Special issue: review

Received: 13 February 2013, Accepted: 5 March 2013 Published online in Wiley Online Library: 22 April 2013

(wileyonlinelibrary.com) DOI 10.1002/bmc.2911

Chromatographic methods in the study

of autism
Ewa Żurawicz, Joanna Kałużna-Czaplińska* and Jacek Rynkowski
ABSTRACT: Research into biomarkers of autism is a new means of medical intervention in this disease. Chromatographic
techniques, especially coupled with mass spectrometry, are widely used in determination of biomarkers and assessment of
effectiveness of autism therapy owing to their sensitivity and selectivity. Among the chromatographic techniques gas
chromatography and liquid chromatography, especially high-performance liquid chromatography, have found application
in clinical trials. The high-performance liquid chromatography technique allows an analysis of liquid samples with a wide
range of molecules, small and large, providing an opportunity to perform advanced assays within a short time frame.
Gas chromatography with the appropriate preparation of samples (gaseous and liquid) and a selection of analysis conditions
enables the separation of thermally stable, volatile and non-volatile organic substances in short runtimes. The chromatographic
techniques that are currently used in metabolic studies in autism are designed to identify abnormalities in three areas: the
metabolism of neurotransmitters, nutritional and metabolic status and manifestations of oxidative stress. This review presents
a necessary theoretical introduction and examples of applications of chromatographic studies of disorder markers in autism.
Copyright © 2013 John Wiley & Sons, Ltd.

Keywords: autism; chromatography; HPLC; GC-MS; biomarker

Introduction allows an analysis of various substances. Clinical analysis is based

According to the Diagnostic and Statistical Manual of Mental
Disorders, autistic disorder is a pervasive developmental disorder
and is characterized by social, verbal and nonverbal communi-
cation abnormalities. Manifestations of autism typically occur
before the age of 36 months (American Psychiatric Association,
2000). The report presented by the Autism and Developmental
Disabilities Monitoring Network for 2008 presents the average
prevalence of autism as 11.3 per 1000 (1 in 88) children aged
8, and the diagnosis is more frequent in males than females,
with ratios from 3.7:1.0 to 7.2:1.0 (Centers for Disease Control
and Prevention, 2012). The etiology of autism remains unknown,
although interactions of genes and environmental factors have
been suggested (Hertz-Picciotto et al., 2006).
The use of chromatographic techniques and metabolomics in
the study of autism is a result of the broad spectrum of this dis-
ease, which goes far beyond the purely developmental disorder.
A number of different disorders associated with autism have
been found, with metabolic disorders taking an important place
(Table 1). The study of abnormal accumulation or deficit of
specific metabolites in a defined pathway can provide clues into
relevant candidate genes and/or environmental exposure in
autism. Such knowledge can also be helpful in targeted inter-
vention strategies to restore metabolic balance and potentially
improve the symptoms of autism (Melnyk et al., 2012). A more
complete understanding of metabolic conditions is possible
owing to the studies of biomarkers. According to International
Union of Pure and Applied Chemistry nomenclature, a bio-
marker is an indicator signaling an event or condition in a bio-
logical system or sample and giving a measure of exposure,
effect or susceptibility (Duffus, 1993). Because of the possibility
of using a large number of metabolites as biomarkers, in clinical
on the fact that metabolic changes associated with specific dis-
orders can cause changes in the biochemical profile of a specific
body fluid (Fig. 1), so when the disease is suspected, specific
parameters are tested in certain body fluids (e.g. serum, plasma,
whole blood, and urine; Lindon et al., 2007). Blood, coming into
contact with all organs of the body, is a source of various
biomarkers, therefore it has the longest track record for use in
modern diagnostic procedures. It is possible to see the whole
spectrum of different biomarkers, but this makes the biomarkers
found in blood less specific and generally they may not be con-
fined to emanating from a single organ. Urine and cerebral spi-
nal fluid (CSF) are very amenable to biomarker analysis. CSF is
quite specific to the central nervous system (CNS). CSF samples
are taken by means of a lumbar puncture (LP), which may not
be possible for all patients with a CNS disease, and it is generally
not applicable. Urine seems a more suitable biofluid than blood
or CFS because it can be obtained in large quantities by non-
invasive sampling. Urine is also more organ-specific than blood.
It can be obtained in large volumes (deciliter to liter amounts)
and can be collected over a period of time, even offering the
* Correspondence to: Joanna Kałużna-Czaplińska, Institute of General and
Ecological Chemistry, Department of Chemistry, Lodz University of Tech-
nology, Zeromskiego 116, 90–924 Lodz, Poland. E-mail: jkaluzna@p.lodz.pl
Institute of General and Ecological Chemistry, Department of Chemistry,
Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland
Abbreviations used: 5-HIAA, 5-hydroxyindoleacetic acid; AA, amino acids;
CFS, cerebral spinal fluid; CNS, central nervous system; DA, D-arabinitol;
IAG, indolyl-3-acryloylglycine; LA, L-arabinitol; LP, lumbar puncture; PUFA,
plasma polyunsaturated fatty acid; SAH, S-adenosylhomocysteine; SAM,
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analysis it is extremely important to adapt a technique that S-adenosylmethionine; SLOS, Smith–Lemli–Opitz Syndrome.

Biomed. Chromatogr. 2013; 27: 1273–1279 Copyright © 2013 John Wiley & Sons, Ltd.
 E. Żurawicz et al.

Table 1. Conditions related to autism

Psychiatric, behavior and Metabolic disorders Genetic disorders

neurodevelopmental disorders
Attention-deficit hyperactivity disorder
(Yates and LeCouteur, 2009)
Tourette syndrome/tic disorder
(Yates and LeCouteur, 2009)
Dyspraxia/developmental coordination
disorder (Yates and LeCouteur, 2009)
Dyslexia (Yates and LeCouteur, 2009)
Obsessive–compulsive disorder
(Yates and LeCouteur, 2009)
Specific phobias (Yates and
LeCouteur, 2009)
Anxiety (Yates and LeCouteur, 2009)
Depression/mood disorder (Yates and
LeCouteur, 2009)
Sleeping difficulties (Yates and
LeCouteur, 2009; Williams et al., 2004)
Feeding difficulties (Yates and
LeCouteur, 2009)
Anorexia nervosa (Zafeiriou et al., 2007)
Abormalities of purine metabolism
(Celani, 2003)
Creatine deficiency (Zecavati and
Spence, 2009)
Biotinidase deficiency (Zecavati and
Spence, 2009)
Succinic-semialdehyde dehydrogenase
deficiency (Zecavati and Spence, 2009)
Cerebral folate deficiency (Zecavati and
Spence, 2009)
Smith–Lemli–Opitz syndrome (Zecavati
and Spence, 2009)
Infantile ceroid lipofuscinosis (Zecavati
and Spence, 2009)
Histidinemia (Zecavati and Spence, 2009)
Urea cycle defects (Zecavati and
Spence, 2009)
Gastrointestinal Candida overgrowth
(Shaw et al., 2010)
Fragile X syndrome (Zafeiriou
et al., 2007)
Tuberous sclerosis complex (Zafeiriou
et al., 2007)
Down syndrome (Zafeiriou
et al., 2007)
Angelman syndrome (Zafeiriou
et al., 2007)
Neurofibromatosis type 1 (Zafeiriou
et al., 2007; Celani, 2003)
Phenylketonuria (Zafeiriou et al.,
2007; Celani, 2003)
Aristaless Related Homeobox
Syndrome (Zafeiriou et al., 2007)
Charge, Goldenhar and Moebius
syndromes (Zafeiriou et al., 2007)
Chromosome 2q37 deletion
syndrome (Zafeiriou et al., 2007)
Cohen syndrome (Zafeiriou et al., 2007)
Mitochondrial disorders (Zafeiriou
et al., 2007)

ability to collect multiple samples within 24 h. Urine analysis
allows a very detailed monitoring of the patient’s therapeutic
response (Good et al., 2007).
A very important task of the clinical analysis involves separa-
tion of species in complex matrices, which are the body fluids
(Varcoe, 2001). The problem of separation can be solved through
1274
the use of chromatography, which is the most powerful, flexible
and simple analytical technique in separation science, especially
in separation of complex organic mixtures (Bruner, 1993).
Through the use of chromatography, it is possible to carry out
a simultaneous separation and quantitative analysis of the
substance. Separation efficiency is related to the type of the
chromatographic column (hydrophobic/hydrophilic) and solvent
system and column pressure. An appropriate selection of chro-
matographic techniques for the analysis is dependent on the
nature and amount of sample, the objective of the separation
and the limitations of available time, equipment and expertise.
Simple economical and effective chromatographic techniques
such as paper chromatography and thin layer chromatography,
but also higher-sensitivity and more expensive high-performance
liquid chromatography (HPLC) and gas chromatography (GC) find
broad application in clinical investigations (Rao et al., 2009). Gas or
liquid chromatography helps in the analysis of various categories
of metabolites such as volatiles, steroids, steroids, organic acids
and amino acids as well as similar compound types (Agilent
Technologies I2, 2007) from physiological fluids (urine, serum,
amniotic fluid, cerebral spinal fluid, etc.). Providing selectivity and
sensitivity, which are necessary for clinical trials, chromatographic
techniques contribute to the success of the analytical process
(Vékey et al., 2007). The application of clinical research with
the use of chromatographic techniques with subsequent multi-
variate statistical analyses provides a well-established strategy
for differential metabolic pathway profiling and chemical con-
stituents that may serve as markers for the presence of disease
or its absence.
Gas chromatography is a powerful tool for separating volatile
and thermally stable compounds. It can also be suitable for
compounds of low volatility or those with the polar functional

Figure 1. The metabolic profile as reflection of the organism state. group after chemical derivatization. Derivatization performed

wileyonlinelibrary.com/journal/bmc Copyright © 2013 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2013; 27: 1273–1279
Chromatographic methods in the study of autism
by alteration of functional groups provides increased sample
volatility, improved selectivity, chromatographic efficiency and
enhanced detection. Combination of GC with mass spectrome-
ter (GC-MS) is recommended as a technique for metabolic
profiling, mainly owing to its high sensitivity and specificity
and access to extensive mass spectral databases. GC-MS instru-
mentation is available for many clinical laboratories, although
GC-MS analysis is relatively expensive in terms of equipment,
personnel and time required (Kwong, 2002).
High-performance liquid chromatography (sometimes referred
to as high-pressure liquid chromatography) is the chromato-
graphic technique most commonly used in the studies of autism
markers. As opposed to GC, liquid chromatography enables an
analysis of polar and thermally labile compounds (Agilent
Technologies I2, 2007). Using a photodiode array detector or mass
spectrometer detector coupled with HPLC it is possible to obtain
structural information, which is essential in the identification of
unknown compounds (Kwong, 2002). For each HPLC system the
achievement of high resolution at high flow rates is characteristic.
Important advantages of HPLC are better resolution, reproducibil-
ity and excellent within- and between-run precision (Gooding and
Regnier, 2002).
Appropriate sample preparation is essential for the successful
chromatographic analysis of specimens which include biological
samples such as blood and urine. Body fluids in particular need
to be processed prior to analysis in order to remove any interfer-
ing compounds that could cause either contamination or block-
age of the column (especially blood), giving inaccurate results.
Sample preparation can involve pH adjustment, ultrafiltration,
enzymatic reactions, extraction methods like liquid–liquid ex-
traction (solvent extraction), solid-phase extraction, solid-phase
microextraction, and a very important step in the GC-MS analy-
sis, derivatization (Hempel, 2004). According to the reports,
blood is the most common body fluid in the chromatographic
studies (Fig. 2), despite its complex matrix.
This review presents a necessary theoretical introduction and
examples of applications of chromatographic studies of disorder
markers in autism.
The metabolic profile, a link between the
biological system and diagnosis
With well-prepared samples of body fluids, chromatographic
techniques offer a possibility of measuring metabolic end points
Figure 2. Diagram of report frequency (2005–2012) using chromatography in analysis of biological fluids. Bibliographic research related to the use of
chromatography in blood, plasma, urine and cerebrospinal fluid analysis (PubMed). A total of 64,697 reports were found.
Biomed. Chromatogr. 2013; 27: 1273–1279 Copyright © 2013 John Wiley & Sons, Ltd.
(metabolic profiles), which are determined by host genetic
and environmental factors. Figure 3 shows the stages of work
in the study of metabolic profiles. Metabolic profiling, or
metabolomics, is the study of low-molecular weight metabo-
lites and their intermediates found in biological fluid samples.
A metabolic profile illustrates the dynamics of the biological
system as a response to genetic modification (e.g. transgenic
or viral) as well as physiological (e.g. gender), pathophysiological
(e.g. disease morbidity) and/or developmental stimuli (e.g. aging;
Clarke and Haselden, 2008). Metabolic profiles can provide
information about mitochondrial diseases, neurotransmitter
metabolites, nutritional and metabolic status, oxidative stress
and environmental impact.
Finding interesting metabolites requires a search for statis-
tically significant variations in abundance within a set of
experimental and control samples. The next step in the
study of metabolic profiles is identification, which means
determination of the chemical structure of these metabolites
after profiling and finally interpretation, which involves find-
ing connections between the metabolites discovered and
the biological processes or conditions. Statistically significant
variations can only be seen by analyzing large numbers of
samples, which is an additional challenge in the case of
autism research. The principal chromatographic technologies
(GC and HPLC) that have been developed to study metabolic
profiles are usually based on spectroscopy and mass spec-
trometry (MS).
Figure 3. The scheme of work in the study of metabolic profiles.
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 E. Żurawicz et al.

Chromatographic analysis in autism healthy ones were found, which indicates a reduced risk of
free radical disease. In younger autistic subjects, similarly high
Neurotransmitter metabolites plasma vitamin concentrations to those in older cases were
observed. The results indicate that consumption of fruit and
An analysis of catecholamines can be a problem because of their
vegetables in autism was optimal in this study group. The
presence at low concentrations, their susceptibility to oxidation
results also showed that average plasma concentrations of
and the fact that they are unstable at basic pH. The use of HPLC
vitamins E and A in autistics were below threshold values,
allows the determination of neurotransmitters and the levels of
which indicates lower consumption of food rich in vitamins A
their metabolites in platelets, blood and CSF in autistic subjects
and E (Krajcovicova-Kudlackova et al., 2009). Sensitive liquid
(Israngkun et al., 1986; Moretti et al., 2005; Coutinho et al.,
chromatography with tandem mass spectrometry detection
2007). In the case of 13 autistic subjects under study the levels
(LC-MS/MS) made it possible to detect significant changes in
of plasma dopamine, norepinephrine, and epinephrine concen-
leukocyte sulfur amino acids metabolism in autistic children.
trations were significantly higher compared to controls, also
Leukocytes from autistic children contained significantly lower
whole blood serotonin, tested on the same subjects, was found
concentrations of S-adenosylmethionine (SAM), and elevated
to be significantly higher in autistic subjects (Israngkun et al.,
levels of intracellular homocysteine content. Additionally, the
1986). With HPLC analysis high urinary serotonin and low
levels of intracellular total cysteine and glutathione were
whole blood serotonin in autism and age effects on the levels
reduced. The results pointed to altered metabolism in immune
of serotonin were found (Hérault et al., 1996). Further
cells and involvement of inflammation in autism (Suh et al.,
studies showed that urinary excretion of serotonin metabolite
2008). The next study provides an application of LC-MS for
5-hydroxyindoleacetic acid (5-HIAA) in autistic patients under
the analysis of plasma amino acids (AA) levels in autistic
study was similar to that reported for healthy individuals. An
children. Seven of the 20 measured AA were significantly
analysis of 5-HIAA in the urine of 20 autistics was made using
decreased in autistic children: glutamine, threonine, aspara-
high-performance liquid chromatography with fluorescence
gine, citrulline, serine, tyrosine and leucine. Glutamate was
detection (HPLC-FLD; Mulder et al., 2005). The results of GC-MS
the only AA for which the levels were higher in the autistic
analysis of urine demonstrate significant differences in the level
group compared with the control group. An age-dependent
of homovanillic acid and vanillylmandelic acid between 20
decrease in plasma glutamate as well as aspartic acid was
autistic and 36 healthy children. These results explain some
observed only in healthy children. Effects of gender, ethnicity,
disruptions of functioning of the dopaminergic system, such as
time of blood draw, medications, cognitive function on AA
repeated behaviors, mood disorders, aggression attacks and
levels within each group or of the diagnosis (i.e. autism vs per-
disorders of social relationships, observed in the case of autistic
vasive developmental disorder – not otherwise specified), as
children (Kałużna-Czaplińska et al., 2010a). Two further studies
well as any associations between clinical scores, namely Autism
applied chromatography to test the relationship between child-
Diagnostic Interview, Revised and Autism Diagnostic Observation
hood and brain opioid activity. In the first study urinary opioid
Schedule, and any AA levels in the autistic group were not
peptides were examined by HPLC coupled with matrix-assisted
observed in this particular study group (Tirouvanziam et al.,
laser desorption ionisation time-of-flight mass spectrometry
2012). Another chromatographic HPLC-FLD analysis of plasma
(Cass et al., 2008), while in the second study they were tested
amino acids was performed in the group of autistic and Asperger
by liquid chromatography coupled with ultraviolet and mass
Syndrome patients, their siblings and parents. In this group six of
spectrometric detection (Hunter et al., 2003). Opioid peptides
the 17 measured amino acids were raised. These were glutamic
were not detected in autistic subjects, which is why these
acid, phenylalanine, asparagine, tyrosine, alanine and lysine
studies are in opposition to the opioid peptide excess theory
(Aldred et al., 2003). In another study of plasma amino acid
for the cause of autism. Another study shows the use of HPLC
analysis was performed by HPLC coupled with tandem mass
with fluorescence detection in the analysis of CSF levels of
spectrometry (MS/MS) technique. Twenty primary amino acids
indoleacetic acid, which is the metabolite of tryptamine, a
(proteinogenic) and 21 secondary amino acids with amino acid
central neuromodulator. There was no significant difference
metabolites were analyzed. Considering the primary amino
in the levels of CSF indoleacetic acid between the autistic
acids, the autistic children had significantly lower levels of tryp-
and healthy groups (Anderson et al., 1988).
tophan and higher levels of glutamate, slightly higher serine,
and slightly lower tyrosine and phenylalanine in plasma. The
analysis of secondary amino acids showed that the autistic
Nutritional and metabolic status
group had a higher level of b-amino isobutyrate and a lower
Feeding habits of children with autism are very specific. These level of taurine in plasma (Adams et al., 2011). GC-MS technique
children may have problems in three areas: food selectivity made it possible to determine the levels of homocysteine in
based on type, color and texture; food refusal; and disruptive the urine of autistic and healthy children. The results showed
mealtime behaviors. A selective, deficient diet can significantly increased levels of homocysteine in the urine of autistic children.
exacerbate the symptoms of the gastro-intestinal tract and The higher level of homocysteine in autistic children may indicate
nervous system. Chromatographic techniques allow the quanti- deficiencies of folic acid and vitamins B6 and B12 in nutrition of
tative determination of both nutrients and their metabolites, these children (Kałużna-Czaplińska et al., 2011a). GC-MS analysis
which in turn makes it possible to set an individual uptake of was used to determine the level of urinary tryptophan in autistic
nutrients and to find metabolic abnormality markers. Selected children. It showed that they had significantly lower urinary
antioxidants were analyzed with HPLC for both autistic and levels of tryptophan compared with healthy children. Defi-
healthy children. The study included vitamins C, E, A, carotenoids ciency in tryptophan can lead to the worsening of autistic
b-carotene and lycopene. In older autistic children significantly symptoms such as mild depression and increased irritability
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higher plasma values of vitamin C and b-carotene than in the (Kałużna-Czaplińska et al., 2010b). Two discordant reports show

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Chromatographic methods in the study of autism
the use of chromatographic techniques for analysis of indolyl-
3-acryloylglycine (IAG) in the urine of autistic subjects. In the
first study IAG levels were detected with HPLC coupled with
UV detection. The results of analysis showed very significant
increases in the levels of urinary IAG in the autistic group.
According to the authors, urinary IAG may constitute an objec-
tive diagnostic indicator for autism (Bull et al., 2003). On the
other hand, the results of other studies carried out with the
use of HPLC-MS/MS suggest that there is no association
between the presence of IAG in urine and autism, so it can
be used neither for diagnosis nor for therapeutic intervention
in the diet (Wright et al., 2005). GC-MS was used for the analysis
of urinary dicarboxylic (succinic, adipic and suberic) acids as
markers of metabolic disorders in children with autism before
and after vitamin B2, vitamin B6 and magnesium supplementa-
tion. The levels of all acids were significantly higher in the
group of children before supplementation. The reduction of
urinary dicarboxylic acids was associated with the improve-
ment of some autistic symptoms such as the ability to concen-
trate and make eye contact (Kałużna-Czaplińska et al., 2011b).
Plasma fatty acid profiles of autistic patients and age-matching
control subjects were obtained with a gas chromatograph with a
flame ionization detector. Fatty acids in the plasma of Saudi
autistic patients were increased, especially in most of the satu-
rated fatty acids except for propionic acid, and decreased in most
of the polyunsaturated fatty acids. The results were related to
oxidative stress, mitochondrial dysfunction and a high concen-
tration of lead (Pb) previously reported in Saudi autistic patients
(El-Ansary et al., 2011). The aim of the next study was to investi-
gate brain energy metabolism in autistic children through mea-
suring plasma polyunsaturated fatty acids (PUFAs), serum
carnitine and plasma lactate, which is an index of mitochondrial
function, in relation to autistic severity and clinical manifestations.
The determination of the levels of polyunsaturated fatty acids in
plasma was made by gas chromatography. Autistic patients had
significantly lower plasma PUFAs (except linoleic acid) than con-
trols. Severely autistic patients had lower values of plasma PUFAs
than patients with mild and moderate autism, but these differences
were statistically insignificant. Hypotonic autistic children (20%)
had significantly lower serum docosahexaenoic acid than
normotonic autistics (Mostafa et al., 2005). With GC-MS it was pos-
sible to detect a marked increase in analogs of Krebs cycle metab-
olites and arabinose in the urine of two brothers with autistic
features. The metabolites which were elevated were citramalic,
tartaric (3-OH-malic), and 3-oxoglutaric acids and compounds ten-
tatively identified as a citric acid analog and partially identified as a
phenylcarboxylic acid. The authors present three explanations for
such results. The first is that these siblings have a new genetic dis-
ease in which some abnormal metabolites characteristic of yeast
metabolism are excreted coincidentally but are not causally
related to autism. The second explanation is that these siblings
can be infected with one or more yeasts and/or bacteria but this/
these infection(s) are secondary to immune deficiency. The third
explanation is the infection of siblings with one or more microor-
ganisms. These microorganisms produce metabolites that are
causally related to the disease through inhibition of mitochondrial
Krebs cycle activity by citramalic, carboxycitric, 3-oxoglutaric and
tartaric acids, and (or) by interference in normal carbohydrate
metabolism caused by high concentrations of arabinose (Shaw
et al., 1995). The aim of another study was to establish whether
autism might have a distinctive chromatographic profile on uri-
nary analysis. Metabolic profiles were performed with HPLC over
Biomed. Chromatogr. 2013; 27: 1273–1279 Copyright © 2013 John Wiley & Sons, Ltd.
prepubertal boys. The results showed that the laboratory was able
to identify correctly the urine of boys with autism in over half of
the cases. It was also found that the results were markedly
improved when the analysis was restricted to those of normal abil-
ity or mild learning disability (Alcorn et al., 2004). Owing to the
chromatographic analysis of plasma sterols, it was possible to de-
termine the prevalence of autism in children with Smith–Lemli–
Opitz Syndrome (SLOS). Plasma cholesterol, 7-dehydrocholesterol
and 8-dehydrocholesterol were measured by capillary-column
gas chromatography. Most children with SLOS (characterized by
abnormal levels of sterols in plasma) had some variant of autism,
which may mean that SLOS appears to have the most consistent
relationship with autism of any single gene disorder (Sikora
et al., 2006). Liquid chromatography–electrospray ionization-
mass spectrometry (LC-ESI-MS) on time-of-flight instruments
made it possible to discover proteins altered by the disease. A
total of 6348 serum peptide components were analyzed. As a
result, five peptide components showed the biggest differences
between the autistic and typical groups, four of which corre-
spond to three proteins involved in the complement pathway
(Corbett et al., 2007). GC-MS was also used for the analysis
of the D-arabinitol, L-arabinitol and D-arabinitol/L-arabinitol
(DA/LA) ratio in the urine of autistic children as a biomarker of
candidiasis. The study included quantification before and after
the probiotic therapy. The results show that the level of DA
was significantly higher in the urine of autistic children before
and after probiotic supplementation. The probiotic supplemen-
tation caused a significant decrease in DA and DA/LA and an
improvement in the ability for concentration and carrying out
orders (Kałużna-Czaplińska and Błaszczyk, 2012).
Oxidative stress
Oxidative stress is a general term used to describe an imbalance
between the production and manifestation of reactive oxygen
species and the ability of the biological system to readily detox-
ify the reactive intermediates or to repair the resulting damage.
When the normal redox state of tissue is affected, the system
can respond through the toxic effect, that is by the production
of peroxides and free radicals, which damage all components
of the cell, including proteins, lipids and DNA. The task of
chromatographic techniques is the identification and quantifica-
tion of markers of oxidative stress and antioxidants in the body
fluids and tissues. An example of application of chromatography
in the study of oxidative stress in autism is LC-MS/MS analysis
of reduced and oxidized glutathione in plasma. Also, high-
performance liquid chromatography with fluoroscence detec-
tion (HPLC-FLD) analysis of plasma taurine was applied to study
the plasma of autistic patients. The results show significantly
decreased levels of the plasma taurine, reduced glutathione
and increased levels of the transsulfuration metabolite of oxi-
dized glutathione. Increased levels of plasma oxidized glutathi-
one were correlated with increasing mercury-associated urinary
porphyrins (Geier et al., 2008). HPLC coupled with an electro-
chemical detector was applied for the analysis of amino acids
to investigate the dynamics of an integrated metabolic pathway,
essential for cellular antioxidant and methylation capacity.
The results show that methionine and SAM were significantly
decreased, and S-adenosylhomocysteine (SAH) and adenosine
were significantly increased in the case of autistic children.
The SAM/SAH ratio, an indicator of methylation capacity, was
1277
decreased in children with autism. In the case of total cysteine,
wileyonlinelibrary.com/journal/bmc

Figure 4. Diagram of report frequency (2005–2012) using chromatography and biomarkers in the study of autism. Bibliographic research related to
the use of chromatography and biomarkers in autism investigations (PubMed). A total of 530 reports were found.
its levels were lower in children with autism. Cystine (was
considerably elevated in autistic children, and the plasma free
cysteine/cystine redox ratio was significantly lower. One can
conclude that the deficit in antioxidant and methylation capacity
is specific to autism and may promote cellular damage and
altered epigenetic gene expression (Melnyk et al., 2012).
Conclusions and significant predictions for
the future
As autism is not a pure genetic disease, the chromatographic
analysis of metabolites makes it possible to find the source of
the symptoms observed in this disease. However, the descrip-
tion of the human metabolome (a set of all metabolites) will
be an even more difficult challenge than describing the genome
and proteome. The reason is the large number of metabolites,
among which there may be one, or a few, that can answer
the question about the cause of autism. Chromatographic
techniques, especially GC-MS, GC-MS/MS, LC-MS, LC-MS/MS
and liquid chromatography–nuclear magnetic resonance–mass
spectrometry, are very important tools in clinical chemistry. In
contrast to the conventional biochemical analysis, chromato-
graphic techniques provide high reliability in trace analysis.
They contribute to the determination of concentrations of a
number of organic substances in biological samples, such as
body fluids, tissues and breath. Coupled techniques provide
specificity and sensitivity of the analysis. Analytical procedures
are rapid, easy and, in most cases, do not require complicated
sample pretreatment. There is higher reliability for chromato-
graphic determination of trace concentrations in biological
samples than in classic biochemical investigations.
According to the trend shown in Fig. 4, it can be expected that
the importance of chromatography and biomarker studies of
autism will continue to grow. So far, in the studies of autism,
chromatographic techniques have been applied in many areas.
An example is the identification and quantitative determination
of markers of neurological and metabolic disorders. With the
chromatographic techniques, it is possible to determine the
progression of the disease and to find relationships between
metabolite levels and the age of autistic children. It is becoming
1278
increasingly important to determine the nutritional status in
wileyonlinelibrary.com/journal/bmc Copyright © 2013 John Wiley & Sons, Ltd.
E. Żurawicz et al.
children with autism, which in turn enables an individual diet
and supplementation. The next step in the chromatographic
studies of the nutritional status in autism is the monitoring of
changes in metabolism during diet and supplementation. A very
important tool in the diagnosis of autism may also be determin-
ing the influence of environmental factors on children’s develop-
ment. These factors may be compounds that are foreign to a
living organism (e.g. drugs, carcinogens and various compounds
introduced into the environment by artificial means).
The most common limitations of chromatographic studies
connected with autism are relatively small and uneven sizes
of patients and control groups, lack of matching by gender
and imprecise matching for ethnicity and age. Such limitations
make the generalization of the results difficult and uncertain.
One of the priorities in the investigation of autism should be
a search for early biomarkers of this disease, and here
chromatographic techniques can play a key role. Rapid identi-
fication of high-risk children would give the opportunity to
monitor metabolic and neurological changes that predict the
beginning of behavioral symptoms of autism, thus making it
possible to counter the early symptoms.
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