2-hydroxyglutaric acid) in the urine of autistic children was
described by Zafeiriou and co-worker [73]. The highest values
of 3-hydroxyglutaric acid for a child with autism were found by
Shaw et al. [61]. According to Shaw, this metabolite is associated
with the genetic disease – glutaric aciduria type I, which results
from the deficiency of glutaryl CoA dehydrogenase, an enzyme
involved in the breakdown of lysine, hydroxylysine, and trypto-
phan.
Shaw [74] collected the data showing that many autistic chil-
dren had frequent urination of small volume and found that the
phenomenon was associated with higher levels of oxalates. He also
found that these children often manifested gastro-intestinal symp-
toms such as diarrhea and stomach pain. They may also experience
pain in the urinary tract. That pain is relieved when a low oxalate
diet is introduced. Shaw also found that children improved their
cognitive, academic and motor skills once the amount of oxalates
in their diets was sharply reduced.
The levels of p-hydroxyphenylacetic acid were significantly
higher in autistic children and this may reflect increased gut
metabolism of tyrosine secondary to bacterial overgrowth. d-
Arabinitol is a metabolite of the most pathogenic Candida species
and urinary excretion is elevated in autistic patients [60,61]. The
use of probiotics can be effective in reducing the level of d-
arabinitol in the urine of children with autism. d-Arabinitol is
five-carbon sugar alcohol, which is a metabolite of the most
pathogenic Candida species, in vitro as well as in vivo. There are
reports which show the improvement of some physiological and
behavioral symptoms of autism after the use of antifungal prepa-
rations such as nystatin [60].
d-Mannitol is a monosaccharide, which is relatively poorly
absorbed by the human intestine. The higher level of d-mannitol
in the urine of autistic children can be a potential marker of leaky
gut [75].
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6 J. Kałużna-Czaplińska et al. / J. Chromatogr. B xxx (2014) xxx–xxx
Ribonic acid is an oxidation product of ribose by the enzyme
ribose 1-dehydrogenase. Moreover, this acid is a tryptophan pre-
cursor [76]. Elevated levels of this acid and decreased levels of
tryptophan in the urine of autistic children observed in this study
may indicate disturbance of synthesis of tryptophan in these chil-
dren or can be connected with diet and nutrition.
According to Pasca et al. [77], the levels of homocysteine in
serum were significantly higher in autistic children compared to
the control group of non-autistic children. This fact correlated with
the deficit of B12 vitamin. Their results are consistent with other
observations of autistic children presented in literature [78–80].
These observations suggest that hyperhomocysteinemia might be
present in autism.
Another amino acid which occurred at elevated levels in the
urine of autistic children was l-tyrosine. Tyrosine is formed from
the essential amino acid phenylalanine in a reaction catalyzed by
the enzyme phenylalanine hydroxylase and it is also contained
in food [69]. Inborn errors characterized by the accumulation of
tyrosine in body fluids and tissues are tyrosinemias [81]. Impaired
tyrosine metabolism was linked to mental disorders [82]. Tahiroğlu
et al. [83] described elevated levels of tyrosine in the urine of a
two-year boy with autism. Studies of tyrosine in body fluids made
it possible to diagnose tyrosinemia type III. The boy had mental and
motor retardation and hyperactivity symptoms, which resolved
after a therapeutic low-tyrosine diet and special infant food. Subse-
quent studies showed decreased excretion of tyrosine in children
with Kanner’s syndrome [84]. In this study, however, we assume
that the elevated levels of this amino acid observed in the urine of
children with autism may be the result of their unvaried diet.
4.2. Metabolites excreted in lowered amounts
The levels of other potential biomarkers were significantly lower
in the group of autistic children than those in the non-autistic
children. Propionic acid (PPA) is an intermediate of normal cellu-
lar metabolism and is produced in the gut as a result of bacterial
fermentation of diet carbohydrates and proteins. Mortensen and
Clausen [85] indicate that propionic acid may be a factor of Can-
dida species involved in the development of some types of ASD.
Literature describes the transport of propionate and butyrate across
the blood–brain barrier. These intracerebroventricular infusions
can cause behavioral, electrophysiological, biochemical and neu-
ropathological disorders in adult rats, which are compatible with
those seen in autism [86–89]. The low level of PPA in plasma of
autistic patients can be a reflection of the high rate of uptake by
brain [90,91].
Low urinary levels of phosphoric, sebacic (decanedioic) and
butanoic acids are observed. Phosphate urinary excretion is directly
proportional to dietary intake. Excess phosphate is also associated
with hyperparathyroidism, vitamin D-resistant rickets, immobi-
lization following paraplegia or fracture due to bone resorption.
Low urinary phosphate occurs in vitamin D deficiency [92,93].
There are data regarding a possible connection between ASD and
vitamin D [94]. Sebacic acid is a saturated, straight-chain natu-
rally occurring dicarboxylic acid with 10 carbon atoms. In humans
sebacic (C10) acid derives from the ␤-oxidation of a longer chain
of dicarboxylic acids [95]. This acid is a normal urinary acid. Only
in the case of patients with multiple acyl-CoA-dehydrogenase defi-
ciency (MADD) or glutaric aciduria type II (GAII), biochemical data
show an increase in urine sebacic acid excretion [96]. However, to
our knowledge, the lower level of sebacic acid in urine of autis-
tic children was observed for the first time in this study. Butanoic
acid (butyric acid), a four-carbon fatty acid, is formed in the human
colon by bacterial fermentation of carbohydrates (including dietary
fiber), and putatively suppresses colorectal cancer (CRC). People
who eat a diet low in carbohydrate have lower amounts of butanoic
Please cite this article in press as: J. Kałużna-Czaplińska, et al., J. Chromatogr. B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.01.041
acid in their colon [97]. Shaw and co-workers [60] observed the
presence of increased 3-OH-butyric acid in the urine of autistic
children. El-Ansary et al. [90] demonstrated the reduced levels of
butanoic acid in plasma of autistic children from Saudi Arabia.
Tryptophan is a precursor to the neurotransmitter serotonin,
which is strongly linked to autism. Low urinary tryptophan was pre-
viously reported as anomalous for autistic children [5]. Lower levels
of tryptophan may lead to the worsening of autistic symptoms such
as mild depression and increased irritability [98].
Our results also show decreased levels of serine in the urine
of autistic children. Serine deficiency disorders are rare defects
in the biosynthesis of the amino acid l-serine. Serine synthesis
deficit may initially be considered a form of non-specific develop-
mental delay. Disorders such as deficiencies of 3-phosphoglycerate
dehydrogenase (3PGPH) and phosphoserine phosphatase (PSPH)
are associated with serine deficiency disorders. 3PGPH and PSPH
failure can lead to neurological symptoms such as congenital micro-
cephaly and severe psychomotor retardation [99]. Ming et al. [100]
depict reduced levels of serine in the urine of autistic children, how-
ever, the importance of reducing the level of this amino acid is not
clear.
5. Conclusions
The difficulty in determining the pharmacotherapy, which could
give a clear benefit in the general population of autism, results from
the wide range of spectrum disorders and existing phenotypic vari-
ation. Metabolic studies of markers of autism seem to be a good tool
to extract the metabolic phenotype and this is the first stage of the
work aiming at establishing effective treatment for autism.
In this metabonomic study, a GC–MS based urinary metabo-
lite analysis combined with a multivariate statistical technique is
able to detect metabolic profile variations between autistic and
non-autistic children. This work suggests that there is a significant
metabolic difference between autistic and non-autistic children.
Moreover, an analysis of different metabolites such as some organic
acids and amino acids can be associated with autism spectrum dis-
orders. In addition, the correlation of some compounds in autistic
children with some autistic symptoms could be used as potential
biomarkers for the diagnosis of autism spectrum disorders. Biolog-
ical markers can also help to define autism subtypes and reveal
potential therapeutic targets.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
We would like to thank the parents of autistic children from the
Navicula Centre in Łódź, Poland for their help in collecting urine
samples from autistic children.
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