Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477

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Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

Review

Biomarkers of Oxidative Stress and Antioxidant Defense

a
Sema Demirci-Çekiç , Gülay Özkan , Aslı Neslihan Avan , Seda Uzunboy , b a a
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]]]]]]
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⁎ ⁎⁎
Esra Çapanoğlu b, , Reşat Apak a,c,
a
Department of Chemistry, Faculty of Engineering, Istanbul University-Cerrahpasa, Avcilar, 34320 Istanbul, Turkey
b
Department of Food Engineering, Faculty of Chemical and Metallurgical Engineering, Istanbul Technical Uviversity, Istanbul, Turkey
c
Turkish Academy of Sciences (TUBA), Vedat Dalokay St. No. 112, Cankaya, 06670 Ankara, Turkey

a r t i cl e i nfo a bstr ac t

Article history: A number of reactive oxygen and nitrogen species are produced during normal metabolism in human body.
Received 29 July 2021 These species can be both radical and non-radical and have varying degrees of reactivity. Although they
Received in revised form 27 October 2021 have some important functions in the human body, such as contributing to signal transmission and the
Accepted 11 November 2021
immune system, their presence must be balanced by the antioxidant defense system. The human body has
Available online 28 November 2021
an excellent intrinsic enzymatic antioxidant system in addition to different non-enzymatic antioxidants
having small molecular masses. An extrinsic source of antioxidants are foodstuffs such as fruits, vegetables,
Keywords:
Biomarkers herbs and spices, mostly rich in polyphenols. When the delicate biochemical balance between oxidants and
Oxidative stress antioxidants is disturbed in favor of oxidants, “oxidative stress” conditions emerge, under which reactive
Lipids species can cause oxidative damage to biomacromolecules such as proteins, carbohydrates, lipids and DNA.
Proteins This oxidative damage is often associated with cancer, aging, and neurodegenerative disorders. Because
DNA reactive species are extremely short-lived, it is almost impossible to measure their concentrations directly.
Antioxidant protection Although there are certain methods such as ESR / EPR that serve this purpose, they have some dis­
advantages and are quite costly systems. Therefore, products generated from oxidative damage of proteins,
lipids and DNA are often used to quantify the extent of oxidative damage rather than direct measurement of
reactive species. These oxidative damage products are usually known as biomarkers. Determination of the
concentrations of these biomarkers and changes in the concentration of protective antioxidants can provide
useful information for avoiding certain diseases and keep healthy conditions.
© 2021 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . 2

1.1. Reactive Oxygen Species (ROSs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . 2
1.2. Antioxidants (AOXs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . 3
2. Metabolic Oxidation Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . 4
2.1. Lipid Peroxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . 4
2.1.1. Lipid Peroxidation Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . 6
2.2. Protein Damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . 9
2.2.1. Oxidation of the Protein Backbone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . 9
2.2.2. Oxidation of Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . 9
2.2.3. Protein Carbonyl Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... 10
2.3. DNA Damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . 10
3. Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... 10
3.1. Enzymatic Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . 10
3.2. Non-Enzymatic Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... 12
4. Analytical Strategies for the Measurement of Oxidative Stress and Related Antioxidant Defense . . . . . . ..... . ..... . ..... . ..... . ..... . ..... 15

⁎
Correspondence to: Istanbul Technical Uviversity, Department of Food Engineering, Faculty of Chemical and Metallurgical Engineering, Istanbul, Turkey.
⁎⁎
Correspondence to: Istanbul University-Cerrahpaşa, Department of Chemistry, Faculty of Engineering, Avcilar, 34320 Istanbul, Turkey.
E-mail addresses: capanogl@itu.edu.tr (E. Çapanoğlu), rapak@istanbul.edu.tr (R. Apak).

https://doi.org/10.1016/j.jpba.2021.114477
0731-7085/© 2021 Elsevier B.V. All rights reserved.
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al. Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477

4.1. Determination of Lipid Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

4.1.1. Determination of Lipid Hydroperoxides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.1.2. Determination of Thiobarbituric Acid Reactive
Substances (TBARS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... . 16
4.2. Determination of Oxidative Protein Damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... . 16
4.3. Determination of Oxidative DNA Damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... . 17
5. Balance Between Oxidants and Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... 18
6. Biomarkers of Oxidative Stress and Antioxidant
Defense in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... 18
6.1. Breast cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... . 18
6.2. Prostate cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... . 19
6.3. Colorectal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... . 19
6.4. Cervical cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... . 19
6.5. Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... . 19
7. Biomarkers of Oxidative Stress and Antioxidant Defense in Neurodegenerative Diseases . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... 19
7.1. Amyotrophic lateral sclerosis (ALS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... . 19
7.2. Parkinson’s disease (PD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... 20
7.3. Alzheimer’s disease (AD). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... 20
7.4. Autism spectrum disorder. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... 20
8. Biomarkers of Oxidative Stress and Antioxidant
Defense in Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... 20
8.1. Type 1 diabetes mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... 20
8.2. Type 2 diabetes mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... 21
8.3. Gestational diabetes mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... 21
9. Conclusions and Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... 21
Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... 21
Declaration of Competing Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... 21
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . ..... . ..... . ..... . ..... . ..... . ..... 21

1. Introduction
1.1. Reactive Oxygen Species (ROSs)
A number of reactive oxygen−containing substances, including
free radicals such as hydroxyl (•OH) and superoxide (O2•-), and non-
radical species such as hydrogen peroxide (H2O2), are commonly
referred to as reactive oxygen species (ROS). There are also certain
reactive species that carry different elements other than oxygen,
such as nitrogen, sulfur and chlorine, so maybe it would be more
appropriate to use the term “reactive species” (RS) instead of ROS.
Generation of RS is an unavoidable part of aerobic life due to the
stepwise uptake of electrons by molecular oxygen from various
substrates, and RS can normally be balanced by antioxidants (AOX)
in a healthy organism [1,2]. Human beings have an immune system
that protects them from disease and keeps them alive, and RS have
an active role to kill infecting pathogens. But after elimination of
threat, prolonging the activation of immune system also has harmful
effects [3]. There are also various studies claiming that RS are ef­
fective in redox signaling in the body. It is also known that RS con­
tribute to adjustment for the situations caused by ischaemia and
heavy exercise. To maintain a healthy life, the task of the human AOX
defense system is to eliminate excessive amounts of RS without
hindering them from maintaining their beneficial role [1]. The cel­
lular AOX system tightly controls the amount of RS to maintain the
natural balance that exists in the cells, known as ‘redox home­
ostasis’. The cellular AOX system consists of a series of components
including enzymatic and non-enzymatic species, mainly superoxide
dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as
enzymatic species and glutathione which is a non-enzymatic AOX.
The function of SOD is catalyzing the dismutation reaction of O2•-
having end-products of O2 and H2O2. CAT and GPx decompose H2O2
into H2O and O2. On the other hand, overproduction of RS cannot be
counterbalanced by AOX, leading to impairment of the balance in
favor of oxidants and oxidative stress. High levels of oxidative stress
may harm cellular macromolecules and increase cell death [4]. In­
terestingly, the term oxidative stress appeared in rubber chemistry
before redox biology [5]. In later years, addition of H2O2 to ery­
throcytes was expressed as “cells were subjected to oxidative stress”
[6]. In 1985, the term began to be used to mean oxidative damage to
cells and organs as we understand it today [7]. In the chapter titled
“Oxidative Stress: Introduction Descriptions” written by Sies (in the
book named ‘Oxidative Stress’) this concept was formulated and
introduced as an imbalance between AOXs and oxidants in favor of
oxidants [8]. Later Sies improved his definition by adding an ex­
pression which stated that oxidative stress causes a disruption on
redox signaling [9]. Considering the new definition of oxidative
stress, this concept should be interpreted beyond the oxidative da­
mage in biomolecules caused by free radicals and should be accepted
as the disruption of the balanced cellular redox state [10]. However,
the oxidative damage generated by RS in the macromolecules such
as DNA, lipid and protein is still an important topic. Damage gen­
erated on these biological elements may result in cell death and
cause serious health problems. Different studies in the literature
point out to a relationship between oxidative stress and some dis­
eases such as cancer, atherosclerosis, Alzheimer’s disease (AD) and
Parkinson’s disease (PD). For example, it is known that cancer cells
have higher amounts of RS compared to healthy cells. There is also
some evidence indicating that protein carbonyls, generated at the
end of oxidative protein damage, are present in Parkinson patients’
brain. There is also a link between oxidative stress and the biology of
Down syndrome. Chromosome 21, which is associated with Down
syndrome, is thought to have certain genes that contribute to neu­
rodegeneration caused by oxidative stress. Other oxidative stress
related health problems worth to mention are diabetes, chronic in­
flammation and urolitiasis [11]. In addition, disruption of metal
homeostasis can lead to various diseases. For example, Wilson’s
disease and thalassemia are related to metal overload. The most
likely reason for this is that an increase in metal ions in the body can
trigger the formation of RS [12]. Thalassemia is a disorder associated
with defects in the synthesis of α- or β-globin subunits of he­
moglobin A, i.e. these sub chains (α or β) can be reduced or absent.
Depending on the severity of the patient’s condition, a regular blood
transfusion may be needed. With or without a blood transfusion,

2
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
iron overload can be seen in thalassemia patients, and oral iron
chelators may be used in disease management [13]. Although during
the normal metabolism iron is bound to certain proteins, after
several transfusions, iron binding proteins are saturated and free
iron (or non-transferrin bound iron) may accumulate in tissues or
blood [14]. On the other hand, in thalassemias that do not require
regular blood transfusion, the production of erythrocytes may cause
an increase in gastrointestinal absorption and deposition of iron in
the liver [15].
According to current knowledge it is a well-known fact that RS are
routinely generated from different sources in most of the cells. Among
the endogenous sources of RS; mitochondria, oxidase enzymes (nico­
tinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine
oxidase (XO), lipoxygenases, cyclooxygenases, polyamine and amino
acid oxidases), other enzymes (cytochrome P450 enzymes, nitric oxide
synthases, flavin-dependent demethylase), heme groups, metal storage
proteins, free metal ions (iron and copper) can be mentioned.
Endogenously produced RS are consumed by enzymatic AOXs such as
SOD, CAT, GPx and certain reductases. There are also different non-
enzymatic, small molecular mass AOXs in the body such as ascorbate,
pyruvate [16]. During aerobic respiration, ATP is produced to provide
the necessary energy for the body as a result of the reaction between
oxygen and glucose in the mitochondria. In this process, O2 undergoes
step-by-step reduction and the generated intermediates are: hydro­
peroxyl radical (HOO•), superoxide anion radical (O2•-), hydrogen per­
oxide (H2O2), hydroxyl radical (•OH), hydroxide anion (OH-), and the
final product is H2O:
According to this reduction scheme, oxygen is reduced by oxidase
enzymes to form superoxide anion radical (O2•-) which is formed in
mitochondrial electron transport chain during oxidative phosphoryla­
tion. While O2•- is a weak oxidant for ascorbic acid and thiols (such as
reduced glutathione, GSH) in aqueous solutions, it is a strong reducing
agent against some iron complexes. The reaction between Fe(III) and
O2•- is called as Haber-Weiss reaction, with the sequence:
Fe3+ + O2•− → Fe2+ + O2
Fe2+ + H2O2 → Fe3+ + OH− + •OH
where the net reaction is:
O2• + H2O2 → •OH + OH− + O2 [17]
−
As can be seen from the above reaction steps, superoxide anion ra­
dical is a potential reductant for Fe(III) ions, and the second step is a
classical Fenton reaction. Haber-Weiss reaction is known as the source of
generation of •OH by means of O2•− in biochemical systems. On the other
hand, the fact that the iron ions are not free but in the form of chelate
complexes may not prevent the reaction but even accelerate it, de­
pending on the stability and coordinative saturation of the chelate [18].
HOO• is the protonated form of O2•-, and is stronger than su­
peroxide radical, both as an oxidant and reductant. The question
“how super is superoxide?” was asked by Sawyer [19] to indicate the
moderate effectiveness of superoxide anion radical as a redox spe­
cies (i.e. both as an oxidizing and reducing agent). Since the pKa
value of HOO• is 4.80, at physiological pH it easily turns into O2•- by
acidic dissociation. In the human body, superoxide dismutase group
enzymes are responsible for consuming superoxide radicals [11].
Superoxide can accept two protons and one electron to easily gen­
erate H2O2. In the presence of certain oxidase enzymes, O2 can
successively accept two electrons to form H2O2. The ground-state
3
Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
electron configuration of molecular oxygen (O2) shows that it is a
diradical with 2 electrons singly occupying the two antibonding
orbitals at parallel spins. Thus, the relative chemical inertness of
oxygen comes from this spin restriction, as a pair of electrons from a
donor with antiparallel spins cannot be accommodated in the anti­
bonding orbitals of O2 each occupied by a single electron. In other
words, oxygen takes up electrons one-by-one in stepwise manner,
initially leading to superoxide anion (as the rate-limiting step) fol­
lowed by hydrogen peroxide [20]. If there are transition metal ions in
the medium (especially at their lower oxidation states) where H2O2
is present, highly reactive radicals such as •OH are formed [21].
Additionally, H2O2 may directly attack heme proteins, inactivate
enzymes; oxidize -SH groups and biomacromolecules (e.g. DNA,
proteins and lipids) [22]. In the human body, H2O2 is consumed by
CAT. CAT converts H2O2 to water while it does not show this effect
for other peroxides [23]. The hydroxyl radical, which can be pro­
duced by the reaction between H2O2 and transition metal ions, is
generally considered to be a highly reactive species that makes the
most important contribution to oxidative stress. For example, the
Fenton reaction between Fe(II) and H2O2 which can be formulated as
“Fe2+ + H2O2 → Fe3+ + •OH + OH-” is one of sources of hydroxyl ra­
dicals. Therefore, the type and amount of free and bound iron in the
body is of particular importance. Iron is replenished as ferric ions in
a Fenton reaction. O2•- is another RS in the body having the ability to
release iron from iron-containing proteins such as ferritin and Fe-S
clusters. In normal metabolism, the level of iron is strictly regulated
by metal-binding proteins. Only a small percentage of iron (i.e. less
than 5% of total cell Fe) is present in the redox-active (labile) iron
pool, essentially representing Fe-bound to low-affinity intracellular
low-molecular-mass ligands [24]. However, the amount of catalytic
iron increases under oxidative stress conditions [25], and this iron
released from its proteins may induce further oxidative damage [26].
Although with the simplest representation, Fenton reaction seems as
an outer-sphere electron transfer reaction, there are strong evi­
dences indicating that it occurs through an inner-sphere mechanism
including the formation of a transient L-Fe(H2O2)2+ complex. Here L
can be a biological chelator (or phosphate or EDTA) [27,28].
In this case, the reaction steps can be shown as:
2+
L-Fe + H2O2 → L-Fe(H2O2)2+
2+
L-Fe(H2O2) → L-Fe3+ + •OH + OH- or
2+
L-Fe(H2O2) → L-Fe4+ + 2OH- or
L-Fe(H2O2)
2+
+ R → L-Fe3+ + OH- + ROH
R is the substrate and can be oxidized to ROH.
After the formation of the L-Fe(H2O2)2+ transient species, it can
yield different products, either Fe(III) or Fe(IV), through the reaction
between ligand-bound Fe(II) and H2O2.
1.2. Antioxidants (AOXs)
The AOX concept is a common interest for different disciplines
such as bioanalytical chemists, physicians, dieticians and maybe
just for consumers. Therefore, the term AOX should be in its most
basic form, simple and the same for all relevant groups. Probably
the most appropriate definition is that an AOX is a substance
capable of impeding or retarding the unwanted oxidation of bio­
logically relevant substrates [29]. AOXs have the ability of reducing
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
oxidant species [2] usually by donation of electrons or H-atoms.
For food chemists, AOXs serve to prevent foods against deteriora­
tion by means of inhibiting oxidation [30]. The use of AOXs to
prevent spoilage and prolong the lifespan of foods dates back to
ancient times [31]. Apak mentioned two traditional Turkish foods
as examples of this phenomenon in his study. These are a soup
named “tarhana” and herby cheese which have been known and
made for over two hundred years. Both of them are made using
certain herbs, vegetables and spices that are rich in phenolic AOX.
The aim here is to prepare these foodstuffs in the summer and
store them during the winter when there are no fresh herbs and
vegetables, and consume them at this time [29]. Although the term
AOX generally has positive connotations, there are some evidences
to the contrary in the literature. Especially Halliwell and Gutter­
idge stated in their various studies that supplemental formulas
containing AOX are not as beneficial as they thought in terms of
health [32,33]. Another important point is that in vitro and in vivo
activities of some AOX compounds such as polyphenols can be very
different from each other. Studies on the effects of polyphenols
using cell culture indicate that they can act as prooxidants in
certain conditions. Prooxidants may have both reducing and oxi­
dizing character; they can induce oxidative stress, either by gen­
erating reactive oxygen/nitrogen species (RONS) or by inhibiting
antioxidative defenses of the organism [34]. For example, under
certain conditions, polyphenolic antioxidants may reduce transi­
tion metal ions to their lower oxidation states thereby triggering
Fenton reactions giving rise to ROS. On the other hand, a mild
oxidative stress can be beneficial since it may trigger endogenous
AOX defense system (e.g., by increasing GSH level). Therefore, if
there is no nutritional deficiency, excessive consumption of AOXs
should be avoided [25]. During the normal functioning of meta­
bolism, the prooxidant/antioxidant balance slightly favors proox­
idants to cause a mild oxidative stress and allows endogenous AOX
(and repair systems) to interact to reduce disease incidence. It is
still a controversial subject whether the use of synthetic AOXs is
effective to prevent some diseases [11]. Foodstuffs such as fruits,
vegetables, grains and herbs, which are used as spices or beverages
with a number of antioxidative ingredients (such as polyphenols,
tocopherols and carotenoids) usually serve better than only one
specific type of AOXs because of the collaborative action of AOXs.
Measuring plasma total antioxidant capacity (TAC) and its changes
may give useful hints to evaluate the health status of an individual
person. Since the amount of AOX is directly proportional to redu­
cing the rate of oxidants and/or suppressing (or delaying) oxida­
tion, there is usually an negative correlation between the
concentration of TAC and total RS in the human body [35]. For
example, Bean et al. [36] successfully demonstrated this negative
correlation between RS and TAC for bladder tissue undergoing
ischemia/reperfusion using the cupric ion reducing antioxidant
capacity (CUPRAC) method. To briefly explain the CUPRAC method
introduced by Apak et al. [37], the copper(II)-neocuproine (Cu(II)-
Nc) reagent as the chromogenic oxidant can oxidize antioxidants,
while itself being reduced to the yellow-orange Cu(I)-Nc chelate.
The developed color is measured as absorbance at 450 nm against
a reagent blank. The protons released during the reaction are
neutralized by an ammonium acetate buffer at pH 7 [37]:
2+ + +
Cu(Nc)2 + AOX → Cu(Nc)2 + oxidized AOX + H
Bean et al. determined that while the level of free radicals in­
creased, there was a decrease in protective AOX levels, measured by
two different assays [36]. The authors stated that they were able to
demonstrate this negative correlation between RS and TAC using the
CUPRAC method [37], but the Ferric Reduction Antioxidant Power
assay (FRAP) [38] method, which was the other method they tried,
was not successful in this regard. The FRAP method, originally
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Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
developed by Benzie and Strain (1996), depends on the AOX oxi­
dizing ability of ferric iron in the presence of 2,4,6-tripyridyl-s-
triazine (TPTZ) as ligand. Fe(III) can oxidize certain antioxidants in
acidic medium, while itself being reduced to the blue-colored Fe(II)-
TPTZ chelate, the absorbance of which is measured at 593 nm [38].
The health beneficial effects of AOXs are usually associated with
their ability of removing reactive species. From this point of view,
biochemists, medicinal chemists and nutritionists often refer to sub­
stances that can quench metabolically produced (in vivo) reactive
species as AOX. Therefore, these scientists are more focused on oxi­
dative stress biomarkers in understanding health protection, and con­
sequently they are less concerned with TAC which is not exactly
correlated to disease prevention. On the other hand, some food sci­
entists oriented at classifying and screening foodstuffs may understand
AOXs as substances having high TAC and radical quenching ability in
vitro [39]. It can be concluded that although RS and TAC are the two
essential parts of redox biology, biological scientists are usually inter­
ested in RS assays while food chemists are interested in TAC assays.
There are a few studies in the literature aiming to fill this gap between
these two types of assays. An example of this is the work of Çekiç et al.
The authors developed a method depending on the determination of
certain RS using N,N-dimethyl-p-phenylenediamine (DMPD) reagent,
where RS in the reaction medium caused the formation of pink colored
DMPD•+ radical cation from DMPD and the color intensity was directly
proportional to the amount of RS. The observed color may be explained
by an extended π-conjugation caused by a good electron donor (N atom
of DMPD) and an acceptor (RS) system created by oxidation. However,
since these RS could be scavenged by AOXs, the color intensity de­
creased proportionally with AOX concentration. In this manner, both RS
and TAC could be measured by using the same procedure [35]. This
method, which was applied in a solution environment, was later
turned into a sensor application by using a Nafion cation-exchange
membrane as a solid support material for capturing DMPD•+ [40]. Di­
rect determination of RS (especially very short-lived free radicals) is
difficult and needs sophisticated and expensive instrumentation such
as electron spin (paramagnetic) resonance (ESR/EPR). Therefore, in­
direct techniques based on the determination of products formed from
the reaction between RS and selected biological macromolecules (DNA,
proteins and phospholipids) are generally preferred. The RS causing
oxidative stress, specifically H2O2 and superoxide radicals, can be
sensed by voltammetric and amperometric techniques [41], while RS-
induced oxidative conversion products on DNA and proteins can also be
measured by electrochemical techniques.
When RS cannot be counterbalanced by AOX molecules, a series
of irreversible chemical modifications occur in the body. The pro­
ducts generated by means of these modifications may be considered
as “oxidative stress biomarkers”. Currently there are measurable
biomarkers which are not clinically relevant yet [42]. Biologists re­
searching free radicals have adapted this term from molecular epi­
demiology [43], related to oxidation products generated from DNA,
proteins and lipids and AOX consumption [44]. These biomarkers
particularly include malondialdehyde (MDA) or isoprostanes for
lipid oxidation; protein carbonyls for protein oxidation, and 8-hy­
droxydeoxyguanosine (8-OHdG) for nucleic acid oxidation [45].
2. Metabolic Oxidation Products
As a result of oxidative stress conditions, changes occur in the
structure and function of biomolecules such as DNA, lipids and
proteins [46], which will be disussed in this section.
2.1. Lipid Peroxidation
Lipids are essential components of cellular units such as plasma
membrane, Golgi apparatus, endoplasmic reticulum. Cell mem­
branes composed of lipid bilayers provide structural integrity by
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
Fig. 1. Chemical structures of linoleic and arachidonic acids.
surrounding the cell. The fluidity of a membrane significantly affects
its functions. The presence of double bonds in lipids containing
unsaturated fatty acids increases membrane fluidity. The more
double bonds a polyunsaturated fatty acid (PUFA) contains, the more
fluid it will be. At the same time, this property makes PUFAs sus­
ceptible to autoxidation [47] based on the arrangement of the double
bonds within the molecule. Hydrogen abstraction is governed by the
bond dissociation energy (BDE) of the covalent C-H bond, which is
weakened by adjacent electron-rich –C]C– bonds in PUFAs that are
particularly open to H-atom abstraction due to the presence of single
or multiple CH2-interrupted hydrogens with lower BDEs. After the
formation of lipid radicals, the conjugated π-electrons of the double
bond stabilize the free radical through delocalization over the diene
structure and enable rapid reaction with triplet oxygen to form
peroxyl radicals which can abstract another H-atom from the lipid
structure leading to hydroperoxide formation and free radical chain
propagation [48].
In biological systems, lipid peroxidation can be induced by exo­
genous sources such as air pollution, smoking, UV light or by free
radicals generated by endogenous enzymatic systems such as
NADPH oxidase and XO [49]. As a result of the peroxidation of
phospholipids in the cell membrane with ROS, reactive lipid hy­
droperoxides and secondary carbonyl compounds such as 4-hy­
droxy-2-nonenal, malondialdehyde, acrolein, crotonaldehyde,
methylglyoxal are formed [50].
Lipids can undergo peroxidation, in particular, PUFAs with more
than two cis-double bonds and their esters are easily oxidized by
free radical−induced chain oxidation. Major PUFAs are linolenic
acid (HOOC(CH2)7CH=CHCH2CH=CH(CH2)4CH3), docosahexaenoic
acid (cis-4,7,10,13,16,19-docosahexaenoic acid), arachidonic acid
(cis-5,8,11,14-eicosatetraenoic acid) and eicosapentaenoic acid
Fig. 2. Steps of lipid peroxide generation from a polyunsaturated fatty acid (PUFA).
5
Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
(cis-5,8,11,14,17-eicosapentaenoic). Linoleic acid and arachidonic
acid are shown in the Fig. 1 [51].
Lipid peroxidation takes place in three steps: initiation, propa­
gation and termination [52]. Lipid peroxidation can be initiated by
removing a hydrogen atom from the methylene group of the PUFA
side chain by a free radical. With the abstraction of a hydrogen atom
from the fatty acid, an unpaired electron remains on the carbon. The
unstable fatty acid radicals formed turn into conjugated diene
structure in order to become thermodynamically more stable. In the
propagation step, fatty acid radicals also uptake oxygen to form the
lipid peroxyl radicals which can start the chain reaction by ab­
stracting hydrogen from another PUFA [53,54]. Generation of lipid
peroxides from a PUFA was shown in Fig. 2.
In the termination step, non-radical products such as alkanals
and alkenals are formed. These compounds exhibit toxic properties
due to forming a Schiff base with amino groups, inactivating various
enzymes, and interacting with thiol groups. As a result of lipid
peroxidation in the cell membrane, the membrane transport system
is affected, intracellular and extracellular ion balances are disrupted,
membrane permeability changes [53].
The chain reactions can also terminate with the interaction of
AOXs with lipid radicals. The AOX radical formed as a result of the
interaction of the AOX with the lipid peroxyl radical may turn into
non-radical species by dimerizing or reacting with another lipid
peroxyl radical [55].
Lipid peroxidation occurs through two different mechanisms:
non-enzymatic peroxidation and enzymatic peroxidation [55]. Non-
enzymatic peroxidation of lipids can be initiated with a radical as
described above or by generating radical species from non-radical
molecules [54]. In biological systems, this occurs through Fenton and
Haber-Weiss reactions in the presence of various transition metals,
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
especially iron and copper [56]. Lipid peroxides can rapidly de­
compose in the presence of iron salts and form alkoxyl radicals and
peroxyl radicals [57].
Enzymatic peroxidation of lipids occurs by enzymes such as li­
poxygenases (LOXs), cyclooxygenases (COXs) and cytochrome P450
(CYPs). COXs, CYPs, LOXs are responsible for the synthesis of lipid
endoperoxides, epoxyieicosatrienoic acids and lipid hydroperoxides,
respectively [54].
2.1.1. Lipid Peroxidation Products
2.1.1.1. Isoprostanes. Isoprostanes (IsoPs) are widely used as
biomarkers in the assessment of lipid peroxidation in vivo due to
their stability, selectivity and specificity (Fig. 3). IsoPs are
prostaglandin-like molecules formed by the non-enzymatic
peroxidation of arachidonic acid (AA). Various IsoP compounds are
formed by oxidation of AA. Firstly, the arachidonyl radicals formed
by the peroxidation of AA undergo endocyclization to produce four
positional bicyclic endoperoxide intermediate isomers, known as
5-, 12-, 8-, or 15-series, according to the carbon to which the
hydroperoxyl in the side chain is attached. These intermediates are
then reduced to form F2-isoprostan (F2-IsoP) regioisomers [58–60].
Fig. 3. The oxidation and peroxidation products of arachidonic acid.
6
Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
IsoPs, initially bound to phospholipids, are freely found in bio­
logical fluids after being released from cell membranes by phos­
pholipases [61,62]. The determination of IsoPs in biological fluids is
important in monitoring the treatment of diseases, as they are as­
sociated with various diseases. Levels of F2-IsoPs, have been noted to
be significantly increased in asthma [63], neurodegenerative dis­
eases such as multiple sclerosis [64], AD [65,66] and cardiovascular
disease [67].
Since docosahexaenoic acid (DHA) contains more double bonds
than AA, it is more susceptible to free radical-mediated peroxidation.
Peroxidation of DHA, the most abundant fatty acid in the central
nervous system, forms isoprostane-like compounds called neuro­
prostanes (NPs). The bicyclic endoperoxide intermediates formed by
the peroxidation of DHA are reduced to form F-ring NPs (F4-NPs)
(Fig. 4) [58]. While AA is found in all cells, DHA is mostly found in
neuronal membranes. Therefore, the determination of F4-NPs
formed by the oxidation of DHA allows the determination of oxi­
dative damage in neuronal membranes specifically [68].
Additionaly, as oxygen tension increases, isofurans (IsoFs) and
neurofurans (NFs) can be formed, respectively, by the peroxidation
of AA and DHA. The plausible mechanisms for the formation of NFs
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al. Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477

Fig. 4. DHA oxidation products.

include cleavage of cyclic peroxides and hydrolysis of epoxides
[69,70]. The formation mechanism of these compounds occurs si­
milarly to F2-IsoPs [58].
2.1.1.2. Lipid Hydroperoxides and Lipid Hydroxides. Hydroperoxides
are primary products formed as a result of peroxidation of PUFA and
cholesterol (Ch) by lipoxygenase or free radical mediated peroxidation.
Linoleates are the most abundant PUFAs in vivo, and their radical-
mediated oxidation results in hydroperoxyoctadecadienoic acids
(HPODEs). There are four HPODE isomers (Fig. 5) 13-hydroperoxy-
9(Z), 11(E)-octadecadienoic acid (13-(Z,E)-HPODE); 13-hydroperoxy-
9(E), 11(E)-octadecadienoic acid (13-(E,E)-HPODE); 9-hydroperoxy-
10(E), 12(Z)-octadecadienoic acid (9-(E,Z)-HPODE) and 9-
hydroperoxy-10(E), 12(E)-octadecadienoic acid (9-(E,E)-HPODE) [62].
Hydroperoxides formed as a result of the peroxidation of PUFAs are
reduced by glutathione peroxidases and selenoprotein P to their

Fig. 5. Hydroperoxyoctadecadienoic acid (HPODE) isomers.

7
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
corresponding hydroxides and form reactive secondary degradation
products [51].
Some reactive secondary degradation products are formed by the
decomposition of lipid hydroperoxides, which are the primary pro­
ducts of the peroxidation of PUFAs. Hydroxy acids are formed by the
reduction of lipid peroxides. Lipid hydroxides can be produced by
reduction of hydroperoxides, and can also be formed by enzymatic
peroxidation, singlet oxygen peroxidation, or radical mediated per­
oxidation. While cis-trans hydroxides are formed by all three per­
oxidation pathways, trans-trans hydroxides are formed only as a
result of free radical mediated peroxidation; thus, the latter serve as
specific biomarkers of free radical induced lipid peroxidation [51].
Due to their relative chemical stability and consistency of measured
levels in human fluids and tissues, lipid hydroxides may be con­
sidered to be more suitable as biomarkers than hydroperoxides [51].
Hydroperoxyeicosatetraenoic acid (HPETE), which is formed by the
peroxidation of AA, and hydroperoxyoctadecadienoic acid (HPODE),
which is the peroxidation product of linoleic acid, can be reduced to
hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic
acids, respectively, with a strong reducing agent [54].
2.1.1.3. Aldehydes. As a result of lipid peroxidation, PUFAs can form
various short chain aldehydes, including 2-alkenals, 4-hydroxy-2-
alkenals and ketoaldehydes (Fig. 6) [58]. These are secondary
metabolites of lipid peroxidation. Aldehydes are the most common
degradation products of lipid peroxides [54]. The main ones are 4-
hydroxynonenal (4-HNE) and MDA.
MDA may be formed as a result of enzymatic and free radical
peroxidation of PUFAs containing at least three double bonds [71]. In
addition, MDA is formed in prostaglandin synthesis [72]. MDA is
frequently used as a lipid peroxidation marker. MDA can mainly
react with lysine (Lys) residues of proteins to form adducts with
proteins and DNA. It plays a role in the pathogenesis of various
diseases such as diabetes mellitus [73] and neurodegenerative dis­
eases [74]. It has been reported that MDA reacts with DNA bases to
form deoxyguanosine (dG), deoxyadenosine (dA) and deoxycytidine
Fig. 6. Secondary oxidation products of lipids.
8
Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
(dCyd) adducts, and these DNA-MDA adducts have mutagenic effects
in bacteria and mammalian cells [72].
Phospholipids containing linoleic acid and AA are considered the
main source for 4-HNE production [75]. 4-hydroxy-2-nonenal and 4-
hydroxy-2-hexenal (HHE) are formed as a result of peroxidation of
ω6 and ω3 fatty acids, respectively. 4-hydroxy-2-alkenals are highly
reactive due to their three functional groups (carbonyl, -C = C-
double bond and secondary alcohol) they have, and under the in­
fluence of these groups they can interact with biomolecules con­
taining amino and thiol groups through Michael additions and Schiff
base formation [71]. In addition to biochemical modifications, it may
cause changes in the biophysical properties of membranes, such as
membrane fluidity, depending on its concentration [76].
Lipid peroxidation products such as 4-HNE and MDA can be
found in free form in metabolism; they can also be covalently bound
to various molecules such as proteins and nucleic acids due to their
high reactivity [46]. It has been stated that the formation of these
products is associated with inflammation, apoptosis, and various
degenerative diseases [77]. Acrolein, one of the highly reactive
products of the metal catalyzed oxidation of PUFAs, can also induce
neuronal damage by forming addition products with proteins [68].
2.1.1.4. Oxysterols. Ch is a sterol lipid found in the lipoproteins and
membranes of eukaryotic cells. Since Ch is a monounsaturated lipid,
it is less sensitive to peroxidation than substances containing
polyunsaturated fatty acids. Peroxidation of Ch can occur in
enzymatic or radical-mediated modes. Peroxidation in the cyclic
part of Ch is generally radical-mediated, while peroxidation in the
side chain is usually enzymatic [78]. Ch peroxidation products used
as biomarkers of oxidative stress are called oxysterols. These
oxysterols (Ch oxides) include hydroperoxides, diols, epoxides and
ketones. As a result of the peroxidation of Ch, different oxidation
product such as 7α- and 7β-hydroperoxycholesterol (7α-OOHCh and
7β-OOHCh), 7α- and 7β-hydroxycholesterol (7α-OHCh, 7β-OHCh), 5α,
6α- and 5β, 6β-epoxycholesterol and 7-ketocholesterol (7-KCh) are
formed (Fig. 7) [62]. Ch can also be oxidized by enzymes, most of
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
Fig. 7. Cholesterol peroxidation products.
which belong to the cytochrome P450 class. 27-hydroxycholesterol,
which is an intermediate product in bile synthesis from CYP27A1
enzyme in the inner membrane of mitochondria, 24(S)-
hydroxycholesterol is produced from CYP46 enzyme, especially
found in neurons, and 7α-hydroxycholesterol is produced from
CYP7A1 enzyme. 25-hydroxycholesterol and 7α-hydroxycholesterol
can be formed by both enzymatic and radical mediated
peroxidation [79].
2.2. Protein Damage
Proteins become an important target for oxidizing agents due to
their abundance in cells, plasma and tissues and their high reaction
rate with various oxidants [80]. The interaction of proteins with
reactive oxygen species can result in oxidation of amino acid side
chains or the protein backbone, the formation of carbonyl-derived
compounds, and protein-protein crosslinking [81]. Since proteins
can contain different amino acids in their side chains, a large number
of radicals can occur on proteins. Amino acids with a thiol group and
aromatic side chain, namely cysteine and tyrosine, respectively, are
the most sensitive moieties to oxidation [82,83].
In metal-catalyzed protein oxidation, transition metal ions such
as Fe (II) and Cu (I) bind to the metal binding site in proteins and
then react with H2O2 to form •OH radical that attacks amino acid
residues near this site [84].
9
Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
As a result of oxidative damage of proteins, some changes may
occur in their physical or chemical properties such as solubility,
structural integrity, and enzyme activity [81]. It has been reported
that the levels of protein oxidation products increase in various
diseases related to aging such as AD [85], diabetes [86], and catar­
acts [87].
2.2.1. Oxidation of the Protein Backbone
In the first stage, as a result of the interaction of the amino acid
with the radical, a hydrogen atom is removed from the α-carbon, and
a carbon-centered radical is formed. Subsequently, an alkylperoxy
radical is formed by the addition of O2. At this stage, the carbon
centered radical can react with another carbon centered radical in
the absence of oxygen and form protein-protein crosslinks. As a
result of the reaction of the alkyl peroxy radical with HO2• or Fe2+
and H+, alkyl peroxide is formed. The hydrolysis of the imino deri­
vative, which is formed by the decomposition of the alkyl peroxide,
forms an aldehyde or an α-keto acid [88].
2.2.2. Oxidation of Amino Acids
Cysteine and methionine are very sensitive to oxidative mod­
ification due to the nucleophilic character of the thiol and thiol ether
groups, respectively, that they contain. The oxidation of cysteine
residues begins with the formation of sulfenic acid and sulfinic acid
and can continue with the formation of sulfonic acid, an irreversible
further oxidation product. The oxidation of cysteine to sulfenic and
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
sulfinic acid derivatives is reversible [89]. Since these species are
unstable, they can form oxyacids by hydrolysis, and can also form
disulfide bonds by reacting with another thiol group or in the pre­
sence of a donor such as glutathione [81]. Cystine, in which the two
cysteines are linked by disulfide bonds, is an important cysteine
modification. The formation of these disulfide bonds is effective in
stabilizing proteins.
As a result of the oxidation of methionine residues in the side
chains of proteins, a mixture of stereoisomers of methionine sulf­
oxide is formed. Methionine sulfoxide can be reduced back to me­
thionine by the methionine sulfoxide reductase enzyme [90]. As the
further oxidation product of methionine sulfoxide, methionine sul­
fone is formed. However, this product cannot be reduced again by
methionine sulfoxide reductases [89].
In addition, the tyrosyl radical formed by the oxidation of tyr­
osine side chains can form dityrosine by dimerizing with another
tyrosyl radical, or it can also form 3,4-dihydroxyphenylalanine by
interacting with hydroxyl radicals [89]. As a result of the oxidation of
proteins; nitrotryptophan or kynurenine from tryptophan residues,
2-oxohistidine from histidine residues, 3-, 4-, 5- hydroxyleucine
derivatives from leucine residues can be formed. Some amino acid
residues such as Lys and arginine also turn into various carbonyl
derivatives [90,91].
2.2.3. Protein Carbonyl Derivatives
The carbonylation of proteins is irreversible and can cause pro­
teins to lose their activity since it causes a change in the peptide
chain structure [92]. Carbonyl derivatives are frequently used as
biomarkers in the oxidative damage of proteins and can be formed
mainly in four different ways. The first of these is the direct oxida­
tion of proline, arginine, Lys and threonine residues in protein side
chains. As a result of this, various carbonyl compounds such as γ-
glutamyl semialdehyde, α-aminoadipic semialdehyde, 2-amino-3-
ketobutyric acid are formed [81]. Also, they can be produced by
cleavage of protein backbones by the α-amidation pathway or oxi­
dation of glutamyl residues. Oxidation by α-amidation pathway re­
sults in N-α-ketoacyl derivative compounds [89]. Furthermore,
carbonyl derivative compounds are formed by the Michael addition
to cysteine, histidine and Lys residues of aldehydes, which are sec­
ondary oxidation products of lipids, or by the reaction of Lys residues
with reducing sugars or their oxidation products such as ketoamines
and ketoaldehydes [81,92]. The products formed as a result of a
series of reactions of Lys residues with the carbonyl groups of car­
bohydrates called glycoxidation are advanced glycation end products
and are used as biomarkers in the oxidative damage of proteins [93].
2.3. DNA Damage
The interaction of DNA with reactive oxygen species can cause
DNA-protein cross-linking, the formation of sugar and base mod­
ification products, intra-strand and inter-strand cross-linking, single
or double strand-breaks [94]. Damage to DNA caused by ROS origi­
nating from endogenous or exogenous sources may increase the risk
of developing various diseases such as cancer, cardiovascular dis­
eases, and neurodegenerative diseases [95].
A large number of sugar and base modification products have
been described, depending on the reaction conditions [96]. Of the
DNA bases, guanine is sensitive to oxidation due to its lowest redox
potential [97] and highest oxidizability; it can be easily oxidized to
8-oxo-7,8-dihydrodeoxyguanine via single-electron transfer to ROS
such as H2O2, superoxide and hydroxyl radicals [98]. DNA mod­
ification causes various mutations, mainly GC-TA transversion
mutation, due to mispair [42]. Two important products resulting
from the guanine modification are 8-hydroxyguanosine (8-OH-G)
in the presence of oxygen and 2,6-diamino-4-hydroxy-5-for­
mamidopyrimidine (FapyG) in the presence of reducing agents.
10
Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
Similarly, 8-hydroxyadenine (8-OH-Ade) and 4,6-diamino-5-for­
mamidopyrimidine (FapyA) are formed from adenine. By radical-
induced modification of thymine, compounds such as thymine
glycol, 5-(hydroxymethyl) uracil, and 5-formyluracil (5-FoU) are
formed. Some modifications of the cytosine base are cytosine glycol,
5-hydroxycytosine (5-OHC) and 5-hydroxyuracil (5-OHU). The re­
lated DNA oxidation products are shown in Fig. 8 [96].
The hydroxyl radical reacts with the sugar portion of DNA to
generate carbon-centered sugar radicals, which results in breaking
the DNA strand, forming various sugar products and abasic sites (i.e.
apurinic/apyrimidinic sites as common lesions in DNA) [99]. DNA-
protein cross-linking can occur by adding the DNA base radical
(formed by the interaction of DNA with radicals) to the aromatic
amino acid of proteins or amino acid radical [100].
3. Antioxidants
Considering that, free radicals, produced in the body, may cause
oxidative damage on biomacromolecules such as lipids, proteins and
DNA, the importance of AOXs that delay or prevent this damage by
scavenging reactive species cannot be underestimated. AOXs can
have different functions depending on the type of radical species
produced and of the target molecule protected [101]. As AOXs can
reduce the risk of degenerative disease development by counter­
balancing an excess of reactive species, they are vital for human
health [102]. Furthermore, AOX intake from exogenous sources is
very important as oxidants in the body may increase in various
disease conditions. While some AOXs can be naturally synthesized in
the body, some are taken up through nutrition. Among the various
classes of AOXs, enzymatic AOXs such as SOD, CAT and glutathione
peroxidase; hydrophilic ones such as urate, ascorbate, glutathione
and flavonoids; lipophilic ones such as tocopherols, carotenoids and
ubiquinol can be mentioned [103]. Aside from their health-beneficial
effects, AOXs in foods, food supplements and pharmaceuticals pro­
long their shelf life. AOXs can be classified as primary or secondary
AOXs depending on their mechanism of action. Primary AOXs sca­
venge radical species by H-atom or electron transfer, while sec­
ondary AOXs inactivate prooxidant catalysts. Although synthetic
AOXs are known as primary AOXs, it has been reported that they can
be toxic and carcinogenic depending on their concentrations [102].
AOXs are also divided into three subgroups according to their de­
fense mechanisms: (i) preventive, (ii) radical scavenger and (iii)
enzyme repairing. Plant-based AOXs are generally hydrophilic,
however tocopherols, vitamin K and carotenoids are hydrophobic.
Uric acid and ascorbic acid are water-soluble AOXs [104]. The con­
jugate base (salt) form of phenolic AOXs is more soluble in the
aqueous phase than the parent free acid (at pH above pKa of
acid-form).
3.1. Enzymatic Antioxidants
SOD, CAT, GPx are the best-known AOX enzymes. Since they have
high affinity for ROS, they provide more effective protection in case
of oxidative stress [103].
Coenzyme Q10 is a lipid-soluble AOX and, apart from its radical
scavenging effect, also plays a role in the regeneration of various
vitamins A (retinol), C (ascorbic acid) and E, and in metabolic events
such as inhibiting the degradation of biomacromolecules such as
lipids, DNA and proteins [105].
SOD, which is a metalloenzyme, is one the most effective enzy­
matic AOXs found in all subcellular compartments and acts as a
protective agent against the toxic effects of high amounts of ROS
[106]. SOD catalyzes the breakdown of superoxide into oxygen and
hydrogen peroxide. According to their metal cofactors, there are
three types SOD enzymes in the human body: cytosolic CuZn-SOD,
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al. Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477

Fig. 8. The main oxidation products of DNA bases.

mitochondrial Mn-SOD and Fe-SOD [103,106]. It helps prevent the

transition metal-catalyzed formation of hydroxyl radicals [106].
Catalases are enzymes with tetrameric heme group and are in­
volved in the detoxification of H2O2 produced in peroxisomes by
oxidases involved in various catabolic events [106]. It catalyzes the
decomposition reaction of hydrogen peroxide into the nontoxic
products of water and oxygen (via the reaction: 2 H2O2 → 2 H2O +
O2) [107]. This enzyme, which can break down about 6 million H2O2
molecules per minute, has almost the highest turnover rate [106].
In addition, CAT has also been reported to be effective on some
toxic compounds via peroxidative reaction [103]. Catalases (hydro­
peroxidases) have been studied extensively to date, containing more
than 300 types, namely monofunctional catalases, bifunctional
catalases and manganese containing catalases [107]. Fig. 9. Glutathione reductase and involved cellular redox cycles (GSH: reduced glu­
On the other hand, transition metals such as Fe, Cu and Mn in tathione, GSSG: oxidized glutathione, ASA: ascorbate, NAD(P)H: nicotinamide adenine
dinucleotide phosphate.
different oxidation states can act both as prooxidants and AOXs.
These metals can transfer their electrons to form radicals and cause
oxidative stress. At the same time, since these metals constitute the decomposes hydrogen peroxide into water. Especially when free or
active redox centres of AOX enzymes, they play an important role in weakly-complexed Fe and Cu ions are present in excess, they can
the biological AOX defense system. As being the active centres of cause peroxidation and act as prooxidants through Fenton and
metalloenzymes, Cu, Zn and Mn in superoxide dismutase convert Haber-Weiss reactions, but metals that are more tightly bound to
superoxide anion to hydrogen peroxide, while Fe in catalase protein structures such as ferritin prevent these structures from

11
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
Fig. 10. Chemical structure of β-carotene.
Fig. 11. Chemical structure of α-tocopherol.
acting as prooxidants [108]. It should be noted that although Fe in
ferritin is relatively tightly bound, its release under severe condi­
tions (such as redox-mediated chelation) is possible. There are stu­
dies in the literature indicating that certain agents such as
superoxide anion can promote iron release [109].
Glutathione reductase, which is commonly found in chloroplasts,
plays an important role in the ascorbate-glutathione cycle. This
enzyme catalyzes the reduction reaction of glutathione, a low-mo­
lecular-mass AOX, and plays a role in many metabolic events. As can
be seen from Fig. 9, glutathione reductase not only plays a role in the
reduction of glutathione, but also protects the ascorbate-glutathione
cycle and acts as a ROS scavenger [106].
The glutathione-S-transferase enzyme mainly catalyzes the re­
action of tripeptide glutathione with electrophilic xenobiotic sub­
strates. In addition, it is involved in the conversion of toxic
substances such as hydroxyperoxide and pesticides into nontoxic
metabolites in hormone homeostasis [106].
3.2. Non-Enzymatic Antioxidants
Vitamin E, carotenoids, polyphenols and ascorbic acid are non-
enzymatic AOXs that have important physiological functions, and
since they cannot be synthesized in the body, they should be sup­
plied by nutrition. Vitamin E exists in eight different forms, four
tocopherols and four tocotrienols, the essentially used form of which
is α-tocopherol. Coenzyme Q10 (CoQ10) or ubiquinone is found in
the biological system which is synthesized by the conjugation of
benzoquinone ring with an isoprenoid chain, but it can be supple­
mented through diet to further energize the body through ATP
production. Nutrition and diet are an important part of the AOX
defense mechanism [103]. Glutathione, uric acid and bilirubin are
also small molecular mass AOX present in human fluids [110]. Vi­
tamins C and E are known as AOX vitamins, together with β-carotene
(actually a provitamin A carotenoid) (Fig. 10), and play an important
role in preventing some diseases caused by oxidative damage [111].
Carotenoids are an important group found in fruits and vege­
tables that can prevent cancer due to their AOX functions [110], and
they are natural pigments which synthesized by plants, algae and
12
Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
photosynthetic bacteria [112]. Carotenoids with strong AOX prop­
erties are divided into two main groups: (i) carotenes (ii) xantho­
phylls. These two groups differ from each other in terms of their
chemical structures. While the first group contains carbon and hy­
drogen in its structure, the second group compounds contain at least
one oxygen atom [113]. This group contains lipid-soluble AOXs, and
its another important role is to form vitamin A by enzymatic clea­
vage in the body [110]. There are more than 600 carotenoids in
nature, of which the most abundant is β-carotene found in orange
and yellow fruits, and green leafy vegetables. Studies have also
proven that carotenoids can scavenge 1O2 [106,114]. There have been
many studies on β-carotene, lycopene, lutein, and zeaxanthin and
their protective effects, especially against cancer and eye diseases
[115]. Zeaxanthin and lutein are the main components of the eye-
protecting macular pigments [116]. Foods rich in carotenoids are
usually colorful, and the deterioration in color is associated with
reduced or oxidized forms of these carotenoids [117]. Their AOX
activity show differences according to the location of double bonds
in the molecule, their coexistence with other AOXs, and their con­
centration. Similar to most other AOXs, they can act as prooxidant
when they are in excessive amounts [115].
Another compound that is an AOX precursor, such as carotenoids,
is glutamine and is particularly beneficial for the liver, intestinal
functions, and the immune system [115]. N-Acetyl cysteine, the
source of sulfhydryl, is a thiol compound that acts as a radical sca­
venger [105]. Also, N-Acetyl Cysteine, which is a glutathione pre­
cursor, can increase its AOX effect by increasing the glutathione
level. It has also been used as a criterion to investigate the effect of
ROS on various diseases [115].
α-Lipoic acid is a low-molecular-mass cyclic disulfide and soluble in
both lipid and water, and takes part in various metabolic events such as
regulating glucose metabolism and the circulation of various anti­
oxidants in blood [105]. In the human body, lipoic acid is reduced to
dihydrolipoic acid by means of lipomide dehydrogenase enzyme. It is
known that this reduction may be completed with the aid of other
cellular reducing systems [118]. Lipoate may also enhance the favorable
effects of other AOX systems such as SOD, CoQ10 and glutathione, and
may regenerate the oxidation state of vitamins C and E.
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
Glycine is another non-enzymatic AOX precursor amino acid that
takes part in the synthesis of glutathione and proteins acting as
AOXs, and is especially involved in protecting the kidneys. Studies
have also showed that glycine has a protective effect against
ROS [115].
At high concentrations, vitamins C and E can act as prooxidants
[108]. There are 8 types of vitamin E; α, β, γ, and δ-tocopherol; α, β, γ,
and δ-tocotrienol. α-tocopherol is the most effective one among
them (Fig. 11). The reason for this is the -OH group attached to
carbon number 6 in its structure [119]. The H-atom of the -OH group
in the chromanol ring can be given to a free radical, giving rise to a
delocalized and stabilized unpaired electron; the resulting vitamin E
radical is kinetically inert so as to terminate the chain reaction of
lipid peroxidation [120] Vitamin E is a fat-soluble and chain-
breaking AOX owing to its reaction ability against lipid peroxide
radicals, and prevents this radical from reacting with a new PUFA.
Vitamin E radical, which takes part in the defense against lipid
peroxidation, is converted into vitamin E by vitamin C at the water-
lipid interface [108], enabling the regeneration of vitamin E by vi­
tamin C via favourable redox potentials. Another important role of
vitamin E is to prevent the formation of N-nitroso compounds in the
stomach, thus having a protective effect against various diseases
[110]. So, Vitamin E can both act as a direct radical scavenger and as
an AOX by regulating various enzymes such as SOD, CAT, GPx [115].
Ascorbic acid is a water soluble AOX widely available in plants; it
is a highly effective radical scavenger and reducing agent in aqueous
medium [106,108]. One of the important tasks of vitamin C is to
prevent the formation of N-nitroso compounds in the stomach and
support the formation of collagen. For this reason, it has a protective
effect against various diseases, including cancer [110]. Vitamin C can
also act as a direct ROS scavenger as well as recycler of α-tocopherol,
which plays an important role in lipid peroxidation. In addition, it is
important for the prevention of neurodegenerative diseases due to
its role in the transport of drugs that cannot cross the blood-brain
barrier [115]. For example, Qui et al. worked on the design and
synthesis of ibuprofen prodrugs combined with ascorbic acid [121].
In addition, it was stated in literature that the conjugation of lipo­
some ligands with ascorbic acid (i.e. ascorbic acid thiamine disulfide
delivery system) may be an effective strategy to enhance central
nervous system drugs’ delivery ability into brain [122].
GSH is a water-soluble, low-molecular-mass antioxidant. GSH
enables the reduction of hydroperoxides by some hydroperoxins and
GPx, thus playing an important role in the cell defense system. At the
end of the reaction, some reactions catalyzed by glutathione-S-
transferase are observed and GSH is oxidized to a GSSG form. GSH
can be reversibly oxidized to GSSG (glutathione disulfide) by the
action of oxidants and free radicals. There is a delicate balance be­
tween the sulfhydryl and disulfide forms of glutathione, where they
play a protective role in cellular redox homeostasis called ‘dynamic
thiol/disulfide homeostasis’, and the ratio of reduced-to-oxidized
forms of glutathione (GSH/GSSG) is considered as a well-being in­
dicator important for human health. Redox homeostasis is generally
understood as a dynamic equilibrium between electrophiles and
nucleophiles to maintain an optimal redox steady state [120]. In
general, antioxidant biothiols may react with free radicals while
being oxidized to thiyl radicals (RS•) and show a protective effect by
preventing the target cell from being damaged, the effect being
dependent on the reactivity of the RS• formed [115].
Proline is known as an inhibitor of programmed cell death (PCD).
This is why it is referred to as a powerful AOX that reduces oxidative
stress. In addition, it has been proven in scientific studies that pro­
line is a hydroxyl radical scavenger [106].
Histidine is both a singlet oxygen (1O2) scavenger and a chelator
capable of complexing Cu(II) ions. It is an amino acid that acts as an
13
Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
AOX, because it can inhibit lipid peroxidation by complexing the Cu
(II) ion. Nicotinamide also acts as an AOX in preventing lipid per­
oxidation caused by photosensitization [115].
The bioavailability of non-enzymatic dietary AOXs depends on
many factors such as food processing, AOX structure and its lipo­
philic-hydrophilic balance, solubility and stability, stabilizing effect
of food matrix, etc. [103]. After it was reported that synthetic AOXs
may have toxic effects, studies were focused on phenolic com­
pounds, which are sources of natural AOXs [102].
The AOX properties of phenolic compounds, which constitute a
large part of the non-enzymatic AOX class, depend on their struc­
ture. Phenolic compounds have aromatic ring structures containing
at least one OH group. Simple phenols, phenolic acids, acet­
ophenones and phenyl acetic acids, hydroxycinnamic acids, hydro­
xyanthraquinones, stilbens, flavonoids, lignans, biflavonoids and
tannins are the main phenolic structures [123]. Different structures
of flavonoids such as flavonols, flavones, flavanols, flavanones, an­
thocyanidins and isoflavonoids are formed with the differences in
the C ring (Figs. 12 and 13) [124]. Plant polyphenols have anti­
bacterial, antiviral, antitumor and AOX properties. Polyphenols are
multifunctional, and can be classified as primary AOXs found as
electron donors, H-atom donors and radical scavengers. The ability
to chelate metal ions is another important feature of polyphenols, as
the presence of transition metal ions catalyzes reactions that can
initiate oxidative degradation, such as the Fenton reaction [125].
Flavonoids are taken through the daily diet, and onions, cab­
bage, broccoli, parsley, citrus fruits, grapes and legumes are accepted
as good sources. Due to its AOX properties, they prevent lipid per­
oxidation and have protective effects against oxygen
radicals. Considering that oxygen radicals have important effects on
atherosclerosis, cancer and chronic inflammation, the importance of
flavonoids taken through food becomes clear once again [127]. It has
been reported that AOXs taken through diet, especially in the form of
fruits and vegetables, have a protective effect against many diseases.
Although this protective effect is mostly attributed to the vitamin C
and β-carotene content, plant-derived phenolics such as flavonoids
also have an important role [124].
The chemical structure of flavonoids depends on the degree of
hydroxylation, presence of other substitutions, distribution of con­
jugated double bonds, and the structural class in which they are
found. The hydroxyl group or glycosylation of the C3 group in the C
ring can change the photostability of the molecule. As mentioned
before, changes in A, B and C rings in the flavonoid structure create
the structure of different classes. The biological activities of flavo­
noids and their metabolites are mainly affected by changes in this
chemical structure. Quercetin having an optimal combination of the
listed AOX properties is one of the most abundant flavonols in fla­
vonoid-rich food plants [128].
The AOX mechanisms of flavonoids mainly proceed in three dif­
ferent ways: Radical scavenging effect by H-atom transfer or electron
donation, prevention of metal-catalyzed formation of reactive spe­
cies via metal chelating effect, and interaction with other
AOXs [123].
Phenolic compounds can be distributed differently in plant fa­
milies. Flavonols, and to a lesser extent, flavones are found in most
plants along with hydroxycinnamic acid derivatives. Flavanones and
flavones are found together in most plants and connected by en­
zymes. However, most plants rich in flavanone have almost no an­
thocyanins [124].
Some phenolic compounds such as butylated hydroxyanisol
(BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone
(TBHQ), 2-tert-butyl-4-methylphenol (TBMP) are called synthetic
AOXs which are used as food additives. These synthetic AOXs
transfer their hydrogen atoms to lipid radicals to prevent the radical
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al. Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477

Fig. 12. Chemical structures of flavonoids.

chain reaction involved in lipid peroxidation, as shown with the
reaction equations:
ROO• + ArOH→ ROOH + ArO•
ROO• + RH → ROOH + R• (+O2→ ROO•)
TBHQ, which is a synthetic AOX used in some foods, is espe­
cially useful for the stabilization of unsaturated oils. However, it
has side effects up to DNA damage when taken at high doses. BHA,
which prevents radical formation and lipid oxidation, is a heat-
resistant synthetic AOX used as a preservative in dry cereals,
mixtures or desserts, and especially in fried products. It has been
reported that it can be a tumor initiator if taken at high doses.
TBMP has been used as an intermediate of AOXs and UV stabilizers
used to reduce oxidative processes. Propyl gallate (PG), which is as
effective as vitamin E, plays a protective role in lipid peroxidation.

14
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
Fig. 13. Stereochemistry of flavanones (naringenin).
It acts as a preservative in foods, especially in polyunsaturated
fats [129].
4. Analytical Strategies for the Measurement of Oxidative Stress
and Related Antioxidant Defense
As mentioned earlier, both RS and AOX can be beneficial or
harmful depending on certain conditions, and the balance between
these two types of compounds is crucial in the human body.
Although there are different methodologies for detection of RS in
vitro or in animal studies, to apply these strategies to patients is not
easy. Therefore, oxidative stress status is usually determined by
measuring the products (biomarkers) produced by the oxidation of
certain molecules in biological samples such as blood or urine
[42,50]. The most well-known biomarkers used to detect oxidative
stress are MDA, protein carbonyls and 8-OHdG, which are terminal
products of oxidation reactions. At this point, it can be argued
whether we measure “oxidative stress” or “oxidative damage”.
Ghezzi classified oxidative stress biomarkers into four subgroups.
According to this classification, the oxidative stress biomarkers
showing oxidative damage of lipid, proteins and nucleic acids are
collected as ‘type 1’ biomarkers. The molecules that decrease at the
end of the oxidation with RS (e.g., glutathione (GSH)) are also in­
cluded in type 1. It should be mentioned here that although the ratio
of reduced-to-oxidized forms of glutathione (GSH/GSSG ratio) is not
an exact oxidative stress biomarker (because of the reversibility of
thiol oxidation to disulfide), it reflects cellular redox homeostasis
and is usually accepted as an indicator of well-being. On the other
hand, there are certain enzymes in the human body that are able to
produce RS like H2O2. Molecules that activate RS-generating en­
zymes are accepted as ‘type 2’ biomarkers. Uric acid (UA) and al­
lantoin, which are stable products generated by means of XO
enzyme, are type 2 biomarkers. Hypochlorous acid (HOCl), which
shows phagocytic H2O2 production by means of myeloperoxidase, is
another type 2 biomarker. The enzymes and small molecular mass
biological AOXs such as vitamin C, E and bilirubin can scavenge RS in
the human body. They are defined as ‘type 3’ biomarkers and the
levels of RS producing enzymes such as NADPH oxidases can also be
measured within this type of biomarkers. And finally ‘type 4’ bio­
markers include the degree of mutations of different enzymes re­
sponsible for RS formation or scavenging which may indicate the
role of oxidative stress in a certain disease [45].
There are different analytical strategies for in vitro determination
of oxidative stress. They can be classified according to the bioma­
cromolecules exposed to RS. Additionally, determination of AOX
level can also indicate the oxidative status in human body [130]. But
increased TAC in plasma or serum sometimes indicates oxidative
stress, for example, elevation of uric acid related to a certain disease
such as renal failure can elevate TAC [131]. On the other hand, a
decrease in the plasma or serum TAC level may cause decreased
production of RS [132]. In this case, urate-free TAC level of serum
gains importance, as urate is an AOX molecule compensating for a
15
Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
significant proportion of serum TAC. Serum or plasma TAC con­
centrations may reflect both RS overload and the level of dietary AOX
intake [130]. Oxidative stress biomarkers can be used as beneficial
tools to assess in vivo AOX activity/capacity, Apak et al. mentioned
this concept in their comprehensive review on AOX activity/capacity
measurement [133]. The most vulnerable targets for RS in the
human body (namely lipids, proteins and DNA) can be used as rea­
listic probe materials for determining the activity or capacity of AOX
substances. Because AOX can prevent or delay the oxidation of these
probes, the degree of this inhibition (or delay) can be used to eval­
uate the efficacy of the AOX compounds tested. For example,
Uzunboy et al. used DNA as probe material. The authors created
oxidative damage on DNA by means of radical species obtained by
Fenton-type reactions and were able to determine the oxidative
damage products with the CUPRAC method, which they modified in
accordance with the purpose. They also added AOX compounds to
the reaction medium during radical formation and measured the
decrement in the formation of oxidative damage products, which is
directly related to the concentrations of AOX compounds in the
system [134,135].
4.1. Determination of Lipid Oxidation
Lipid peroxidation is a radical chain reaction consisting of three
basic steps: I- initiation (formation of lipid radical, L•); II- propaga­
tion (producing lipid peroxyl radicals through oxygen uptake by lipid
radicals); this can result in the formation of new lipid radicals; III-
inhibition and termination by formation of nonradical products or of
much less reactive radicals. Especially in the presence of chain-
braking AOX compounds via the reaction between lipid oxidizing
radicals and AOX, the more stable AOX radicals (such as ArO•) gen­
erate [136].
The initial product of lipid peroxidation is peroxyl radical, which
can be detected directly by using ESR technique. In this method, the
spin signal of unpaired electron is used, but since this radical species
is very unstable (transient), a radical spin trap should be used [137].
However, ESR is not a highly sensitive technique and needs steady-
state radicals in the high micromolar concentration range [138].
Although ESR can be applied in vitro, it is not suitable for use in vivo.
One of the reasons for this phenomenon is the need to use spin
trapping agents such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO),
which have high levels of unknown toxicity in vivo. The other pos­
sible reason is the scavenging of DMPO-radical adducts by the en­
zymatic metabolism and endogenous AOXs such as ascorbate [139].
Among the main indicators of oxidative damage on lipids, ethane
and pentane in exhaled gas, lipid hydroperoxides, conjugated dienes,
and isoprostanes (i.e. prostaglandin isomers generated in vivo from
polyunsaturated fatty acids via a free radical induced mechanism)
can be mentioned [140].
4.1.1. Determination of Lipid Hydroperoxides
According to the reaction between iodide (I-) and hydroperoxides
in acidic medium, I2 (or its aqueous-soluble form, triodide: I3-) is
liberated, which can be determined spectrophotometrically or ti­
trimetrically. Spectrophotometric determination of I3- is conducted
at 360 nm by measuring the absorbance of triiodide complex ion
formed from I2 and excessive I- [141]. For higher concentrations, the
very well-known thiosulfate titration in the presence of starch in­
dicator can be applied. This is one of the oldest approaches to
measure the amount of total hydroperoxides and oxidative rancidity
[133]. There are some modifications reported in the literature on the
determination of liberated I2 (or I3-), such as using potassium iodide-
silica gel reagent [142], conducting potentiometric titration [143]
and automated potentiometric titration. When AOX compounds are
present in the reaction medium, the released I2 concentration is
decreased due to the RS scavenging effect of AOXs, thereby leading
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
to the indirect determination of AOX activity [144]. Çelik et al. had an
interesting approach in this regard by developing a novel colori­
metric sensor for measuring hydroperoxide content using starch-
stabilized gold nanoparticles (ss-AuNPs); upon the addition of ss-
AuNPs and excess I- to a hydroperoxide solution, the formed I3- was
rapidly adsorbed on the surface of nanoparticles causing their ag­
gregation with a concomitant red shift (from 525 nm to 563 nm) of
surface plasmon resonance (SPR) absorption of AuNPs. The con­
centration of hydroperoxides estimated in linoleic acid peroxidation
correlated well with those found by the reference ferric thiocyanate
assay. The method was useful for measuring both hydroperoxide and
peroxyl radical scavenging AOXs [145].
Ferric thiocyanate and ferric xylenol orange assays are also used
for this purpose. These methods are based on the oxidation of Fe(II)
to Fe(III) in the presence of hydroperoxides. Then, the generated Fe
(III) forms colored complexes with thiocyanate [146] and xylenol
orange [147]. The ferric xylenol orange assay is also called the FOX
assay which is used for determination of lipid peroxides in biological
samples [148]. As mentioned previously, the color intensity of gen­
erated complexes decreases in the presence of AOX compound in the
reaction medium [133].
Another analytical method to track lipid peroxidation is ultra­
violet measurement of conjugated dienes. During lipid peroxidation,
after the formation of lipid radicals, conjugated dienes that exhibit
π-π * electronic transitions are formed by rearrangement of bonds in
the molecule. This allows the degree of lipid peroxidation to be
determined by measuring the absorbance produced at 230–235 nm
[133]. Conducted spectrophotometric measurement in UV region
makes the method susceptible to interference from organic com­
pounds absorbing in the same wavelength range. Therefore, the
method needs a pre-separation such as solvent extraction prior to
analysis of biological samples [149]. The method was also applied for
the determination of AOX activity in some synthetic and natural
samples [150,151].
The kinetic analysis of lipid peroxidation can provide useful in­
formation on the absolute or relative rate constants for the lipid
peroxyl radical scavenging ability of AOXs. It is also important for
understanding the effect of AOX action and its radical scavenging
capacity [140].
In regard to electrochemical determination of hydroperoxides,
Adhoum and Monser developed a Prussian blue (PB)-modified glassy
carbon sensor electrode on which hydroperoxides are electro­
chemically reduced; the method was applied to the amperometric
determination of peroxide value (PV) in edible oil. The sensor elec­
trode responded to all hydroperoxides, but the strongest responses
were recorded for H2O2 followed by tert-butyl hydroperoxide, cu­
mene hydroperoxide and linoleic acid hydroperoxide [152].
4.1.2. Determination of Thiobarbituric Acid Reactive
Substances (TBARS)
The thiobarbituric acid reactive substances (TBARS) assay is
probably the most common and most criticised method of de­
termining lipid oxidation. One of the final products of lipid oxidation
is MDA. The method is based on measuring the optical absorbance of
the chromogenic adduct compound consisting of MDA and thio­
barbituric acid (TBA) at 1:2 stoichiometry (MDA-(TBA)2) at 532 nm
spectrophotometrically [153] or at 553 nm fluorometrically [154].
The basic disadvantage of TBARS method is that, especially in
complex matrices, TBA can react with compounds which are not
directly related to lipid oxidation causing erroneous results
[155,156]. To separate MDA from possible interferents, the method
can be combined with FTIR and HPLC to enhance selectivity and
sensitivity [156]. Similar to the other oxidative stress parameters, the
production of MDA decreases in the presence of AOX compounds.
Therefore, TBARS method can also be used for the determination of
16
Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
AOX efficiency [30]. Bakır et al. used the TBARS test in a linoleic acid
emulsion model system, and the authors reported that TBARS assay
was not successful to reflect the relationship between the quantity of
protecting AOXs and the decrease in the amount of formed chro­
mophore [157].
To determine lipid peroxidation products, GC-MS is also used. It
is possible to get more accurate results by chromatography com­
pared to spectrophotometry. However, most of the lipid oxidation
products are not thermally stable, hence a derivatization should be
applied. The other chromatographic techniques used in this field are
electron ionization (EI) MS coupled to selected ion monitoring (SIM)
technique, electron capture (EC) and atmospheric pressure chemical
ionization (APCI) MS (EC-APCI-MS) [158,159].
4.2. Determination of Oxidative Protein Damage
Proteins are the major targets to RS because they contain certain
amino acid residues and thiol groups vulnerable to oxidation. Due to
the effect of RS on proteins, some modifications occur and these can
be used to quantify protein damage. Oxidative damage of proteins
causes some changes in parent amino acid residues which can be
determined by different analytical techniques such as HPLC,
fluorometry and LC-MS [80]. Another oxidative stress biomarker is
oxidation of low-molecular-mass thiols. For example, GSH can be
oxidized to different products such as GSSG, mixed disulfides or
glutathione sulfonamide. Glutathione reductases reduce disulfides
to GSH. This change can be monitored spectrophotometrically by
using 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB, also known as
Ellman’s reagent) at 412 nm. Here, GSH, generated from GSSG by
glutathione reductase, is reacted with DTNB [160]. Erel and Nese­
lioglu designed a DTNB reagent−based automated assay for thiol/
disulfide homeostasis using sodium borohydride as reducing agent
[161]. Görüşük et al. adopted a different approach for determining
both thiols and disulfides with the same 2,2'-azinobis-3-ethylben­
zothiazoline-6-sulfonic acid (ABTS) reagent, where the slower re­
acting disulfides required an elevated temperature incubation for
reducing the ABTS•+ radical cation [162]. As residues of reducing
agents pose additional problems in redox-based assays, Çifteci et al.
developed an AuNPs–based turn-on fluorometric sensor for quanti­
fication of sulfhydryl and disulfide forms of biothiols in relation to
the measurement of thiol/disulfide homeostasis. In this assay, elec­
trostatic adsorption of water-soluble fluorophore Rhodamine–6 G
(Rh–6 G) dye onto citrate–stabilized AuNPs resulted in Förster re­
sonance energy transfer (FRET) in the turn–off mode, but due to the
high surface affinities of biothiols (in both thiol and disulfide forms)
to AuNPs, the fluorescence increment at 552 nm (turn-on mode)
accompanying the release of Rh–6 G dye molecules indicated thiol
content. Due to the greater affinity of AuNPs for thiols, the accom­
panying release of the fluorescent dye Rh-6 G from the nano­
particles’ surface gave rise to an increase in fluorescence. By this
selective adsorption-desorption process onto-from gold nano­
particles, the assay could measure the total amount of thiols and
disulfides, but when free thiols were blocked by the thiol-alkylating
agent N-ethylmaleimide, only disulfides gave the fluorescence re­
sponse, thereby enabling to differentiate disulfides from thiols and
determine thiol/disulfide ratio (as the wellness indicator) fluor­
ometrically [163].
Except determination of changes on amino acid residues; protein
carbonyls, disulfides (−S-S−), sulfinic (−SO2H) or sulfonic (−SO3H)
acids are among the mostly used biomarkers of oxidative protein
damage [140]. Quantification of protein modification by oxidants by
making use of the reactions such as the loss of the parent amino acid
residue, formation of unstable intermediates, and the generation of
stable products was excellently reviewed by Hawkins et al.; the most
common methods to measure protein oxidation products are HPLC-
MS, GC-MS and Western blot/ELISA test [80].
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
The most studied oxidation product is protein carbonyls, which
can be analyzed in different biological samples. Their chemical
stability at appropriate storage conditions is another advantage
(they are stable for 3 months at −80oC) [164]. One of the well-known
methods for the determination of protein carbonyls is based
on the reaction between carbonyl compounds produced by protein
oxidation and 2,4-dinitrophenylhydrazine (DNPH). The formed
2,4-dinitrophenylhydrazones can be determined either spectro­
photometrically (λmax = 370 nm) or using Western blotting ELISA
techniques [80,165]. However, since the colors of the product
(hydrazone) and the reagent (hydrazine) are nearly identical, and
other possible interferents (e.g., heme-containing proteins and
retinoids) absorb light around this analytical wavelength, the
analysis may overestimate the result [166]. Bayarsaikhan et al.
developed an alternative modified CUPRAC method to identify
the products derived from oxidative protein damage and measure
the protective effects of different AOXs on this damage. The authors
used three different model proteins, namely bovine serum albumin,
egg white and fetal bovine serum, for this purpose. In this study,
CUPRAC method was used in two different ways (i.e. in solution and
on a Nafion membrane sensor). They also used the modified DNPH
carbonyl determination method to compare their findings.
According to this modified DNPH assay, the final anionic product was
obtained in strongly alkaline medium in the presence of a cationic
surfactant (cetyl trimethyl ammonium bromide) and after extraction
into acetonitrile, the absorbance was read at 430 nm [167]. The
presence of concentrated NaOH shifted λmax to longer wavelengths
and allowed a direct quantification without a need for lengthy
pretreatments [168].
In another technique used by Ma et al., carbonylated proteins
were determined by frequency-shift based surface-enhanced Raman
spectroscopy (SERS) immunoassay. The authors reported the elim­
ination of possible interferences by using a capture antibody [169].
There are a number of fluorophore compounds produced at the
end of several oxidative and glycoxidative modifications of amino
acid residues, giving way to fluorescence spectroscopy to determine
protein oxidation. The drawback of this method is not to allow in­
dividual analysis of tested compounds as it measures all fluorophore
products in the reaction medium, meaning that substances that
fluoresce but are not a product of protein oxidation will cause in­
terference. Thus, in order to separate protein oxidation biomarkers, a
preliminary chromatographic separation is needed.
Finally, the overall structure of an oxidized protein can be
monitored by using different approaches such as gel electrophoresis,
light scattering analysis and X-ray crystallography. Using these
techniques, it is possible to compare intact and oxidized protein
samples, but these approaches are not capable of showing site-
specific oxidative damage.
There are also different electroanalytical methods in literature
aiming to measure oxidative protein damage. For example, Enache
et al. developed an electrochemical sensor to detect the carbonyl
groups generated by oxidation of proteins with RS. The method was
based on the redox characteristics of DNPH. The model protein used
for the study was bovine serum albumin (BSA). The researchers in­
vestigated electrochemical behavior of intact and oxidized BSA using
differential pulse voltammetry technique and glassy carbon elec­
trode. The authors showed that the hydrazine moiety of the DNPH
reagent was the electroactive center and was responsible for car­
bonyl complexation. DNPH was immobilized on a mixed 4-styr­
enesulfonic acid-Nafion matrix to retain its properties [170].
Sun et al. investigated both protein damage and protective effects
of certain AOX compounds. The authors used the BSA/poly (dia­
llyldimethylammonium chloride) (PDDA) functionalized graphene
nanosheets (PDDA-G) composite film modified glassy carbon elec­
trode (BSA/PDDA-G/GCE) for this purpose. The electrochemical
method chosen was square wave voltammetry (SWV). The model
17
Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
protein, BSA, was oxidized by means of a classical Fenton system
(FeSO4/H2O2). The tested AOXs were four different flavonoids
namely; catechin, kaempferol, apigenin and naringenin. All tested
polyphenols caused a change in the SWV signal of BSA in the solu­
tion containing cobalt(III) tris-2,2’-bipyridine complex. The re­
searchers determined that the most effective AOX among the tested
compounds was catechin [171].
4.3. Determination of Oxidative DNA Damage
Considering the determination of oxidative DNA damage, the first
compound that comes into mind is 8-hydroxy-2-deoxyguanosine (8-
OHdG) as the most examined oxidation product, however, it is not
the only biomarker of oxidative DNA damage. Oxidative attacks to
DNA can cause simultaneous base damages and modifications.
Chromatographic methods, especially GC-MS, is often used to detect
these products [172]. Among the other oxidation damage products of
DNA bases, 2-hydroxyadenine (2-OH-Ade), 5-OHC, 5-FoU, and 5-
OHU can be mentioned [173,174]. In a recent study co-authored by
Halliwell and Dizdaroğlu [175] as the two leading researchers in this
field, it has been reported that the hydroxyl radical has an important
contribution to oxidative DNA damage in vivo. The authors also ex­
pressed that hydroxyl radical, unlike all other RS, generates a large
number of different damage products from all DNA bases. Some of
the other RS either do not react directly with DNA or selectively
target guanine due to its relatively low standard reduction potential
(E0) value. Since DNA has negatively charged phosphate groups,
transition metal cations such as divalent Fe and Cu are bonded to
DNA. On the other hand, H2O2 can cross intracellular membranes
and when it reaches DNA, •OH can be generated by Fenton reaction
and causes ‘site-specific’ damage on DNA. The distribution of DNA
degradation products caused by site-specific and other modes of
hydroxyl radical attack upon DNA through transition metal io­
n−induced •OH generation depends on several factors, including the
site of metal binding to DNA [175].
During the oxidation of DNA, not only damaged bases but sugar
modification products are also generated and these can be mon­
itored by GC-MS or LC-MS techniques. Among other DNA oxidation
biomarkers are oxidized purines (and pyrimidines), strand breaks,
and C-8 hydroxylation products of guanine, 8-oxo-7,8-dihydro-2’-
deoxyguanosine. Since the amount and types of the oxidation pro­
ducts are closely related to reaction conditions and redox status of
the cell, assesment of multiple products could give more realistic
results [133].
Among the most widely used techniques to identify biomarkers
associated with DNA oxidation are electrochemical detection
methods, namely cyclic voltammetry (CV), differential pulse vol­
tammetry (DPV), and square wave voltammetry (SWV), using bare or
suitably modified electrodes. Especially nanostructured materials
(either carbon− or metal−based) are often used as electrode surface
modifiers. There are also studies in which electrodes are directly
modified by single-stranded DNA; alternatively, oxidative DNA da­
mage can be followed with the use of dendrimer, aptamer or anti­
body modified electrodes to achieve high sensitivity and selectivity
at relatively low cost. Recently, they have been miniaturized and
used in automation systems [176].
Some degree of oxidative damage to DNA is often associated with
age-related cancer. So, oxidative DNA damage can be a meaningful
indicator showing a person’s cancer risk. On the other hand, there
are ongoing discussions on the possibility of cancer prevention
through DNA protection against oxidative damage with the aid of
AOX compounds taken up via appropriate diets [172].
In a sample study, the oxidative damage on DNA and protective
effects of AOX compounds were determined by using gas chroma­
tography–tandem mass spectrometry. The authors measured a series
of DNA oxidation products in the presence and absence of V.vinifera
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al. Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477

Table 1
Biomarkers of oxidative stress and antioxidant defense system in various diseases.

Disease Oxidative stress biomarkers Antioxidant status Reference

Cancer
Breast cancer MDA, AOPP, Trx SOD [181]
Breast cancer TBARS, LH, carbonylation of serum proteins FRAP, GSH [182]
Prostate cancer MDA, NO, TPP, OSI TAC [183]
Prostate cancer TBARS, carbonylation of serum proteins CAT, SOD, vitamin C and E, NPSH [184]
Prostate cancer LPO, 8-OHdG CAT, SOD, GST [185]
Colorectal cancer MDA, ox-LDL TAC, SOD, GPx, GST [186]
Colorectal cancer NOX, XO, AGE, AOPP, MDA, 8-OHdG SOD, CAT, GPx, GR, GSH [187]
Cervical cancer 8-OHdG, carbonylation of serum proteins, MDA SOD, CAT, GPx [188]
Lymphoblastic leukemia TBARS, carbonylation of serum proteins CAT, SOD [189]
Lung cancer TBARS CAT, SOD, TS-H, NPSH
Neurodegenerative disease
Amyotrophic lateral sclerosis MDA, 8-OHdG GPx, GR, SOD, TAS, GSSG, GSH [190]
Parkinson’s disease LPO, MDA, CRP SOD, CAT, TRAP, SH [191]
Parkinson’s disease MDA, AOPP, 4-HNE [192]
Parkinson’s disease AOPP, TBARS SOD, CAT, FRAP, vitamin C, NPSH [193]
Alzheimer's disease TBARS, MDA, homocysteine FRAP [194]
Autism MDA GSH, CAT, PON1, TAC [195]
Diabetes
Type 1 diabetes mellitus MDA, NO, FBS, HbA1c SOD, CAT, GPx [196]
Type 1 diabetes mellitus LPO, MDA, carbonylation of serum proteins, HbA1c GSH, GPx, α-tocopherol, β-carotene [197]
Type 1 diabetes mellitus HbA1c Cu/Zn-SOD, CAT, TAS [198]
Type 2 diabetes mellitus MDA GSH, GR, GPx, SOD, CAT [199]
Type 2 diabetes mellitus LPO, MDA FRAP, protein thiols, GSH, CAT [200]
Gestational diabetes mellitus TOS, OSI Irisin, TAS [201]
Gestational diabetes mellitus MDA, 8IsoP, XO, LPO SOD, TAC, GPx, vitamin C, vitamin E [202]

(var. alphonse) extract rich in AOXs, and concluded that the use of
the extract caused a significant decrease in the amounts of DNA
oxidation products [177].
Instead of detecting biomarkers, studies on the determination of
RS directly by using electrochemical sensors to monitor oxidative
stress have been mentioned by Deshpande et al. in a recent review.
Although using glassy carbon electrodes is possible for this purpose,
there are a number of restrictions for live tissues. The best solutions
to overcome this situation is to use small sized electrodes (size:
5–10 nm, diameter: ~100 mm) having the ability of measuring RS in
the proximity of cells [41]. For example, using this approach H2O2
and O2•– could be detected by means of Au-nanocones modified
carbon fiber microelectrodes (CFME) in a single drop of blood. Here
the authors used coupling of the small sized CFME and a selective
synthetic receptor. The technique used here was DPV [178].
In another example study to determine O2•–, the suitable redox
potential of O2/O2•– couple vs. NHE (330–140 mV) was exploited.
This value allowed direct determination of O2•– by electrochemical
oxidation using platinized microelectrodes or with nitrogen-doped
carbon AgNPs electrodes [179].
Beissenhirtz et al. developed a new type of superoxide sensor
based on a multilayer cytochrome c (cyt. c) electrode. To develop the
gold wire electrode, it was modified by alternating layers of cyt. c
and poly(aniline(sulfonic acid)). The authors concluded that the in­
crease in the layers contributed to electrochemical activation of cyt.
c. The developed electrode was used in the determination of en­
zymatically generated superoxide. The authors concluded that the
multilayer sensor showed a superior sensitivity for O2•– determi­
nation compared to monolayer electrode systems. Additionally, the
researchers reported the good thermal stability of the modified
electrode [180].
5. Balance Between Oxidants and Antioxidants
The aim of this part of the study is to give detailed information
about oxidative stress and antioxidant status (AS) biomarkers to be
measured in each pathogenesis. Then, the balance between oxidative
stress and AOX defense system in patients were also discussed by
comparing that of control groups. Biomarkers of oxidative stress and
AOX defense in various diseases are covered in Table 1.
MDA, malondialdehyde level; SOD, superoxide dismutase ac­
tivity; AOPP, advanced oxidation protein product; Trx, thioredoxin;
GSH, reduced glutathione; TBARS, thiobarbituric acid reactive sub­
stances; LH, lipid hydroperoxide; FRAP, ferric reducing antioxidant
potential; 8-OHdG, 8-hydroxydeoxyguanosine; LPO, lipid hydroper­
oxides; GST, glutathione S-transferase; GPx, glutathione peroxidase;
ox-LDL, oxidized-low density lipoprotein cholesterol; GR, glu­
tathione reductase; AGE, advanced glycation end products; TAC, total
antioxidant capacity; TAS, total antioxidant status; NPSH, non-pro­
tein thiols; TS-H, total thiols; GSSG, glutathione disulfide; SH, sulf­
hydryl group; TRAP, total radical-trapping antioxidant parameter; 4-
HNE, 4-hydroxynonenal; XO, xanthine oxidase; NOX, NADPH oxi­
dase; NO, nitric oxide; PON1, paraoxonase 1 activity; FBS, fasting
blood sugar; HbA1c, glycated hemoglobin; TOS, total oxidative
stress; OSI, oxidative stress index; 8IsoP, 8-isoprostane.
6. Biomarkers of Oxidative Stress and Antioxidant
Defense in Cancer
6.1. Breast cancer
Breast cancer is a major public health issue and is the primary
reason of cancer related death among women worldwide. In a re­
presentative study on breast cancer, it was stated that oxidative stress
is responsible from promotion and progression of various cancers,
such as breast cancer [203]. Therefore, in order to reveal the asso­
ciation between the disease and oxidative stress biomarker levels, 23
surgically treated women with breast cancer (against 13 healthy
controls) were involved in the study [181]. Blood samples of the pa­
tients were analyzed in terms of malondialdehyde (MDA) levels as an
index of lipid peroxidation, superoxide dismutase (SOD) activities,
advanced oxidation protein product (AOPP) and thioredoxin (Trx)
levels. The results of this study indicated that MDA, SOD, AOPP as well
Trx levels of the patients were found to be significantly higher than
those of the controls. As the data pairs of serum parameters in control
group and breast cancer patients, Trx, MDA, AOPP and SOD levels
were (4.12; 141 ng/mL), (3.08; 6.58 nmol/mL), (60.2; 148.7 µmol/L),

18
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
(6.76; 22.2 Units (U) /mL), respectively, patients having higher levels
than controls in all parameters [181]. Additionally, the oxidative stress
caused by different treatments during breast cancer was also in­
vestigated in a recent study; the degree of oxidative stress produced
by adjuvant chemotherapy, radiotherapy or hormonal therapy treat­
ments in seventy women with breast cancer was examined. After
collecting blood samples, GSH, TBARS, lipid hydroperoxide (LH) level
and carbonyl level measurements were conducted as oxidative stress
analysis, and FRAP assay was used to detect serum TAC levels. The
FRAP values were associated with diet quality only at the baseline (at
p0); oxidative stress parameters showed an increase after adjuvant
chemotherapy (at p1), but they were not associated with diet
quality [182].
6.2. Prostate cancer
Prostate cancer is another serious worldwide health problem and
it is the second most common malignancy in aging men [204]. Due
to the fact that the cause of oxidative stress relies on an imbalance
between reactive oxygen/nitrogen species or lipid peroxidation
products and the cell’s detoxification defense system [205], a case-
control study was designed with 120 patients with prostate cancer
(against 100 healthy controls) to assess the levels of blood MDA, NO,
total plasma peroxide (TPP), TAC as well as oxidative stress index
(OSI) [183]. Results showed with statistical significance (set at
p < 0.05) that the patients with prostate cancer had significantly
higher MDA (0.48 and 0.29 µmol/L respectively), NO (4.6 and
2.4 µmol/L, respectively), TPP (25.3 and 17.2 µmol/L H2O2, respec­
tively) and lower TAC levels (3.5 and 3.9 mmol/L, respectively) in
comparison with control groups. In order to investigate the presence
or absence of bone metastasis in the levels of oxidative damage and
AOX defense, biological damage caused by free radicals in lipids and
proteins was determined by TBARS and carbonylation of serum
proteins in 55 patients with prostate cancer against 55 healthy men.
Moreover, AS of the patients was examined by enzymatic (CAT and
SOD) and non-enzymatic AOXs (non-protein thiols (NPSH), vitamin
C and vitamin E) [184]. Results indicated that TBARS and protein
carbonyl levels were increased for both patients with localized
prostate cancer and bone metastatic disease. While SOD activity was
increased only in patients with localized prostate cancer, NPSH le­
vels were increased in patients with bone metastasis. Besides, vi­
tamin C and E contents as well as CAT activity were reduced in both
groups in comparison to the control. Similar findings were reported
by Shukla et al. [185], indicating higher 8-OHdG levels to quantify
the DNA damage and lipid hydroperoxides (LH) as well as lower GST
value. However, there was no significant variation in CAT and SOD
levels [185].
6.3. Colorectal cancer
Colorectal cancer is the third and the second most frequent type
of cancer in men and women, respectively [206]. It has been stated
that increased homocysteine concentration [207], higher levels of
oxidative stress and increased or decreased AOX enzyme activities
are among the risk factors of colorectal cancer [208]. It has been
indicated that hyperhomocysteinemia (homocysteine concentration
≥ 14 µmol/L) admits overproduction of free radicals and hydrogen
peroxide, and may further cause gene mutation and cancer. In ad­
dition to this, high homocysteine level also affects cysteine which is
responsible for the rate-limiting impact on the synthesis of GSH and
GSH-dependent AOX defense system [209]. In a study on the re­
lationship between high homocysteine levels and increased risk of
colorectal cancer [186], plasma homocysteine, cysteine, pyridoxal 5’-
phosphate (PLP), folate and reduced glutathione values, oxidative
stress indicators including MDA and oxidized-low density lipopro­
tein cholesterol as well as AS measuring TAC, SOD, glutathione
19
Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
peroxidase (GPx) and GST levels in one hundred sixty eight patients
with colorectal cancer were investigated. A glance at the results
revealed the significantly higher homocysteine, cysteine, folate and
glutathione levels in patients with colon or rectal cancer when
compared to those of control. While higher homocysteine con­
centration was associated with the risk of colorectal cancer; cy­
steine, PLP and folate had no effect on the risk of colorectal cancer.
Moreover, 151 colorectal cancer patients had significantly higher ox-
LDL levels and SOD activities as well as significantly lower GPx and
GST activities than those of 188 healthy controls. In the AOX defense
system, SOD and GPx are considered to be the primary AOX enzymes
and GST plays as the secondary AOX enzymes. The raised SOD ac­
tivity could catalyze the dismutation of superoxide into oxygen and
hydrogen peroxide. Later, catalase and GPx activities manage the
elimination of hydrogen peroxide. However, in this study, excessive
amount of hydrogen peroxide or cellular toxicity of cancer resulted
with the decreased GPx and GST activities [186]. These results are in
agreement with Zińczuk et al. [187] who showed that there is an
association between the subjects with colorectal cancer and enzy­
matic/non-enzymatic redox imbalance, increased oxidative damage
to proteins, lipids and DNA. Redox homeostasis was defined as the
balance between TAC and total oxidant status (TOS). For the ex­
amination of redox status, pro-oxidant enzymes including NADPH
oxidase (NOX) and XO; AOX enzymes such as SOD, CAT, GPx, glu­
tathione reductase (GR) and GSH as well as oxidative damage pro­
ducts like advanced glycation end products (AGE), AOPP, MDA and 8-
OHdG were determined in normal and cancerous tissue of twenty-
nine patients with colorectal cancer. According to the results, NOX,
XO, SOD, CAT and TAC levels as well as oxidative damage products
were found to be significantly higher in cancer tissue than in normal
colon mucosa [187].
6.4. Cervical cancer
With regard to serum oxidative stress and AOX biomarker levels
in cervical cancer patients, 8-OHdG, carbonylation of serum proteins
and MDA of oxidative damage products as well as SOD, CAT, GPx, and
total AS of AOX defense mechanisms were studied. Results high­
lighted the significant increase in oxidative damage markers and
significant decrease in AOX parameters in cervical cancer patients
compared to controls [188].
6.5. Leukemia
Similar trends were obtained for lymphoblastic leukemia pa­
tients [189]. Increased oxidative damage products including TBARS
and serum protein carbonyls and reduced AOX levels (CAT, SOD)
were found in the patients compared to controls.
On the other hand, Zanini et al. [210] reported higher non-en­
zymatic thiols (NPSH and total thiol groups (TS-H)) levels and an­
tioxidant enzymes (CAT and SOD) activities as well as lower TBARS
values in the patients compared to controls. It was explained by
increased tumor size, progression of cancer and overproduction of
ROS, because it can be assumed that rapidly dividing cells are prone
to build an oxidant-antioxidant status appropriate for their growth;
for instance, high level of AOX and low substrate of peroxid­
ability [211].
7. Biomarkers of Oxidative Stress and Antioxidant Defense in
Neurodegenerative Diseases
7.1. Amyotrophic lateral sclerosis (ALS)
Amyotrophic lateral sclerosis (ALS) is the most common motor
neuron disease and defined by degeneration of upper and lower
motor neurons in the brain and spinal cord [212]. Blasco et al. [190]
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
carried out a pilot study with ten ALS patients to present a link
between ALS disease and oxidative stress by measuring GPx, GR,
SOD, TAS, MDA, 8-OHdG, glutathione status (e.g. glutathione dis­
ulfide (GSSG)/reduced glutathione (GSH)), vitamins (B9, B12, A, E, C)
and metals (Cu, Zn, Mn, Se). The authors reported a significant in­
crease in 8-OHdG and MDA levels and a significant decrease in TAS
levels of ALS patients compared to controls, indicating an imbalance
between the production of ROS and the defense system to remove or
repair the damage. On the other hand, there was no significant
change in AOX enzyme activities (SOD, GPx, GR), vitamin and mi­
neral values, however, glutathione status was altered in ALS patients
resulting in a significantly higher GSSG/GSH ratio. Reduced GSH level
is related with an increase in its oxidized form, which could ad­
versely affect the disease evolution [213]. The results of this study
showed that oxidative stress is an appropriate factor in the evalua­
tion of ALS [190].
7.2. Parkinson’s disease (PD)
PD is the second most common neurodegenerative disorder, in
which the lowered dopamine levels as well as motor and non-motor
manifestations occur due to the injured dopaminergic neurons in the
substantia nigra pars compacta [214]. In addition to the oxidation of
dopamine by monoamine oxidase, chronic inflammatory process
and high concentrations of iron in the brain lead to RS generation
and cellular dysfunction, resulting in neurodegenerative diseases
[215]. Since the increased damage to lipid, protein and DNA might be
tracers of oxidative/ nitrosative stress in this pathogenesis [192],
current studies aim to investigate the relationship between oxida­
tive/nitrosative stress parameters and AOX defense status. For in­
stance; de Farias et al. [191] indicated a higher correlation between
increased LPO, MDA, SOD activities as well as lower total TRAP,
sulfhydryl group (SH), CAT activity and PD disease. It has been re­
ported that higher lipid peroxidation indicators, including LPO and
MDA, are linked to lower AOX concentrations, high substrate levels
for oxidation and unidentified plasma pro-oxidants in cases with PD.
Moreover, while enhanced SOD activity may arise from higher pro­
duction of superoxide radicals, resulting in scavenging of ROS but
H2O2 generation, lower levels of CAT might be detected due to in­
creased usage to decompose H2O2 [191]. With regard to analyze
oxidative stress−antioxidant defense balance in PD subjects, the
oxidative stress biomarkers such as MDA, AOPP and 4-HNE as well as
prooxidant–antioxidant balance were determined. Results indicated
that these markers are found to be gender- and age-related [192]
Indeed, it was reported that elder male subjects are more susceptible
to this disease [216]. In another study, Medeiros et al. [193] explored
the peripheral iron, ferritin, and transferrin levels, oxidative stress
biomarkers and AOX activity in PD patients. Results revealed that
iron levels were found to be significantly lower in PD individuals
(67.5 μg/dL) rather than that of controls (78 μg/dL), whereas there
was no significant difference in ferritin and transferrin concentra­
tions. Although high level of iron in the substantia nigra is regarded
as a risk factor in PD, decreased level in peripheral blood is possibly a
problem for the function of neuronal enzymes as well as ferritin
reservoir in neurons, causing accumulation of free iron in the sub­
stantia nigra [217]. Furthermore, decreased AS including vitamin C,
NPSH and FRAP and increased oxidative stress levels such as TBARS
and AOPP were obtained in the PD cases in comparison with con­
trols. On the other hand, there were no significant variation in SOD
and CAT activities of PD patients and controls [193].
7.3. Alzheimer’s disease (AD)
AD is an irreversible brain disorder, which could be induced by
dyshomeostasis of biochemical parameters. AD is the most common
form of irreversible dementia pertaining to elderly stages of life, and
20
Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
there is an increasing risk in the formation of dementia according to
the transition from mild stage to moderate and severe stages of
cognitive disruption [218]. In this context, Negahdar et al. [194]
analyzed the risk factors in one hundred twenty patients with mild
or moderate to severe cognitive impairment. Serum oxidant/anti­
oxidant status were determined by measuring trace elements
(copper, manganese and zinc), homocysteine, TBARS, MDA and FRAP
levels. Results showed that there is an association between the in­
creased oxidative stress as well as decreased AS and cognitive im­
pairment stage. In addition to these, there was an increasing trend in
homocysteine levels, which is responsible for oxidative stress, de­
pending on aging. However, there was no statistical variation in the
levels of trace elements in both patients and control group [194].
7.4. Autism spectrum disorder
In addition to ALS, PD and AD, autism spectrum disorder (ASD) is
another neuro developmental disorder and characterized by diffi­
culties in communication and social interaction, repetitive and ste­
reotypic behaviors and defects in cognitive abilities [219]. It was
reported that oxidative stress, genetic and environmental factors as
well as immune dysregulation have a role in this disorder [220].
Abdel-Salam et al. [195] indicated a significant increase in MDA le­
vels and decrease in AS, including CAT, paraoxonase 1 (PON1), GSH
and TAC values in the serum of 3 − 12 years aged individuals with
autism spectrum disorder compared to the control group. Moreover,
nuclear factor kappa B (NF-κB), which is a protein transcription
factor, was found to be significantly higher in children with autism
rather than that of control group, suggesting activation of NF-κB
with increased status of oxidative stress [195].
8. Biomarkers of Oxidative Stress and Antioxidant
Defense in Diabetes
Diabetes mellitus (DM), one of the major disorders, is char­
acterized by hyperglycemia along with carbohydrate, lipid, and
protein metabolism abnormalities [221]. DM is predicted to affect
592 million people by 2035 [222]. It was reported that one of the
pathogenesis associated with diabetes may be related to oxidative
stress [223]. Free radicals that are produced at elevated amounts in
diabetes owing to glucose autoxidation process as well as simulta­
neous decrease in AS may deteriorate the lipids, proteins, cellular
organelles and DNA, resulting in insulin resistance [224,225].
8.1. Type 1 diabetes mellitus
Type 1 diabetes mellitus (T1DM) is an autoimmune disorder
caused by defect of pancreatic–beta cells [226] and involves ap­
proximately 5–10% of all diabetes cases [196]. In a recent study, as­
sociation between oxidative stress levels including MDA, NO, fasting
blood sugar (FBS) and glycated hemoglobin (HbA1c) and AOX en­
zyme status such as SOD, CAT and GPx in T1DM patients were
analyzed. Results indicated that significantly higher FBS
(173.09 ± 17.80 mg/dL) and HbA1c concentrations as well as MDA
and NO levels were obtained in T1DM cases rather than those of
healthy individuals. Moreover, serum CAT, SOD and GPx activities
were found to be statistically lower by 16.7%, 72.8%, and 15.3%, re­
spectively in T1DM subjects in comparison with those in controls,
indicating a decrease in the AOX enzyme activities due to glycation
of enzymatic proteins [196]. Similarly, Codoñer-Franch et al. [197]
investigated oxidative stress by measuring the primary (LPO) and
secondary (MDA) lipid peroxidation products as well as level of
carbonylation of serum proteins. While endogenous AOX defense
system was evaluated by determining the GSH and GPx, exogenous
AS was determined by means of α-tocopherol and β-carotene. Sta­
tistically elevated levels of lipid (LPO and MDA) and protein
S. Demirci-Çekiç, G. Özkan, A.N. Avan et al.
(carbonylation of serum proteins) oxidation markers were detected
in the patients with T1DM in comparison with control groups. Be­
sides, GPx activity, which is responsible from protection of cell
proteins and membranes against oxidative damage, were decreased
in T1DM patients, whereas there was no variation in the GSH con­
centration among the groups. With respect to the results of exo­
genous AOX activities, lower α-tocopherol/total Ch ratio and β-
carotene concentrations were found in the subjects with T1DM
when compared with control groups, suggesting usage of AOXs due
to higher oxidative stress [197]. In another study, Salmonowicz et al.
[198] investigated the effects of trace elements which have an im­
pact on the AOX defense system in children with T1DM. According to
the results, participants with T1DM showed lower levels of Mg
(19.27 μg/mL), Zn (88.35 μg/100 mL), higher concentrations of Cu
(126.19 μg/100 mL) and decreased TAS (0.894 mmol/L) levels com­
pared to those of of control groups (20.54 μg/mL Mg, 97.00 μg/
100 mL Zn, 110.55 μg/100 mL Cu and 1.170 mmol/L TAS), suggesting a
relation between damaged AOX defense system and deficiency of Mg
and Zn and increased Cu levels (the indicated metal ions may be
assumed to be divalent, though not indicated in the source litera­
ture) [198].
8.2. Type 2 diabetes mellitus
Recently, it has been reported that complications associated with
generation of T2DM may be related to the same oxidative stress
pathways [227]. In the context of the relation between AS and oxi­
dative stress levels in patients with T2DM, Siddiqui et al. [199] de­
monstrated that participants with newly diagnosed T2DM had
significantly higher amount of MDA (2.66 ± 1.11 nmol/mL), in­
creased GR (1.78 ± 0.85 U/mL) and SOD activity (867.65 ± 259.08
units per gram of haemoglobin, U/gHb) and lower GSH levels
(15.53 ± 3.78 μmol/gHb) than those of the subjects (2.13 ± 0.72
nmol/mL MDA, 1.57 ± 0.78 U/mL GR, 754.11 ± 170.75 U/gHb SOD
and 17.35 ± 3.6 μmol/gHb GSH) with normal glucose tolerance [199].
Glycated plasma proteins possess a crucial role in diabetes [228].
This process causes formation of different glycation intermediate
products and protein modifications depending on the glycation
stage, including fructosamine, amino or carbonyl intermediates,
advanced glycation end-products and amyloid fibrils [228–230].
Tupe et al. [200] investigated the glycated plasma proteins from
various glycation stages, oxidative stress and AOX defense system
parameters in the patients with T2DM. Results showed that while
there was no significant variation in fructosamine levels, protein
carbonyl content, AGEs and plasma amyloid groups were sig­
nificantly increased in the patient group compared to the control
group. Moreover, AS detected as protein thiols and FRAP were found
to be lower in the diabetic group [200].
8.3. Gestational diabetes mellitus
Gestational diabetes mellitus (GDM) has an increasing pre­
valence in pregnant women, which is characterized by abnormal
blood sugar levels, leading to fetal abnormalities and maternal
complications [231]. In addition to these, GDM may induce gesta­
tional hypertension, preeclampsia, fetal macrosomia, shoulder dys­
tocia and rate of cesarean [231,232]. Although the pathophysiology
of GDM has not been clearly understood, it has been indicated that
there is a link between oxidative stress status and GDM-related in­
sulin resistance among pregnant women, as in T1DM and T2DM
[233]. Therefore, there has been various studies that examine the
association between oxidative stress and AS in pregnant women
with GDM. For instance; Usluoğullari et al. [201] showed that par­
ticipants with GDM had significantly higher serum OGTT (oral glu­
cose tolerance test), OSI, TOS and insulin levels, whereas TAS and
irisin content, which has a role in the regulation of glucose and lipid
21
Journal of Pharmaceutical and Biomedical Analysis 209 (2022) 114477
levels, insulin sensitivity and low-grade inflammation, were found to
be significantly lower in the GDM patients in comparison with
control group [201]. Besides, Shang et al. [202] investigated the AOX
defense system and oxidative stress in pregnant woment with GDM
by measuring MDA, 8-isoprostane (8IsoP), XO, SOD and TAC levels in
maternal, cord and placenta. Results demonstrated that oxidative
stress status including MDA, XO and 8IsoP amounts were found to be
significantly increased, while AOX defense system in terms of SOD
and TAC levels were significantly decreased in subjects with GDM
compared to the control group [202].
9. Conclusions and Perspectives
Because of their instability, RS are often measured by indirect
methods. An excess of RS, which cannot be counter-balanced by AOX
compounds, causes damage to biomacromolecules such as DNA,
proteins and lipids in the body. One of the most common strategies
for detection and quantification of RS is to measure their damaging
effects on these biomacromolecules called oxidative stress (OS)
biomarkers, where antioxidant defenses can help protect these
biomarkers from oxidative damage. There is a clear (inverse) re­
lationship between increasing amount of RS and decreasing AOX
concentration. OS has been shown to be associated with the pro­
gression of various diseases including diabetes, neurodegenerative
disorders and cancer, and OS is best reflected in the observed oxi­
dative modifications on biomacromolecules, opening the way to
better diagnose these diseases before they are clinically evident. The
clinical use of in vitro tests on biomarker concentrations and TAC in
direct disease diagnosis is a controversial issue. However, it is gen­
erally an established fact that oxidative stress parameters rise and
AOX defense parameters decline in certain OS−originated diseases. A
particular concentration level of biomarkers can provide useful in­
formation about a person’s health status. Although it is difficult to
make generalizations on large groups, the sudden change in the
concentrations of these substances, especially for a given individual,
may provide important clues for diagnosis and treatment of OS-
originated diseases. On the other hand, both RS and TAC are im­
portant issues for a variety of scientific disciplines, including bio­
chemists, clinical chemists and food chemists. Although several
techniques like UV–vis spectrometry, fluorometry, HPLC and GC with
MS, electrochemical or diode array detectors, ESR/EPR, etc. have been
used to detect the specific biomarkers of the oxidative stress, there is
no single instrumental method to meet all requirements. The same is
true for the selection of biomarkers for specific purposes. In prin­
ciple, OS-biomarkers seem to concern mostly food chemistry and
biochemistry, but this concept can be used for almost all materials
prone to oxidative damage. Although kept beyond the scope of this
review essentially focused on in vitro tests measuring oxidative
stress and related antioxidant defense, in vivo OS-biomarkers may be
envisaged to gain more popularity in the future.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
The authors declare that they have no known competing fi­
nancial interests or personal relationships that could have appeared
to influence the work reported in this paper.
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