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Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb
a r t i c l e i n f o a b s t r a c t
Article history: Healthy body is characterized by the presence of a dynamic and balanced equilibrium between the pro-
Received 30 July 2015 duction of reactive oxygen species (ROS) and the antioxidant capacity. In oxidative stress this balance
Received in revised form is switched to reactions of oxidation leading to increased production of ROS, exceeding the capacity of
17 December 2015
physiological antioxidant systems. Oxidative stress is known to be linked to many disturbances, disor-
Accepted 18 December 2015
ders and diseases. One of these is the autism spectrum disorder (ASD). ASD is a neurodevelopmental
Available online xxx
disorder manifested by abnormalities in social communication and interaction, as well as by occurrence
of repetitive, restricted patterns of behavior or activities. It is believed that adequate knowledge about
Keywords:
Autism
the oxidative stress biomarkers and the possibility of their reliable measuring could be useful in broad-
Biomarkers ening knowledge on various diseases including ASD. A high number of compounds have been proposed
Chromatography as biomarkers of oxidative stress. Some of these are connected with the severity of ASD. The present
Mass spectrometry review gives a summary of the chromatographic techniques used for the determination of biomarkers
Oxidative stress for oxidative stress in autism, and of other compounds important in this context. The first part of the
review focuses on the correlation between oxidative stress and autism. The second part describes appli-
cations of chromatographic and mass spectrometric methods to the analysis of different metabolites
connected with oxidative stress in biological fluids of autistic children. Advantages as well as disadvan-
tages of the application of these methods for the analysis of different types of oxidative stress biomarkers
are discussed.
© 2015 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jchromb.2015.12.035
1570-0232/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: J. Kałużna-Czaplińska, J. Jóźwik-Pruska, Chromatographic and mass spectrometric techniques in
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Please cite this article in press as: J. Kałużna-Czaplińska, J. Jóźwik-Pruska, Chromatographic and mass spectrometric techniques in
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disorders, or diseases. Nowadays, a great number of databases mal conditions, NADPH-dependent enzyme glutathione reductase
for those compounds are available. One of these is Human (GR) converts oxidized glutathione (GSSG) to GSH maintaining an
Metabolomic Database [27]. optimal GSH/GSSG redox ratio. Yet, under conditions of elevated
oxidative stress, GSSG is exported by the cell in order to regain
2.2. Consequences of oxidative stress in ASD intracellular redox homeostasis. This may result in a decrease in the
total amount of GSSC available to recycle to GSH. Moreover, GSH
It is well known that molecular oxygen is essential for aero- participates in the elimination of toxins, including xenobiotics and
bic life, yet excessive amounts of its metabolic by-products are heavy-metals, from cells [39,40]. According to the literature GSH
toxic. This “oxygen paradox” raises the theory of oxidative stress concentration and GSH/GSSG ratio are decreased, whereas GSSG
[28]. Free radicals participate in cellular signaling, mitosis and concentration is increased in ASD patients [31,38,41].
physiological immunological responses. However, because of their The regeneration of methionine to homocysteine occurs in the
instability and the presence of unpaired electrons, they can dam- methionine cycle. Methionine, activated by methionine adenosyl-
age lipids, proteins, nucleic acids and carbohydrates [29]. Due to the transferase, forms S-adenosylmethionine (SAM), which is the basic
fact that especially brain requires large amounts of oxygen, oxida- methyl donor for most cellular methyltransferase reactions, includ-
tive stress is thought to be a significant factor in the pathogenesis ing the methylation of proteins, RNA, DNA, and neurotransmitters.
of psychiatric disorders [30]. The role of oxidative stress in autism The methyl group from SAM can be transferred to various accep-
is also considered. Obtained results seems to be promising and may tors, leading to the formation of S-adenosylhomocysteine (SAH)
become an important element both in pointing biomarkers of this and adenosine. However, homocysteine may also be remethylated
disorder as well as in establishing proper therapy aiming to the to methionine or be removed from the methionine cycle. A decrease
mitigation of occurring symptoms [21,22,30,31]. in methionine results in a decrease in the synthesis of SAM, as well
One of the disorders that have been linked to elevated oxida- as in a decreased synthesis of cysteine and GSH [31]. Methylation
tive stress and lower anti-oxidant capacity is autism [1,20,31]. abnormalities in children with ASD are reported in the literature
Oxidative stress influences membrane phospholipids, the immune [38,42,43] (Scheme 2).
system, metabolic pathways involved in energy, and excitotoxicity. Homocysteine also serves as a biomarker for possible deficien-
It is believed that this influence results in clinical symptoms and in cies of some vitamins, including vitamins B6 , B12 , and folic acid.
the pathology of ASD [30]. A high number of cell membrane pro- This amino acid is metabolized via remethylation to methionine or
teins are attached to membrane phospholipids or embedded in its transsulfuration to cysteine [44–46]. The former involves folic acid
structure. Thus, membrane phospholipid abnormalities affect the and vitamin B12 , while the latter requires vitamin B6 . Accumulation
functioning of these proteins. Additionally, the production of lipid of homocysteine occurs when either of these pathways is defected
peroxides and their by-products leads to the loss of integrity and [46].
functions of the cell membrane [32]. 8-Hydroxy-2’deoxyguanosine (8-OHdG) can be measured in
Literature also reports on impaired immune system response urine or in DNA by various methods. This compound is biomarker
and increased inflammatory response in ASD patients connected for oxidative damage to DNA [21,47]. It is commonly determined
with oxidative stress [33,34]. Excitotoxicity is considered to con- despite there are confounding factors and intra individual varia-
tribute to oxidative stress, simultaneously being the result of the tions. Additionally, the results obtained from independent studies
process. An increased concentration of extra-cellular glutamate are inconsistent [21]. Literature reports that the elevated levels of
(confirmed in autism) and a reduced concentration of gamma- urinary 8–OHdG in ASD patients are not significant [20,21].
aminobutyric acid (GABA) are reported to increase excitotoxicity Isoprostanes are prostaglandin-like molecules produced in vivo
[35]. via nonenzymatic free radical-catalyzed peroxidation of arachi-
One of the most commonly found abnormalities in autism is donic acid [48,49]. The first reports on the formation of these
mitochondrial dysfunction which is marked by impaired energy compounds by auto-oxidation of polyunsaturated fatty acids date
production. Mitochondrial dysfunction may result from many fac- back to the mid-1970s [50]. Yet, only about 20 years later it was
tors such as vitamin B6 or iron deficiency, elevated nitric acid, proved that they are formed in vivo in human body [48]. The F2 -
exposure to environmental toxicants, or oxidative stress [21]. isoprostanes group may consist of up to 64 compounds that are
Patients with ASD are often supplemented with antioxidants in isomeric in the structure to the cyclooxygenase-derived PGF2 ␣
order to minimize some symptoms of this disorder. Supplements [51]. Measurement of F2 -isoprostanes is now considered one of the
such as vitamin C, vitamin B6 (especially in combination with mag- most reliable tools to assess the status of oxidative stress in vivo.
nesium), zinc, and fish oil are included in the diet [36]. These substances have been found to be elevated in ASD patients
Scheme 1 shows the relationship linking oxidative stress to ASD. [13]. However, it should be mentioned that some F2 -isoprostanes,
notably 8-iso-PGF2 ␣, may also be produced from arachidonic acid
2.3. Oxidative stress biomarkers in ASD by the cyclooxygenase, and that PGF2 ␣ may also be formed nonen-
zymatically from arachidonic acid.
Human body is not fully protected against oxidative damage. Plasma 3-chlorotyrosine (3CT), a measure of myeloperoxi-
Some of its constituents are at risk of injury by ROS. The reaction dase activity, is well known biomarker of chronic inflammatory
of ROS with biomolecules results in the formation of oxidatively response. 3CT is formed by the reaction of p-tyrosine with
modified products. Unlike the ROS, their reaction products with hypochlorous acid (HOCl) [52]. It is reported that the concentra-
biomolecules are chemically stable and are therefore often used as tion of plasma 3CT is connected with mitochondrial dysfunction
biomarkers of oxidative stress [1]. and increases with age [53]. The literature [53] reports that plasma
Children with ASD are known to suffer from metabolic abnor- levels of 3CT in ASD children are significantly higher as compared
malities which are related to the interconnected pathways of with healthy children. Moreover, autistic patients who met crite-
glutathione, methionine and folate metabolism [37,38]. Among the ria for probable or define mitochondrial disease were characterized
compounds which have been found to be important in ASD, GSH is by higher levels of 3CT than autistic patients without any biological
one of the most frequently studied. GSH is composed of cysteine, markers or symptoms of mitochondrial disease. However, elevated
glutamate and glycine and serves as a major intracellular redox concentration of 3CT in ASD children may suggest the presence of
buffer. Under conditions of chronic ROS formation, GSH can become chronic inflammation.
depleted leading to disruption in redox homeostasis. Under nor-
Please cite this article in press as: J. Kałużna-Czaplińska, J. Jóźwik-Pruska, Chromatographic and mass spectrometric techniques in
studies on oxidative stress in autism, J. Chromatogr. B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.12.035
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3-Nitrotyrosine (3NT) is as a specific marker for the nitrating The major antioxidant proteins synthesized in some tissues
species such as peroxynitrite from which it is formed. Additionally, including the brain are ceruloplasmin and transferrin. Cerulo-
it can serve as a biomarker of neuron death and a measure of chronic plasmin is ␣2-serum glycoprotein responsible for the transport
immune activation. Although it has been widely studied with the of copper ions in blood. Its role in the metabolism of copper is
use of various techniques, its levels are reported to differ 30-fold essential. Moreover, in red blood cell membranes ceruloplasmin
among different studies [54]. It can be explained by the assumption protects polyunsaturated fatty acids form ROS. Transferrin can be
that 3-NT can be generated easily ex vivo in the process of sample found in body fluids, yet its higher concentrations occur in serum.
preparation and analysis [54,55]. Its measurement has been cor- Its main role is the transport of iron to proliferating cells and it
related with the measurement of the cognitive function, behavior serves as an antioxidant reducing the level of free ferrous ions [56].
and the development for patients with ASD and mitochondrial dys- Literature reports mainly about their quantification using a colori-
function, but not for the patients with ASD without a mitochondrial metric method for ceruloplasmin described by Karl et al. [57]. and
dysfunction [53]. Buglanov et al. [58]. method for transferrin Plasma levels of ceru-
Please cite this article in press as: J. Kałużna-Czaplińska, J. Jóźwik-Pruska, Chromatographic and mass spectrometric techniques in
studies on oxidative stress in autism, J. Chromatogr. B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.12.035
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loplasmin and transferrin were shown to be decreased in autistic then just coming to the market, appeared to be a key to success
patients [56,57,59]. [74,75].
SOD is found in the cytosol of most cells. Ceruloplasmin and SOD The first coupling of chromatography was explored in 1955
are reported to play a significant role in some neurodegenerative by Roland Gohlke and Fred McLafferty of Dow Chemical Com-
diseases such as Parkinson’s disease, Alzheimer’s disease, Down’s pany, who combined homemade gas chromatograph with a
syndrome [60]. In the older literature [61], an increased activity time-of-flight (TOF) instrument. In 1968 Victor Tal’rose published
of superoxide dismutase was found. Yet, in more recent reports information (in Russian Journal of Physical Chemistry) about the
[62] the activity of SOD in erythrocytes has been noticed to be sig- first attempt to connect LC–MS. In 1973 Baldwin and McLafferty
nificantly lower in ASD patients. Other studies present normal SOD developed direct liquid introduction (DLI) LC–MS interface, which
activity in the plasma of ASD subjects compared to controls [60,63]. was capable of generating a stable ion beam in the mass spectrom-
The most studied product of polyunsaturated fatty acids perox- eter [74,75].
idation is malondialdehyde (MDA) [64,65]. The literature reports Chromatographic methods are among the most frequently used
that the mean plasma level of MDA is significantly higher among coupled techniques in the field of study of oxidative stress in autism.
patients with ASD compared to controls, but only for patients being Chromatographic techniques are characterized by many advan-
younger than 6 years of age. In older subjects there is no significant tages especially in describing the composition of many biological
difference between these two groups regarding MDA [65]. matrix including body fluids.
It should be highlighted that levels of some metabolites may GC–MS has been employed in clinical biochemistry and urinary
be related to autism severity. Scientists [66] associated several analysis for more than 30 years. Generally, this technique has slight
of the biomarker groups, including vitamins (vitamin B6, vita- sample requirements and enables rapid analyses and reduced use of
min C, n-methyl-nicotinamide), minerals (calcium, iron, zinc), expensive labeled substrates. Additionally, it allows obtaining mea-
primary amino acids, secondary amino acids and the group of Sul- sureable results. Yet there are some disadvantages, such as lengthy
fation/Methylation/Glutathione/Oxidative Stress, with severity of sample preparation (hydrolysis/derivatization), and incapability of
this disorder (assessed using the Pervasive Development Disorder detection of non-volatile, polar and thermally labile compounds
Behavior Inventory (PDD-BI), Autism Treatment Evaluation Check- [76–78]. LC–MS and LC–MS/MS are among the most powerful tech-
list (ATEC), and Severity of Autism Scale (SAS)). niques used in the study of organic compounds. LC–MS has been
applied in the study of urine for about 30 years. The improvement of
liquid chromatography by the development of ultra-high-pressure
2.4. Positioning chromatographic and mass spectrometric or ultra-performance techniques resulted in high chromatographic
methods in the studies of oxidative stress in autism peak resolution and reduced time of analysis while maintaining
high-quality mass spectrometry [76,80].
Many potential biomarkers have been considered in oxidative GC–MS/MS, UPLC–MS, HPLC–TOF–MS, LC–TOF–MS have also
stress assessment and many analytical techniques have been pro- been used for the measurement of oxidative stress biomarkers. Yet,
posed and used for its measurement. The Yagi [67] method is GC–MS, HPLC–MS, LC–MS, and LC–MS/MS are of particular interest.
widely applied in both clinical and experimental studies, and is one The most common coupling technique for chromatography is
of the most famous methods used to evaluate lipid peroxidation. mass spectrometry (MS). In clinical studies almost all mass spec-
The approach is based on the measurement of substances which trometers are single MS or tandem mass (MS/MS) coupled to LC or
react with thiobarbituric acid (TBA), the so called thiobarbituric GC. Tandem mass spectrometry is characterized by high selectiv-
acid reactive substances (TBARS). Recently, chromatography-based ity, sensitivity and minimum of interferences. Yet, when used in
techniques have become a powerful tool in the determination of the same study population, the obtained results were reported to
oxidative stress biomarkers. be similar [79].
In order to ensure reliability of obtained results, some Homocysteine (HCY) was measured with the use of GC–MS in
requirements must be met. For this purpose scientists use a system- urine of autistic (n = 34) and healthy (n = 21) children and found to
suitability test (SST) which is an essential part of chromatography. be higher in the ASD group (2.36 ± 1.24 vs. 0.76 ± 0.31 mmol/mol
Requirements for chromatographic analyses are specified by Euro- creatinine). The method was validated for recovery, precision,
pean Pharmacopoeia (EP) and demonstrate that a chromatograph limit of quantification (LOQ), limit of detection (LOD), and linear-
is fit for the analysis. System suitability tests are run each time an ity. Sample preparation was based on a simultaneous extraction
analysis is undertaken and each SST is specific for an individual (chloroform) and derivatization (pyridine, ethanol and ethylchlo-
method [68]. SST is applied to verify whether a planned analy- roformate). Elevated HCY concentration may correlate with
sis will be performed by a chromatographic system with correct deficiencies of folic acid and vitamins B6 and B12 in nutrition [80].
detection sensitivity, reproducibility and resolution. The quality of Further studies showed the impact of supplementation with either
the results is demonstrated by using quality control (QC) systems folic acid or vitamins B6 and B12 . The concentration of HCY in urine
involving procedures and techniques performed to ensure that samples was measured in autistic patients before (n = 30) and after
quality requirements (see for example Good Laboratory Practice, intake (n = 24) of these supplements or after supplementation with
quality-assurance system (ISO 9001) and testing and calibration vitamins B6 and B12 alone (n = 6). The obtained results revealed that
(ISO 17025) containing general rules [69]). both supplementations led to the decrease in the HCY level, yet the
A wide range of techniques, including for example spectropho- intake of the mentioned vitamins and folic acid was more effective
tometric analyses [59,61,70], ELISA [71–73], radioimmunoassay in lowering the urinary levels of HCY [81]. In order to determine
[73], have been used to measure oxidative stress biomarkers. Not HCY in blood samples, James et al. [31]. employed HPLC. Plasma
only do they allow identification of these markers but they also concentration of HCY was lower in ASD individuals (5.8 ± 1.0 M;
enable their reliable quantification in different biological matrixes n = 20) compared to healthy subjects (6.4 ± 1.3 M; n = 33).
including whole blood, plasma, and erythrocyte. Oxidized and reduced glutathione (total glutathione tGSH) in
Mass spectrometry has been used for more than 60 years. By plasma samples of ASD and healthy patients were measured by
the 1950s, this technique was a well-established technology. It HPLC with electrochemical detection. Authors used metaphospho-
enabled analyses of volatile compounds in the petroleum, chem- ric acid to precipitate protein. The tGSH level was significantly
ical and pharmaceutical industries. Yet, the desire for availability lower in ASD individuals (4.1 ± 0.5 vs. 7.6 ± 1.4 M), while higher
of rapid, online separation method was growing. Chromatography, levels of oxidized glutathione in autistic subjects were measured
Please cite this article in press as: J. Kałużna-Czaplińska, J. Jóźwik-Pruska, Chromatographic and mass spectrometric techniques in
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6
Table 1
Determination of the selected biomarkers of oxidative stress in human body fluids with the use of chromatography-based techniques, applied parameters and exemplary results.
Compound Function in human body Disturbance in ASD Reported levels in ASD Matrix Chromatography- Chromatographic conditions Ref.
patients patients based
technique
Glutathione Critical endogenous Decreased 4.1 ± 0.5 mol/L Plasma HPLC Reverse-phase [31]
antioxidant & detoxifer 5-m C18 column
Decreased Data presented in figure Cerebellum and HPLC Reverse-phase [41]
∼5 nmol/mg protein temporal cortex 5-m C18 column; isocratic
elution sodium phosphate
monobasic, monohydrate,
ion-pairing reagent OSA,
acetonitrile, pH 2.7 (85%
phosphoric acid)
ARTICLE IN PRESS
concentration of GSSH 5-m C18 column
is connected with Increased Data presented in figure Cerebellum and HPLC Reverse-phase [41]
decreased levels of ∼1 nmol/mg protein temporal cortex 5-m C18 column; isocratic
glutathione- a major elution sodium phosphate
antioxidant monobasic, monohydrate,
ion-pairing reagent OSA,
acetonitrile, pH 2.7 (85%
phosphoric acid)
Increased 0.447 ± 0.13 nmol/ml Plasma HPLC-MS/MS Not given [65]
Homocysteine Essential for proper Decreased 5.8 ± 1.0 mol/L Plasma HPLC Reverse-phase [31]
development of 5-m C18 column
nervous system. source Increased 2.36 ± 1.24 mmol/mol Urine GC–MS Full scan (m/z 50–500) SIM (m/z [78]
of methyl groups creatinine 56,128,234)
Isoprostanes Formed in vivo by Increased F2␣2-VI: Urine TLC with According to method described by [85]
nonenzymatic free 5.2 ± 0.5 ng/mg creatinine GC–NCI–MS Lawson et al. [89]
radical-catalyzed Increased F2-IsoPs: Plasma GC–NCI–MS Ion m/z 569 to ion m/z 573 ratio [22]
peroxidation of ASD-GID 53.6 ± 24.4 pg/mg method described by Milne [90]
arachidonic acid total protein and
ASD-only Morrow [91]
36.5 ± 13.3 pg/mg total
protein
3NT Biomarker of neutron Increased Data presented in figure Cerebellum and HPLC 5-m C18 column; isocratic [41]
death and a measure of ∼80 pmol/mg protein temporal cortex elution sodium phosphate
chronic immune monobasic, monohydrate,
activation ion-pairing reagent OSA,
acetonitrile, pH 2.7 (85%
phosphoric acid)
Increased 16.6 ± 7.8 g/l Plasma HPLC–MS/MS Not given [65]
3CT Serves as a marker for oxidative Increased Data presented in figure Cerebellum and HPLC Reverse-phase [41]
damage; has been shown to cause ∼70 pmol/mg protein temporal cortex 5-m C18 column; isocratic
endothelial dysfunction and elution sodium phosphate
production of free-radical monobasic, monohydrate,
ion-pairing reagent OSA,
acetonitrile, pH 2.7 (85%
phosphoric acid)
CHROMB-19792; No. of Pages 11
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studies on oxidative stress in autism, J. Chromatogr. B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.12.035
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Table 1 (Continued)
ARTICLE IN PRESS
patients patients based
technique
Methionine Essential amino acid; Decreased 19.3 ± 9.1 mol/L Plasma HPLC Reverse-phase [31]
builds various protein 5-m C18 column
molecules, participates 22 ± 9 mol/L Plasma HPLC with photodiode = 254 nm [81]
in the synthesis of array detection
cysteine
SAM Common cosubstrate Decreased 75.8 ± 16.2 nmol/L Plasma HPLC Reverse-phase [31]
involved in methyl 5-m C18 column
group transfers, 214.5 ± 15 mol/dl RBC HPLC–MS/MS not given [65]
transsulfuration, and
aminopropylation
SAH Formed by the Increased 28.9 ± 7.2 nmol/L Plasma HPLC Reverse-phase [31]
demethylation of SAM; 5-m C18 column
intermediate in the 44.6 ± 8.0 mol/dl RBC HPLC–MS/MS Not given [65]
synthesis of cysteine
and adenosine.
Adenosine Plays important role in Increased 0.39 ± 0.2 mol/L plasma HPLC Reverse-phase [31]
energy transfer and 5-m C18 column
signal transduction; 23.2 ± 5.9 10–8 mol/l plasma HPLC–MS/MS Not given [65]
neuromodulator;
regulates a blood flow
7
8
Table 2
Pitfalls in the determination of the selected biomarkers of oxidative stress with the use of various methods.
Compound Pitfalls/interferences in Description Studied body fluid Type of studies Used method Ref.
determination
15(S)-8-iso- Artefactual formation Formation of 15(S)-8-iso-PGF2 ␣ from Plasma In vitro GC–MS/MS [94]
prostaglandin prostaglandin (PG) h synthases
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upon GSH
Artefactual formation Plasma concentration of Plasma In vivo ELISA commercial kit [95]
15(S)-8-iso-prostaglandin F2 ␣
increased by N-acetyl-cysteine (NAC)
(precursor of GSH) and vitamin C.
probable reason may be GSH- and/or
vitamin C-promoted and
pghs-mediated formation of
15(S)-8-iso-prostaglandin F2 ␣
Method validation Low concentrations of compounds Plasma, urine – LC–MS, LC–MS/MS, GC–MS and [93]
challenges cannot be determined because of lack GC–MS/MS allow for accurate,
of sensitivity of applied method sensitive quantification
GSSG Artefactual oxidation Formation of GSSG in GSH-containing Plasma In vitro GC–MS/MS an HPLC–UV [94]
PGHS incubation mixtures. process
dependent upon PGHS concentration
and incubation time.
Artefactual oxidation Sample acidification may result in Body fluids In vitro Chromatography-based techniques [93]
overestimating concentration.
GSH Artefactual oxidation Oxidation during blood withdraw, Blood In vitro Chromatography-based techniques [93]
centrifugation and handling.
8OHdG Artefactual formation Oxidative modification of guanine to HeLa cells vs human In vitro HPLC–ECD, HPLC–MS/MS [93,96]
8OHdG. lymphocytes
(0.55 ± 0.2 vs. 0.32 ± 0.1 nmol/L) were observed [31]. A validated array detection were lower in ASD patients [83]. Literature also
(LOD, accuracy, precision, recovery values were given) HPLC reports on decreased levels of plasma total cysteine (163 ± 15
method with coulometric electrochemical (EC) detection showed vs. 201 ± 17 M), and SAM (75.8 ± 16.2 vs. 96.9 ± 12 nM). In the
decreased levels of GSH and of the GSH/GSSG ratio in both cere- same study, significantly higher levels of SAH (28.9 ± 7.2 vs.
bellum and temporal cortex of autistic children (n = 15) compared 19.4 ± 3.4 nM) and adenosine (0.39 ± 0.2 vs. 0.27 ± 0.1 M) in
to the control group (n = 12). Proposed method eliminates time- plasma samples were observed [31]. Similar results were obtained
consuming precolumn derivatization step [41]. Lower levels of by other scientists by LC–MS/MS. SAM turned out to be signifi-
plasma glutamine and higher levels of plasma glutamate in autis- cantly lower in ASD patients, while SAH did not significantly differ
tic children (n = 23) compared to healthy controls (n = 22) were between ASD vs. control groups. The SAM/SAH ratio was lower in
reported by Shimmura et al. with the use of the same analytical tool autistic individuals. Additionally, adenosine was slightly higher in
[82]. The concentration of plasma glutamic acid measured by HPLC children with ASD [66]. Other compounds determined in ASD indi-
was found to be elevated in ASD children (120 ± 89 vs. 83 ± 35 M) viduals are porphyrins. Specific porphyrin profiles are associated
[83]. Other studies based on the analysis of plasma samples by HPLC with high exposure to mercury and other toxic compounds. Sci-
confirmed these results and demonstrated that the increase of glu- entists studied pentacarboxyporphyrin (5cxP), precoproporphyrin
tamic acid is also maintained in the elderly [84]. Supplementation (prcP), and coproporphyrin (cP) in urine samples and the analy-
with methylcobalamin and folinic acid has been reported to result sis of these compounds in autistic patients (n = 20) and healthy
in the increase in GSH concentrations and the GSH/GSSG redox ratio controls (n = 20) showed their elevated levels in the first group
[42]. (5cxP 5.1 ± 1.3 vs. 4.45 ± 2.09; prcP 21.1 ± 6.8 vs. 17.9 ± 9.3; cP
Plasma methionine was determined by Naushad et al. [83]. 253 ± 74.4 vs. 195 ± 88 nmol/g creatinine). Yet, no significant differ-
with the use of HPLC with photodiode array detection. Children ences were found in non-Hg associated urinary phorphyrins (e.g.,
with ASD were reported to have lower concentrations of methi- heptacarboxyporphyrin) [88]. 8-Oxo-deoxyguanosine, a biomarker
onine (22 ± 9 M, n = 138) in comparison with heathy controls of oxidative DNA damage, was determined in brain tissue of ASD
(28 ± 9 M, n = 138). Similar results were obtained by other sci- individuals and unaffected controls with the use of HPLC–MS. The
entists (19.3 ± 9.1 M in the ASD group vs. 31.5 ± 5.7 M in the level of 8-oxo-deoxyguanosine was elevated in ASD patients and
healthy group) [31]. correlated inversely with GSH/GSSG in the cerebellum [41].
Quantification of tryptophan in plasma samples was performed The studies cited and discussed above clearly indicate the role
with the use of many chromatographic tools. The level of tryp- of oxidative stress in ASD. Reported levels of the biomarkers of
tophan was reported to be decreased in plasma samples of ASD oxidative stress and the compounds differ among the investigator
patients compared to controls (24 ± 11 vs. 41 ± 16 M) as measured groups due to differences in the populations studied, the severity of
by HPLC with photodiode array detection [83]. Others confirmed ASD symptoms and due to other conditions including the analytical
these results with the application of LC–MS/MS [66]. Similar results methods used. Nevertheless, the levels of biomarkers show a clear
were obtained for tryptophan in urine samples using GC–MS. For tendency in differences between ASD and healthy controls.
this purpose authors applied simultaneous extraction and deriva- Table 1 summarizes data for the determination of compounds
tization. Scan range of the total ion chromatogram was from m/z which are significant in oxidative stress with the application of
50 to 550. The mean level of tryptophan in ASD children with a chromatographic techniques.
restricted diet low in casein and gluten (1.98 ± 1.17 g/mL, n = 10), It should be highlighted that analysis and quantification of
as well as without a diet (7.44 ± 1.33 g/mL, n = 23) was lower than biomarkers of oxidative stress (both well-established and poten-
in healthy controls (14.24 ± 2.01 g/mL, n = 21) [85]. tial) involves the risk of occurrence of pitfalls and mistakes. The
The identification and quantification of 3-nitrotyrosine were origin of these difficulties may relate to analytical process, includ-
performed using a wide range of chromatographic methods, includ- ing artefactual generation of biomarkers during sample preparation
ing HPLC, GLC–MS [55]. Studies conducted by Rose et al. [41]. with and the lack of sensitivity, selectivity and specificity of the proposed
the use of HPLC with coulometric electrochemical (EC) detection method. MS-based analytical methods, especially LC–MS/MS and
presented a significant increase in 3NT and 3CT in cerebellum and GC–MS/MS, are known to be more selective and specific. In order to
temporal cortex of autistic patients (n = 15) compared to healthy avoid interferences additional chromatographic steps can be used
controls (n = 12). Researchers eliminated precolumn derivatization (prior to detection). Artefactual formation of biomarkers may also
step. arise from enzyme-catalysed reactions, which are not necessarily
Application of gas chromatography/negative ion chemical associated with oxidative stress. In Table 2 we summarized data
ionization-tandem mass spectrometry (GC/NICI–MS/MS) allowed about pitfalls, which may occur in the determination of oxidative
the determination of F2 -isoprostanes in the erythrocyte mem- stress biomarkers [92–94].
brane of ASD patients (n = 15) and healthy subjects (n = 15), and
revealed significantly elevated levels of these compounds [86]. 3. Conclusions
Consistent results were obtained for urinary analysis of the F2-
isoprostane F2 ␣-VI (ASD group 5.2 ± 0.5; controls 3.1 ± 0.3 ng/mg Chromatographic and mass spectrometric techniques are use-
creatinine) [87]. Validated GC–NICI–MS and GC–MS methods were ful for the quantification of potential oxidative stress biomarkers
used by Gorrindo et al. [22]. to quantify F2 -isoprostanes in plasma in health and disease. The use of these techniques helped identify
samples of four groups ASD-GID (gastrointestinal dysfunction) biological disturbances, new biomarkers and potential factors that
(n = 27), ASD-only (n = 29), GID-only (n = 21) and controls (n = 10). may contribute to the development of ASD. Moreover, these meth-
F2 -Isoprostane plasma levels were elevated in the ASD-GID group ods may help create novel diagnostic methods and improve the
(53.6 ± 24.4 pg/mg total protein) compared to healthy controls therapy of patients suffering from ASD.
(17.3 ± 4.7 pg/mg protein). The ASD-only and GID-only groups
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