#### **2.7. Misfolding and protein oxidation by chronic oxidative stress**

pathways and inhibition of dephosphorylation pathways, which alter intracellular signaling [30, 31], and finally, the loss of regulation of inflammatory response [32], as is shown in **Figure 1**.

**Figure 1.** Effect of chronic oxidative stress on intracellular changes present during the development of Alzheimer's dis‐

The presence of an oxidative stress state has been associated with an aberrant reentry into the cell cycle, a phenomenon associated with the death of terminally differentiated neurons. This mechanism has been observed in samples from Alzheimer's disease and Down syndrome patients [33, 34]. It has been proven that the increase in the cyclin D2 levels due to the loss of FoxO 1a downregulation on this cyclin may take part of the aberrant reentry into cell cycle. In consequence, the mature neurons do not divide activating cell death mechanisms by apoptosis

The increase of free radicals acts upon a series of pathways that lead to transcription of antioxidant enzymes. Transcription factors within these pathways allow the activation of antioxidant response elements (AREs) in the nucleus [36]. The AREs cause the raise in messenger RNA transcription for the increase in antioxidant enzyme synthesis. For instance, FoxO 3a activation stimulates the signaling pathway for the increase in the synthesis of superoxide dismutase enzymes, particularly the manganese superoxide dismutase, MnSOD [35]. The signaling pathway regulated by the transcription factor Nrf2 is also activated by the presence of ROS. Under oxidative stress conditions, Nrf2 transactivates the synthesis of

ease.

84 Free Radicals and Diseases

**2.4. Cell cycle and oxidative stress**

where caspase‐3 might play an important role [35].

**2.5. Antioxidant enzyme regulation in an oxidative stress state**

Oxidation can affect protein structure because the endoplasmic reticulum responds to oxidative stress by affecting chaperons and producing protein misfolding altering their spatial structure. This causes negative effects on the functionality of the proteins and makes them susceptible to aggregation, inducing to cell toxicity [46].

The disassembly in the ubiquitin‐proteasome proteins is a protection mechanism to avoid the damages caused by oxidative stress [47]. A number of studies have reported that from the 26S proteasome complex, the 20S subunit is more resistant to the damage caused by ROS compared to the 19S subunit. It may bind and degrade misfolded oxidized proteins without the need of ubiquitination and ATP expenditure [48]. However, during a chronic oxidative stress state, proteasomal proteins are also modified by oxidation, thus altering their function and causing intracellular and extracellular protein accumulation [49].

#### **2.8. Alterations in endoplasmic reticulum function and signaling by chronic oxidative stress**

The endoplasmic reticulum is the organelle that within their functions include the calcium (Ca2+) storage and protein folding with the participation of different enzymes and chaper‐ ones [50]. The redox state inside the ER lumen is highly oxidant and the changes caused by the presence of ROS affect the correct protein folding. It contributes to the breaking of the disulphide bonds by the binding of reactive species to thiol groups [51]. Misfolded proteins that are formed in the ER cause Ca2+ release in the cytoplasmic space. When the mitochon‐ dria capture excessive amounts of calcium, it loses the regulation of its membrane, creating a mitochondrial transition pore. This results in ATP deficit and ROS increase which induce, in turn, ER Ca2+ release; thus, a vicious cycle in the intracellular regulation of Ca2+ levels is created [52]. Studies performed with cerebral samples from Alzheimer's patients have dem‐ onstrated that the redox modifications caused by oxidative stress inhibit the function of chaperones and the correct folding proteins, causing protein misfolding and ER stress [53]. In Alzheimer's pathology, β‐protein has been reported to induce stress in the ER and change the morphology of the ER and also of the mitochondria. This results in the loss of the mito‐ chondrial membrane potential and the consequent production of ROS [54].

#### **2.9. Mitochondrial damage by oxidative stress**

The mitochondria play a number of cell functions, such as ATP synthesis, calcium homeostasis, and processes of cell survival and death. Additionally, this organelle is the main source of endogenous ROS; therefore, it is constantly exposed to oxidative stress [55]. Several metabolic and mitochondrial abnormalities have been found in hippocampal neurons of patients with Alzheimer's. Mitochondria of AD patients show a reduction in size, DNA alterations, and mitochondrial proteins in vacuoles, suggesting mitochondrial degradation by autophagy [56]. Studies have also observed deficiencies in the production of antioxidant enzymes that play a protective role protecting the mitochondria from the damage caused by ROS; for example, there are the cytochrome oxidase complex IV, MnSOD, and uncoupling proteins [57].

#### **2.10. Loss of regulation of inflammatory response**

The immune defense of the central nervous system (CNS) is composed of a number of small cells known as microglia, astrocytes, and an effective blood‐brain barrier [58]. The presence of an oxidative stress state causes the activation of the microglia and the astrocytes. As a result, inflammatory and neurotoxic factors are released increasing the levels of oxidative stress and causing a chronic neuroinflammatory response. Depending on its duration, the response may cause damage in the brain tissue [59]. Neurodegenerative diseases are characterized by a chronic dysregulation of the inflammatory response that involves glial alteration. This produces alterations in the neuronal metabolism, neuronal survival, and repair that progress to neuronal death and memory disturbances [4].

#### **2.11. Oxidative stress state and dendritic spines**

The chronic oxidative stress state causes alterations in dendritic spines. These alterations consist of a reduction in the size of the spines as well as a decrease in their number. This phenomenon is present in neurodegenerative diseases and in chronic diseases as alcoholism [60, 61]. An explanation of this histologic alteration found in the hippocampus of the patients is the loss of the existing regulation in calcium metabolism during oxidative stress state. There is an increase of extracellular Ca2+ entry, which tries to be regulated by the cells; they send Ca2+ to the ER or the mitochondria to avoid the alteration Ca2+‐dependent cell functions [62]. In addition, some studies have proposed a neuronal defense mechanism in which the cell tries to decrease the membrane surface exposed to free radicals, with the final purpose to try to control the balance in the intraneuronal medium [63]. All these changes affect the number of synapses and the metabolic interaction between astrocytes and neurons.

#### **2.12. Oxidative stress state and neuron‐glia interaction**

disulphide bonds by the binding of reactive species to thiol groups [51]. Misfolded proteins that are formed in the ER cause Ca2+ release in the cytoplasmic space. When the mitochon‐ dria capture excessive amounts of calcium, it loses the regulation of its membrane, creating a mitochondrial transition pore. This results in ATP deficit and ROS increase which induce, in turn, ER Ca2+ release; thus, a vicious cycle in the intracellular regulation of Ca2+ levels is created [52]. Studies performed with cerebral samples from Alzheimer's patients have dem‐ onstrated that the redox modifications caused by oxidative stress inhibit the function of chaperones and the correct folding proteins, causing protein misfolding and ER stress [53]. In Alzheimer's pathology, β‐protein has been reported to induce stress in the ER and change the morphology of the ER and also of the mitochondria. This results in the loss of the mito‐

The mitochondria play a number of cell functions, such as ATP synthesis, calcium homeostasis, and processes of cell survival and death. Additionally, this organelle is the main source of endogenous ROS; therefore, it is constantly exposed to oxidative stress [55]. Several metabolic and mitochondrial abnormalities have been found in hippocampal neurons of patients with Alzheimer's. Mitochondria of AD patients show a reduction in size, DNA alterations, and mitochondrial proteins in vacuoles, suggesting mitochondrial degradation by autophagy [56]. Studies have also observed deficiencies in the production of antioxidant enzymes that play a protective role protecting the mitochondria from the damage caused by ROS; for example,

there are the cytochrome oxidase complex IV, MnSOD, and uncoupling proteins [57].

The immune defense of the central nervous system (CNS) is composed of a number of small cells known as microglia, astrocytes, and an effective blood‐brain barrier [58]. The presence of an oxidative stress state causes the activation of the microglia and the astrocytes. As a result, inflammatory and neurotoxic factors are released increasing the levels of oxidative stress and causing a chronic neuroinflammatory response. Depending on its duration, the response may cause damage in the brain tissue [59]. Neurodegenerative diseases are characterized by a chronic dysregulation of the inflammatory response that involves glial alteration. This produces alterations in the neuronal metabolism, neuronal survival, and repair that progress

The chronic oxidative stress state causes alterations in dendritic spines. These alterations consist of a reduction in the size of the spines as well as a decrease in their number. This phenomenon is present in neurodegenerative diseases and in chronic diseases as alcoholism [60, 61]. An explanation of this histologic alteration found in the hippocampus of the patients is the loss of the existing regulation in calcium metabolism during oxidative stress state. There is an increase of extracellular Ca2+ entry, which tries to be regulated by the cells; they send Ca2+ to the ER or the mitochondria to avoid the alteration Ca2+‐dependent cell functions [62].

chondrial membrane potential and the consequent production of ROS [54].

**2.9. Mitochondrial damage by oxidative stress**

86 Free Radicals and Diseases

**2.10. Loss of regulation of inflammatory response**

to neuronal death and memory disturbances [4].

**2.11. Oxidative stress state and dendritic spines**

The signaling and metabolic changes that occur in the presence of oxidative stress affect the interaction between neurons and astrocytes, and cause the activation of the microglia, inducing and maintaining the loss of regulation of the inflammatory response [64]. Among these alterations, we find the loss of regulation in neurotransmitter metabolism as glutamate, dopamine, as well as alterations in the metabolism of antioxidant systems like glutathione [65, 66]. On the other hand, the changes in the expression of inducible nitric oxide synthase and in the extracellular Ca2+ levels cause the release of proinflammatory cytokines as interleukin (IL)‐ 1, IL‐3, IL‐6, interferon (IFN)‐α, IFN‐β, and tumor necrosis factor (TNF)‐α by the astrocytes (**Figure 2**). This promotes the maintenance of the inflammatory response and the stimulation of the glia, creating a vicious cycle consisting of oxidative stress, cytokine release, inflammatory response, and increase of oxidative stress [67].

**Figure 2.** Microphotography that shows the chronic effect of oxidative stress in hippocampal astrocytes from rats ex‐ posed to low doses of ozone (40×). There are changes in the astrocytes in the samples from animals exposed to ozone.

#### **2.13. Oxidative stress state and disturbance of the blood‐brain barrier**

The blood‐brain barrier finely regulates the entry of substances into the brain. Among its functions is the capability of changing the affinity of the transporters according to the neces‐ sities of the nervous tissue [68]. This barrier is created by a close relationship between neurons, astrocytes, and endothelial cells; the microglia is involved as well [69]. The barrier creates a neurovascular unit that promotes neuronal homeostasis maintenance. Nevertheless, in neurodegenerative diseases and during inflammatory processes, this barrier is broken down and loses its selectivity [70]. We have proven that the blood‐brain barrier is broken and there are changes in the morphology of the end feet of astrocytes in animal models of neurodegen‐ eration caused by oxidative stress in the hippocampus of rats exposed to ozone [71]. Finally, this break is followed by endothelial cell edema, changes in the processes and in astrocytic feet, and an increase of the proinflammatory factors. It promotes the exacerbation of the neurodegenerative process [72]. Oligomer production of β‐amyloid increases oxidative damage of the blood‐brain barrier, attracting astrocytes, glia, and activated microglia [73].

## **3. Oxidative stress and Alzheimer's disease**

#### **3.1. APP processing and β‐amyloid formation**

The break of the amyloid precursor protein (APP) has two phases: a nonamyloidogenic pathway and an amyloidogenic pathway. The amino acid 83 is cleaved by the α‐secretase on the carboxyl‐terminal, producing a long amino N‐terminal ectodomain (sAPPα). The result of this process is the formation of C83, which is retained by the membrane to be cleaved by gamma secretase, creating short p3 fragments. The break down by α‐secretase occurs within the β‐amyloid region, preventing the formation of β‐amyloid [74].

On the other hand, the amyloidogenic pathway is an alternative APP breakdown which leads to the creation of β‐amyloids. This pathway is created by the β‐secretase cleaving in the amino acid 99, allowing sAPPβ release in the extracellular space [75]. In consequence, the breakdown of this fragment between the 38 and 43 residues by the γ‐secretase releases an intact Aβ peptide [76]. The complete β‐amyloid peptide is 40 residues long (Aβ40) and 10% is a 42‐residue variant (Aβ42). This variant is more hydrophobic and easily induces the formation of fibrils. It is also the largest form of this peptides prevailing the β‐amyloid plaques [77].

There is a variety of assembly forms in which the β‐amyloid peptide can be found. This peptide can carry out different physiological or pathological functions depending on the assembly pathway [78]. The β‐amyloid can be deposited in specific brain regions forming amyloid plaques [79, 80].

#### **3.2. Oxidative stress and alteration of the enzymes involved in the processing of APP**

Among the different hypotheses explaining the possible causes intervening in the develop‐ ment of the disease, the participation of oxidative stress is nowadays widely accepted. Results obtained in our laboratory show that oxidative stress in healthy animals is per se capable of producing hyperphosphorylated tau protein and the formation of isoforms of β‐amyloids 1‐ 42 in hippocampal neurons of rats chronically exposes to low doses of ozone [81] (**Figure 3**).

The formation of senile plaques is due to intracellular and extracellular accumulation of insoluble β‐amyloids in the brain. The β‐amyloid peptide is generated by APP cleavage where the enzymes α, β, and γ secretases are involved as previously indicated [82]. Both the enzymes of the amyloidogenic pathway as the enzymes of the nonamyloidogenic pathway are altered by a chronic oxidative stress state. Experiments performed in our laboratory demonstrate that chronic oxidative stress, caused by exposure to low doses of ozone, is capable of inhibiting the enzymes involved in the nonamyloidogenic pathway. It also increases the enzymes in the amyloidogenic pathway in the hippocampus of rats which were exposed to this gas for 4 hours during 60 and 90 days (**Figures 4** and **5**).

Oxidative Stress, Inflammation, and Formation of Beta‐Amyloid 1‐42 in Brain http://dx.doi.org/10.5772/64709 89

feet, and an increase of the proinflammatory factors. It promotes the exacerbation of the neurodegenerative process [72]. Oligomer production of β‐amyloid increases oxidative damage of the blood‐brain barrier, attracting astrocytes, glia, and activated microglia [73].

The break of the amyloid precursor protein (APP) has two phases: a nonamyloidogenic pathway and an amyloidogenic pathway. The amino acid 83 is cleaved by the α‐secretase on the carboxyl‐terminal, producing a long amino N‐terminal ectodomain (sAPPα). The result of this process is the formation of C83, which is retained by the membrane to be cleaved by gamma secretase, creating short p3 fragments. The break down by α‐secretase occurs within

On the other hand, the amyloidogenic pathway is an alternative APP breakdown which leads to the creation of β‐amyloids. This pathway is created by the β‐secretase cleaving in the amino acid 99, allowing sAPPβ release in the extracellular space [75]. In consequence, the breakdown of this fragment between the 38 and 43 residues by the γ‐secretase releases an intact Aβ peptide [76]. The complete β‐amyloid peptide is 40 residues long (Aβ40) and 10% is a 42‐residue variant (Aβ42). This variant is more hydrophobic and easily induces the formation of fibrils. It is also

There is a variety of assembly forms in which the β‐amyloid peptide can be found. This peptide can carry out different physiological or pathological functions depending on the assembly pathway [78]. The β‐amyloid can be deposited in specific brain regions forming amyloid

**3.2. Oxidative stress and alteration of the enzymes involved in the processing of APP**

Among the different hypotheses explaining the possible causes intervening in the develop‐ ment of the disease, the participation of oxidative stress is nowadays widely accepted. Results obtained in our laboratory show that oxidative stress in healthy animals is per se capable of producing hyperphosphorylated tau protein and the formation of isoforms of β‐amyloids 1‐ 42 in hippocampal neurons of rats chronically exposes to low doses of ozone [81] (**Figure 3**). The formation of senile plaques is due to intracellular and extracellular accumulation of insoluble β‐amyloids in the brain. The β‐amyloid peptide is generated by APP cleavage where the enzymes α, β, and γ secretases are involved as previously indicated [82]. Both the enzymes of the amyloidogenic pathway as the enzymes of the nonamyloidogenic pathway are altered by a chronic oxidative stress state. Experiments performed in our laboratory demonstrate that chronic oxidative stress, caused by exposure to low doses of ozone, is capable of inhibiting the enzymes involved in the nonamyloidogenic pathway. It also increases the enzymes in the amyloidogenic pathway in the hippocampus of rats which were exposed to this gas for 4 hours

**3. Oxidative stress and Alzheimer's disease**

the β‐amyloid region, preventing the formation of β‐amyloid [74].

the largest form of this peptides prevailing the β‐amyloid plaques [77].

**3.1. APP processing and β‐amyloid formation**

plaques [79, 80].

88 Free Radicals and Diseases

during 60 and 90 days (**Figures 4** and **5**).

**Figure 3.** Micrography that shows the chronic effect of oxidative stress on the formation of β‐amyloid 1‐42 in rat hippo‐ campus exposed to low doses of ozone for 90 days (100×). Note changes in intracellular accumulation of β‐amyloid 1‐ 42 in neurons of the dentate gyrus.

**Figure 4.** Micrography that shows the chronic effect of oxidative stress on the enzymes of the nonamyloidogenic path‐ way (A: ADAM 9; B: ADAM 10) in dentate gyrus of rat hippocampus exposed to low doses of ozone (40×). Note the decrease in immunoreactivity after 90 days of exposure to ozone compared to the control.

**Figure 5.** Micrography that shows the chronic effect of oxidative stress on the amyloidogenic pathway enzymes (A: presenilin 1; B: presenilin 2) in the dentate gyrus of rat hippocampus exposed to low doses of ozone (40×). Note the increase in immunoreactivity at 90 days for presenilin 1 and presenilin 2 to 60 days in the brains of animals exposed to ozone compared to the control.

The β‐amyloid accumulation in specific compartments of the neuron causes an energy deficit and alterations in protein folding [83, 84]. For its part, the intracellular increase of these oligomers causes the inhibition of the proteasome activity, lowering the possibility of proc‐ essing misfolded proteins and causing their accumulation buildup within the cell [85].

The β‐amyloid accumulation attracts immune system cells such as astrocytes, activated microglia, and macrophages, among others. Glial cells maintain the loss of regulation of the inflammatory response, the inflammation, and the oxidative stress state, creating a vicious cycle that is maintained in time [86].

When the levels of the intracellular β‐amyloid peptide decrease, the internalization of APP is induced. The internalization is mediated by the low density lipoprotein receptor‐related protein 1B (LRP1B), one of the members of the LDL family. This receptor usually binds APP to the plasmatic membrane to prevent the internalization of the β‐amyloid peptide, reducing its production [87]. The failure of these mechanisms by the effect of oxidative stress and the hyperphosphorylation of tau protein induced a disturbance in the formation of microtubules, producing neurofibrillary tangles [88]. Furthermore, tau protein associates with β‐amyloids that might be involved in the internalization of the extracellular protein into the neurons. This produces and externalizes the insoluble isoform of β‐amyloid [89]. During this phase, the synthesis of soluble β‐amyloid is decreased while the synthesis of misfolded insoluble β‐ amyloids is increased. It forms part of the insoluble β‐amyloid plaques [90].

Finally, our laboratory has developed a noninvasive model of oxidative stress using the exposure to low doses of ozone to that effect. Using this model, we have proven that oxidative stress per se is capable of producing progressive neurodegeneration in the hippocampus together with the formation of insoluble β‐amyloid 1‐42 and the accumulation of such peptides.

## **4. Conclusions**

The loss of redox homeostasis accelerates ageing and plays a fundamental role in the patho‐ geny and development of Alzheimer's disease. It alters the cell signaling mechanisms of an important number of metabolic pathways, promotes epigenetic alterations, and alters the posttranslational mechanisms. Finally, the chronic alteration of the redox balance induces the misfolding of proteins by oxidative stress in ER, cholesterol oxidation, and alterations in insulin receptors that lead to changes in neuronal metabolism and survival.

We can infer that Alzheimer's disease is the final manifestation of a series of oxidative altera‐ tions of the metabolism. There is a slow involvement of different biomolecules during the development of the disease. The loss of redox balance plays a crucial role in the formation of hyperphosphorylated tau protein, insoluble β‐amyloids, and the loss of regulation of the immune system.

## **Acknowledgements**

**Figure 5.** Micrography that shows the chronic effect of oxidative stress on the amyloidogenic pathway enzymes (A: presenilin 1; B: presenilin 2) in the dentate gyrus of rat hippocampus exposed to low doses of ozone (40×). Note the increase in immunoreactivity at 90 days for presenilin 1 and presenilin 2 to 60 days in the brains of animals exposed to

The β‐amyloid accumulation in specific compartments of the neuron causes an energy deficit and alterations in protein folding [83, 84]. For its part, the intracellular increase of these oligomers causes the inhibition of the proteasome activity, lowering the possibility of proc‐ essing misfolded proteins and causing their accumulation buildup within the cell [85].

The β‐amyloid accumulation attracts immune system cells such as astrocytes, activated microglia, and macrophages, among others. Glial cells maintain the loss of regulation of the inflammatory response, the inflammation, and the oxidative stress state, creating a vicious

When the levels of the intracellular β‐amyloid peptide decrease, the internalization of APP is induced. The internalization is mediated by the low density lipoprotein receptor‐related protein 1B (LRP1B), one of the members of the LDL family. This receptor usually binds APP to the plasmatic membrane to prevent the internalization of the β‐amyloid peptide, reducing its production [87]. The failure of these mechanisms by the effect of oxidative stress and the hyperphosphorylation of tau protein induced a disturbance in the formation of microtubules, producing neurofibrillary tangles [88]. Furthermore, tau protein associates with β‐amyloids that might be involved in the internalization of the extracellular protein into the neurons. This produces and externalizes the insoluble isoform of β‐amyloid [89]. During this phase, the synthesis of soluble β‐amyloid is decreased while the synthesis of misfolded insoluble β‐

Finally, our laboratory has developed a noninvasive model of oxidative stress using the exposure to low doses of ozone to that effect. Using this model, we have proven that oxidative

amyloids is increased. It forms part of the insoluble β‐amyloid plaques [90].

ozone compared to the control.

90 Free Radicals and Diseases

cycle that is maintained in time [86].

This work was supported by CONACYT under grant 219703 to S. R‐A.

## **Author details**

Selva Rivas‐Arancibia\* , Erika Rodriguez‐Martinez, Angélica Méndez‐García, Mariana Moctezuma‐Salgado, Paola Jiménez‐Espíndola and Ulises López‐Gonzales

\*Address all correspondence to<EMAIL_ADDRESS>
Physiology Department, Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico

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96 Free Radicals and Diseases

#### **Biomarkers in ROS and Role of Isoprostanes in Oxidative Stress Biomarkers in ROS and Role of Isoprostanes in Oxidative Stress**

Mini Chandra, Manikandan Panchatcharam and Sumitra Miriyala Mini Chandra, Manikandan Panchatcharam and Sumitra Miriyala

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64706

#### **Abstract**

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98 Free Radicals and Diseases

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Biomarkers of reactive oxygen species serve as indicators of oxidative stress in the pathology of cardiovascular diseases. This chapter presents an overview of the various biomarkers available to quantify oxidative stress to advance the understanding of the pathophysiology of cardiovascular diseases as well as to serve as an adjunct in their diagnosis and prognosis. The plasma levels of reactive oxygen species themselves are unstable and unreliable markers of oxidative stress. The commonly used stable biomarkers are derivatives of oxygen radicals such as products of lipid peroxidation and protein oxidation, with isoprostanes and malondialdehyde (MDA) being the most widely used biomarkers due to higher specificity and ease of measurement. Recently, micro‐RNA is emerging as stable and specific biomarkers for detection of heart failure. Other biomarkers have a role in certain conditions; for example, advanced oxidation protein products indicate acute inflammation, whereas advanced glycation end products serve as indicators of chronic disease.

**Keywords:** biomarkers, reactive oxygen species, isoprostanes, lipid peroxidation, car‐ diovascular diseases

## **1. Introduction**

Reactive oxygen species (ROS) are formed as by‐products of cellular activity or cellular metabolism or cellular respiration. They have useful function‐serving roles in cell signaling, cell differentiation, cell immunity, etc., when present in low concentrations, all of which are important in maintaining the body's physiological functions known as redox signaling [1].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Their concentration is controlled by the various antioxidants produced in the body such as superoxide dismutases, catalase, and glutathione peroxidase, with the goal to keep ROS concentration low [2]. Oxidative stress is a condition resulting from excessive reactive oxygen species due to either increased production or inadequacy of antioxidants to eliminate them. This increase in ROS results in damage to the cell which includes oxidizing lipids, nucleic acids, and proteins, thus leading to a change or loss in their function and ultimately causes cell death by apoptosis or necrosis. Due to this effect on the cells, oxidative stress has been implicated in aging [3] as well as many diseases including but not limited to cardiovascular disease [4], neurodegenerative diseases [5, 6], cancer [7], and diabetes [8].

Biomarkers are measurable characteristics of a biological condition; in this case, biomarkers of ROS serve as indicators of oxidative stress and how it influences a given disease. Hallmarks of a good biomarker are sensitivity, specificity, ease of obtaining and measuring samples, and cost‐effectiveness. The quantification of oxidative stress with biomarkers is important not only in understanding the pathophysiology of cardiovascular disease but also in the diagnosis, prognosis as well as in designing new therapeutic measures for individual intervention. Oxidative stress plays a major role in the pathology of cardiovascular disease. In the heart, oxidative stress results in the inhibition of Na+‐K‐ pump [9]. The mitochondrial electron transport chain and enzymes xanthine oxidase and nicotinamide‐adenine dinucleotide phosphate (NADPH) oxidase are the main producers of ROS. Risk factors and conditions which predispose to a cardiovascular event such as smoking, hypertension, atherosclerosis, hypercholesterolemia, diabetes, and obesity increase the effect of these enzymes which results in an increased production of ROS [10, 11].

## **2. ROS as biomarkers**

ROS themselves act as biomarkers, their plasma levels being indicators of ROS production. A method known as the reactive oxygen metabolites (ROM) kit is used to measure total oxidative stress and measures superoxides (02‐) and hydroxyl radical (HO) as well as hypochlorous acid (HOCl) and hydrogen peroxide (H2O2) [12] among others. This kit measures reactive oxidants in biological fluids and has been used to assess oxidative stress in animals such as in a study on ewes done by Rizzo et al. [13]. Cytochrome C reduction has been used in many studies to measure the production of superoxide (O2 ‐ ) in the atrium [14], mouse aorta [15], and vessels [16]. Chemiluminescent probes which release photon when in contact with ROS can be detected and used for various ROS measurements with the lucigenin‐enhanced chemilumi‐ nescence being the most commonly used to understand the way superoxide and diseases related to the cardiovascular system are affected in tissues [10]. Electron spin resonance detects free radical by the presence of its unpaired electron. Reactive radicals are detected by addition of probes [10]. As of 2003, the spin traps were not fit to be used in humans due to the potential for toxicity, but they can be used on tissues and body fluids; for example, PBN was used to show free radicals in coronary sinus blood during bypass surgery [17]. Aromatic traps for free radicals such as salicylate have been used in studies to detect superoxide in myocardial infarction [18]. High levels of dityrosine, an oxidation product of ROS, have been used to demonstrate the role of oxidative damage in atherosclerotic plaques [19]. Even though each reactive oxidant can be measured individually, they have drawbacks of being too costly and time‐consuming. Their property of being inherently unstable with short half‐lives of merely seconds, both of which combined with the antioxidants in the circulation, results in very low intracellular concentration of ROS thus making them unreliable markers of ROS.

The derivatives of oxygen radicals such as products of lipid peroxidation and protein oxida‐ tion on the other hand are stable and thus are more commonly used to measure the pres‐ ence of ROS. The serum derivatives are new biomarkers of ROS which are mainly indicators of hydroperoxide levels produced by lipid peroxidation and have been shown to be high in atrial fibrillation [20]. The diacron reactive oxygen metabolites (dROM) test is an inexpen‐ sive analysis which measures ROS in both serum and plasma.

## **3. Peroxidation of lipids biomarkers**

Lipids, especially polyunsaturated lipids, are more susceptible to oxidative damage due to the presence of many double bonds in their molecular structure [21], and thus, the indicators of lipid peroxidation are important indicators of free radicals. The presence of biomarkers in cardiovascular disease confirms the hypothesis that lipid peroxidation contributes to the development of cardiovascular diseases. There are many biomarkers of lipid peroxidation— MDA and isoprostanes being the most widely used. Others are lipid hydroperoxides, oxy‐ sterols, and oxidation resistance assays.

## **4. Isoprostanes**

Their concentration is controlled by the various antioxidants produced in the body such as superoxide dismutases, catalase, and glutathione peroxidase, with the goal to keep ROS concentration low [2]. Oxidative stress is a condition resulting from excessive reactive oxygen species due to either increased production or inadequacy of antioxidants to eliminate them. This increase in ROS results in damage to the cell which includes oxidizing lipids, nucleic acids, and proteins, thus leading to a change or loss in their function and ultimately causes cell death by apoptosis or necrosis. Due to this effect on the cells, oxidative stress has been implicated in aging [3] as well as many diseases including but not limited to cardiovascular

Biomarkers are measurable characteristics of a biological condition; in this case, biomarkers of ROS serve as indicators of oxidative stress and how it influences a given disease. Hallmarks of a good biomarker are sensitivity, specificity, ease of obtaining and measuring samples, and cost‐effectiveness. The quantification of oxidative stress with biomarkers is important not only in understanding the pathophysiology of cardiovascular disease but also in the diagnosis, prognosis as well as in designing new therapeutic measures for individual intervention. Oxidative stress plays a major role in the pathology of cardiovascular disease. In the heart, oxidative stress results in the inhibition of Na+‐K‐ pump [9]. The mitochondrial electron transport chain and enzymes xanthine oxidase and nicotinamide‐adenine dinucleotide phosphate (NADPH) oxidase are the main producers of ROS. Risk factors and conditions which predispose to a cardiovascular event such as smoking, hypertension, atherosclerosis, hypercholesterolemia, diabetes, and obesity increase the effect of these enzymes which results

ROS themselves act as biomarkers, their plasma levels being indicators of ROS production. A method known as the reactive oxygen metabolites (ROM) kit is used to measure total oxidative stress and measures superoxides (02‐) and hydroxyl radical (HO) as well as hypochlorous acid (HOCl) and hydrogen peroxide (H2O2) [12] among others. This kit measures reactive oxidants in biological fluids and has been used to assess oxidative stress in animals such as in a study on ewes done by Rizzo et al. [13]. Cytochrome C reduction has been used in many studies to

[16]. Chemiluminescent probes which release photon when in contact with ROS can be detected and used for various ROS measurements with the lucigenin‐enhanced chemilumi‐ nescence being the most commonly used to understand the way superoxide and diseases related to the cardiovascular system are affected in tissues [10]. Electron spin resonance detects free radical by the presence of its unpaired electron. Reactive radicals are detected by addition of probes [10]. As of 2003, the spin traps were not fit to be used in humans due to the potential for toxicity, but they can be used on tissues and body fluids; for example, PBN was used to show free radicals in coronary sinus blood during bypass surgery [17]. Aromatic traps for free radicals such as salicylate have been used in studies to detect superoxide in myocardial infarction [18]. High levels of dityrosine, an oxidation product of ROS, have been used to

) in the atrium [14], mouse aorta [15], and vessels

‐

disease [4], neurodegenerative diseases [5, 6], cancer [7], and diabetes [8].

in an increased production of ROS [10, 11].

measure the production of superoxide (O2

**2. ROS as biomarkers**

100 Free Radicals and Diseases

More accurate biomarkers of lipid peroxidation are isoprostanes along with its metabolites as stated in a study done by the National Institute of Health (NIH) [22]. In 1990, Roberts and Morrow discovered F2‐isoprostane formed by the peroxidation of arachidonic acid [23] which is polyunsaturated fatty acid found in the cell membrane phospholipids and is one of the many targets of ROS. They are specific indicators of lipid peroxidation both *in vitro* and *in vivo* [24] and are stable compounds which are formed in large quantities *in vivo* following oxidative damage such as with CCl4 which is a producer of free radicals [25] as well as having detectable amounts present even in non‐injured tissue making them reliable as a biomarker of ROS [26]. Isoprostanes are excellent biomarkers and have numerous advantages over other biomarkers of oxidative stress; they are chemically stable markers and are formed *in vivo.* They are specific to lipid peroxidation and are not affected by the dietary lipid content [27]. Since they are detectable in biological fluids, they have the significant advantage over other oxidative stress markers due to the ease of measuring them since they can be measured by noninvasive methods extracellularly in the urine, plasma, and tissue [27, 28]. High levels of F2‐isoprostanes are found in many human diseases such as coronary heart disease [29], obesity, cancer, and even genetic disorders [30]. They are shown to be increased in risk factors such as smoking, obesity, hypercholesterolemia, and myocardial ischemia reperfusion [31–33] as well as in atherosclerosis [34]. They are also observed in bypass [35] and angioplasty. The extent of lipid peroxidation can be measured by calculating the level of F2‐IsoPs which is esterified in phospholipids due to them being initially formed esterified and subsequently gain their free form [27]. In humans, the two major metabolites of isoprostane which are detectable in the urine are 2,3‐dinor‐15‐F2t‐IsoP and 2,3 dinor‐5,6‐dihydro‐15‐F2t‐IsoP [36]. Elevated levels of 8‐isoprostane in the plasma and urine are also observed in cardiovascular disease [37]. There are many other derivatives identified [38]. The method for detecting isoprostanes is gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐mass spectrometry (LC‐MS), but drawbacks in using mass spectrometry is the expense and time required for measurement [22]. ELISA‐based isoprostane detection is currently most reliable indicator of lipid oxidation [39]. There are commercially available kits to measure them using the sandwich ELISA method where the isoprostanes in biological samples compete with isoprostane conjugated with an enzyme to bind to an isoprostane‐specific antibody present in the micro‐ plate. The activity of the enzyme results in increased intensity of color development with increased amount of conjugated isoprostane bound. Isoprostanes are biomarkers of choice, but since the results obtained by these two methods do not corroborate, the result of the immune assays is yet to be given clinical validation [40]. There have been studies showing the variation in isoprostane levels according to the time of day, and thus, this needs to be taken into account when further research is conducted [41, 42].

## **5. MDA**

Malondialdehyde (MDA) is a ketoaldehyde which is produced as an end product of polyun‐ saturated fatty acid and is found in increased concentration in tissue injury. It forms a red pigmentation when it reacts with thiobarbituric acid. This thiobarbituric acid‐reactive sub‐ stance (TBARS) assay can be used to measure lipid peroxidation using spectrophotometry. In relation to cardiovascular disease, elevated levels of MDA are associated with smoking [43]. They are seen to be elevated with the progression of atherosclerosis [44] and are predictors of future cardiovascular events in patients with coronary artery disease [45]. Limitations of this method include low specificity since TBARS includes many other products of lipid peroxida‐ tion other than MDA [46], tendency for inaccurate results due to the varying results generated with different assay conditions used as well as the production of artifacts due to the fact that the MDA measured is mostly generated *in vitro* [47] with most of the MDA formed due to the high temperature used in the procedure [48]. Now, high‐pressure liquid chromatography (HPLC) is preferred over TBARS due to higher specificity [49]. Commercially available ELISA kits are also available for MDA detection which show better specificity [50].

## **6. Isolevuglandins**

Isolevuglandins (IsoLGs) are also produced due to oxidation of arachidonic acid, but unlike isoprostanes, they are highly reactive and react with primary amines for example phosphati‐ dylethanolamine to form lactam and hydroxylactam. The unreacted isolevuglandins are not detected in the tissues or cells. They have been implicated in many disease processes such as atherosclerosis and neurodegenerative diseases [51]. Though methods such as mass spec‐ trometry and immunohistochemical studies have shown increased levels of IsoLG, there is still not enough evidence connecting them with severity of a disease or of their use in predicting the onset of disease. Further studies need to be conducted to determine the utility of IsoLG as clinical biomarkers.

## **7. Oxidation of proteins biomarkers**

#### **7.1. Myeloperoxidase (MPO)**

obesity, hypercholesterolemia, and myocardial ischemia reperfusion [31–33] as well as in atherosclerosis [34]. They are also observed in bypass [35] and angioplasty. The extent of lipid peroxidation can be measured by calculating the level of F2‐IsoPs which is esterified in phospholipids due to them being initially formed esterified and subsequently gain their free form [27]. In humans, the two major metabolites of isoprostane which are detectable in the urine are 2,3‐dinor‐15‐F2t‐IsoP and 2,3 dinor‐5,6‐dihydro‐15‐F2t‐IsoP [36]. Elevated levels of 8‐isoprostane in the plasma and urine are also observed in cardiovascular disease [37]. There are many other derivatives identified [38]. The method for detecting isoprostanes is gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐mass spectrometry (LC‐MS), but drawbacks in using mass spectrometry is the expense and time required for measurement [22]. ELISA‐based isoprostane detection is currently most reliable indicator of lipid oxidation [39]. There are commercially available kits to measure them using the sandwich ELISA method where the isoprostanes in biological samples compete with isoprostane conjugated with an enzyme to bind to an isoprostane‐specific antibody present in the micro‐ plate. The activity of the enzyme results in increased intensity of color development with increased amount of conjugated isoprostane bound. Isoprostanes are biomarkers of choice, but since the results obtained by these two methods do not corroborate, the result of the immune assays is yet to be given clinical validation [40]. There have been studies showing the variation in isoprostane levels according to the time of day, and thus, this needs to be taken into account

Malondialdehyde (MDA) is a ketoaldehyde which is produced as an end product of polyun‐ saturated fatty acid and is found in increased concentration in tissue injury. It forms a red pigmentation when it reacts with thiobarbituric acid. This thiobarbituric acid‐reactive sub‐ stance (TBARS) assay can be used to measure lipid peroxidation using spectrophotometry. In relation to cardiovascular disease, elevated levels of MDA are associated with smoking [43]. They are seen to be elevated with the progression of atherosclerosis [44] and are predictors of future cardiovascular events in patients with coronary artery disease [45]. Limitations of this method include low specificity since TBARS includes many other products of lipid peroxida‐ tion other than MDA [46], tendency for inaccurate results due to the varying results generated with different assay conditions used as well as the production of artifacts due to the fact that the MDA measured is mostly generated *in vitro* [47] with most of the MDA formed due to the high temperature used in the procedure [48]. Now, high‐pressure liquid chromatography (HPLC) is preferred over TBARS due to higher specificity [49]. Commercially available ELISA

Isolevuglandins (IsoLGs) are also produced due to oxidation of arachidonic acid, but unlike isoprostanes, they are highly reactive and react with primary amines for example phosphati‐

kits are also available for MDA detection which show better specificity [50].

when further research is conducted [41, 42].

**5. MDA**

102 Free Radicals and Diseases

**6. Isolevuglandins**

MPO is an enzyme found in inflammatory cells such as macrophages and neutrophils. It generates ROS by the conversion of hydrogen peroxide to hydroxy radical (OH), nitric oxide (ONOO‐NO2), and hypochlorous acid (HOCl) and is a proinflammatory agent responsible for the oxidation of low‐density lipoprotein (LDL) [52]. It is found in abundance in the athero‐ sclerosis plaques [53] and coronary artery disease where it can serve as an inflammatory marker for both the risk of CAD and its existence [54]. MPO concentration is measured in biological samples by ELISA which is commercially available. Its function is measured by spectrophotometry by peroxidase activity assays such as measuring the formation of guaiacol oxidation products [55]. Its levels in the serum can predict risk for acute coronary syndromes [56], for risk of cardiovascular event in patients with chest pain [57] and increased risk of coronary artery disease in seemingly healthy population [58]. It is prone to varying and unreliable results due to the fact that the values are altered during the process of collection and handling as seen in a study done in 2008 by Shih et al. where it was determined that the concentration of MPO varied depending on the collection tube used and the presence of heparin in the patient serum [59]. A concern in the measurement of MPO is the artificial release of MPO from the neutrophils leading to false results showing an increase in MPO. In their study, Shih et al. used nine different types of tubes containing EDTA, citrate plasma, and heparin samples and serum samples. The level of MPO varied in all these tubes, with EDTA and citrate samples showing the lowest concentration and heparin and serum samples showing 10 and 100% higher values, respectively. This suggests that the serum levels of MPO are higher due to their release from leukocytes during coagulation. It has previously been shown by Li et al. that heparin leads to release of MPO from neutrophils during neutrophil activation [60].

### **8. Growth differentiation factor‐15**

It is a cytokine expressed in many cells including cardiomyocytes [61]. It increases in many cardiovascular diseases such as atherosclerosis [62] and heart failure [63]. It has been studied with respect to the progress and outcome of disease since it has a protective role in the heart [64] such as against ischemia reperfusion injury [61] and acute myocardial infarction [65] making it a useful biomarker in clinical settings though more research still needs to be con‐ ducted.

## **9. Oxidized low‐density lipoprotein (OxLDL)**

The use of OxLDL as a biomarker of oxidative stress in cardiovascular diseases has been re‐ ported due to its ability to promote lipid deposition. The oxidation of LDL is linked to the pathology of atherosclerosis by immunohistochemical staining apolipoprotein B‐100 [66]. It is thought to be formed by activated platelets [67]. High‐density lipoproteins (HDL) lead to decreased activation of platelets since it competes with them to bind to oxidized LDL pro‐ tecting against the development of atherosclerosis [68]. Circulating OxLDL is already pro‐ ven to be able to predict the presence of atherosclerosis [69] and coronary artery disease [70]. OxLDL is detected by immune assays in plasma. According to Trpkovic et al., there are cur‐ rently three ELISA assays namely 4E6, E06, and DLH3 developed to detect OxLDL in the blood [71]. Out of these, 4E6 binds to LDL but also detects native LDL and the other two, DLH3 which measures LDL and E06 are used for oxidized phosphatidylcholine [72]. A drawback of E06 method is its non‐specificity to oxidized lipids. In 2001, Holvoet et al. measured circulating LDL levels by ELISA using monoclonal antibody 4E6 which detected higher number of circulating OxLDL in patients with coronary artery disease [73].

#### **10. Allantoin**

Over the years, allantoin has emerged as a reliable biomarker of oxidative damage both *in vivo* and *in vitro.* It is formed by the ROS‐induced oxidation of uric acid [74], where uric acid is converted to allantoin due to overproduction of ROS [75]. In relation to cardiovascular diseases, increased levels of allantoin in the plasma have been shown in people with type 2 diabetes [75] as well as in heavy smokers [76] both of which are risk factors for developing cardiovascular disease. It has been shown to be increased in the plasma in oxidative stress‐ related chronic heart failure [77]. The use of allantoin as a widely applied biomarker is limit‐ ed due to the difficulty of measuring allantoin in the body fluids [78]. The most specific and sensitive method for its measurement is liquid chromatography [79].

## **11. Protein carbonyls**

Oxidation of protein amino acid residues leads to the formation of protein carbonyls by dif‐ ferent means such as deamination of glutamic acid and lysine or due to the resulting break‐ age of protein backbone [80]. They are stable compounds formed early and are usually higher in concentration due to their multiple sources, making them good biomarkers due to the ease of detection as well as no need of expensive equipment. It is a commonly used pro‐ tein oxidation marker, and there are various assays for its detection. They have been shown to increase with age implicating them in the process of aging [81]. They have even been re‐ ported in the human heart following coronary surgery [82]. Assay has been done to observe them in dilated cardiomyopathy [83]. In 1990, Levine et al. were the first ones to determine various methods to measure carbonyls in oxidized protein [84].

A highly sensitive assay is protein carbonyl content (PCC) which has various modifications but in all of them 2,4‐dinitrophenylhydrazine (DNPH) reacts with the protein carbonyls and forms its 2,4‐dinitrophenyl (DNP) hydrazone which is stable and can then be optically measured by immunohistochemistry or by radioactive counting [85]. Spectrophotometric assay can be employed due the ability of this hydrazone product to absorb ultraviolet light which when coupled with high‐performance liquid chromatography, in short HPLC, makes the measurement more specific and sensitive [86]. One sensitive method is to detect carbonyls by first labeling them with tritiated sodium borohydride then separating with SDS‐PAGE [87] or by reducing with tritiated sodium borohydride in solution [88]. An important limitation of carbonyl measurement is that there are different protocols used by researchers leading to variable levels of carbonyls in tissues.

## **12. Advanced oxidation protein products (AOPPS)**

[64] such as against ischemia reperfusion injury [61] and acute myocardial infarction [65] making it a useful biomarker in clinical settings though more research still needs to be con‐

The use of OxLDL as a biomarker of oxidative stress in cardiovascular diseases has been re‐ ported due to its ability to promote lipid deposition. The oxidation of LDL is linked to the pathology of atherosclerosis by immunohistochemical staining apolipoprotein B‐100 [66]. It is thought to be formed by activated platelets [67]. High‐density lipoproteins (HDL) lead to decreased activation of platelets since it competes with them to bind to oxidized LDL pro‐ tecting against the development of atherosclerosis [68]. Circulating OxLDL is already pro‐ ven to be able to predict the presence of atherosclerosis [69] and coronary artery disease [70]. OxLDL is detected by immune assays in plasma. According to Trpkovic et al., there are cur‐ rently three ELISA assays namely 4E6, E06, and DLH3 developed to detect OxLDL in the blood [71]. Out of these, 4E6 binds to LDL but also detects native LDL and the other two, DLH3 which measures LDL and E06 are used for oxidized phosphatidylcholine [72]. A drawback of E06 method is its non‐specificity to oxidized lipids. In 2001, Holvoet et al. measured circulating LDL levels by ELISA using monoclonal antibody 4E6 which detected

higher number of circulating OxLDL in patients with coronary artery disease [73].

sensitive method for its measurement is liquid chromatography [79].

Over the years, allantoin has emerged as a reliable biomarker of oxidative damage both *in vivo* and *in vitro.* It is formed by the ROS‐induced oxidation of uric acid [74], where uric acid is converted to allantoin due to overproduction of ROS [75]. In relation to cardiovascular diseases, increased levels of allantoin in the plasma have been shown in people with type 2 diabetes [75] as well as in heavy smokers [76] both of which are risk factors for developing cardiovascular disease. It has been shown to be increased in the plasma in oxidative stress‐ related chronic heart failure [77]. The use of allantoin as a widely applied biomarker is limit‐ ed due to the difficulty of measuring allantoin in the body fluids [78]. The most specific and

Oxidation of protein amino acid residues leads to the formation of protein carbonyls by dif‐ ferent means such as deamination of glutamic acid and lysine or due to the resulting break‐ age of protein backbone [80]. They are stable compounds formed early and are usually higher in concentration due to their multiple sources, making them good biomarkers due to the ease of detection as well as no need of expensive equipment. It is a commonly used pro‐

**9. Oxidized low‐density lipoprotein (OxLDL)**

ducted.

104 Free Radicals and Diseases

**10. Allantoin**

**11. Protein carbonyls**

Advanced oxidation protein products (AOPPs) are the end products of free radical affected proteins. They have been shown to be linked to many human diseases such as diabetes mel‐ litus [89], coronary artery disease [90], and chronic renal disease [91] among others, and since they have been shown to produce oxidative stress in inflammatory conditions [92], they serve to indicate acute inflammation.

## **13. Advanced glycation end products (AGES)**

They are molecules which are formed as a result of the reaction between reducing sugars and amino groups. Their concentration tends to increase in conditions of oxidative stress. The two main advanced glycation end products are pentosidine and carboxymethyl valine which result from a process known as glycoxidation where the amino acids lysine and argi‐ nine react with carbohydrates as well as the oxidizing effect of ROS on polyunsaturated fatty acids. A precursor of carboxymethyl valine known as glyoxal is formed when RNase incu‐ bates with arachidonate [93]. The presence of AGES has been shown in diseases such as dia‐ betes mellitus and obesity among others [94]. They also have a role in diabetic heart failure as shown by Brouwers et al., where they overexpressed glyoxalase‐I, a glycation precursor detoxifying enzyme in order to reduce AGES, and found that it leads to prevention of diabe‐ tes‐induced oxidative damage in the heart [95]. They are detected after derivatization with 2, 4‐dinitrophenylhydrazine (DNP). The hydrazone formed is then detected using a spectro‐ photometer or by using anti‐DNP antibodies with along with ELISA [96] or by high‐per‐ formance liquid chromatography (HPLC) or by Western blot or immunohistocytochemistry [97]. Out of these methods, HPLC is more specific and can measure carboxymethyl lysine CML [98] and pentosidine [99]. They mainly serve as indicators of chronic diseases [100].

#### **14. Glutathione and glutathione disulfide**

Reduced glutathione (GSH) is present in large quantities in the cells and acts as an inhibitor of lipid peroxidase. Glutathione disulfide (GSSG) is the oxidized form of glutathione. Pro‐ tein glutathionylation regulates cardiovascular function [101]. Its values have been shown to increase in ischemia reperfusion injury [102], atherosclerosis [103], and cardiac hypertrophy [104]. In patients with atherosclerosis obliterans, increased glutathionylation is shown to be related to the progression of the disease proving to be a biomarker at early stage [105]. The ratio of GSH and GSSG is used as an indicator of ROS due to the fact that there occurs a decrease in GSH and increase in GSSG concentration in oxidative stress [106]. There are a number of methods to detect protein s‐glutathionylation. Quantifying the total amount of s‐ glutathionylated proteins is by measuring fluorescence [107]. Labeling glutathione is a method for glutathionylation analysis such as 35s radiolabeling [108], though it is not very sensitive and can only be used in cell culture; furthermore, it cannot detect proteins which have already undergone glutathionylation. Biotinylated glutathione either reduced or oxi‐ dized is superior to the 35s labeling methods, it detects only glutathionylated proteins thus is specific plus it can be analyzed by multiple methods such as fluorescence microscopy [109] or immunoblotting using biotin antibodies [110]. One drawback of this method is that the presence of biotin tag on glutathione may have an effect on the protein function. Anti‐ glutathione antibodies allow the detection of glutathionylated proteins in physiological con‐ ditions. Studies done with antibodies are by using mouse monoclonal antibodies [111–113]. Drawback of antibody method is the lack of specificity, and it can only detect a few proteins in total extract [109] limiting its utility in detecting glutathionylated proteins on a large scale. Recently, the use of liquid chromatography‐couple mass spectrometry using whole proteins is found to be a good method to identify proteins in larger numbers [114].

Recently, the role of micro‐RNA (miRNA) in the generation of ROS and its consequences such as inflammation, angiogenesis, cell proliferation, and apoptosis has been a subject of research. They are found intracellularly and outside cells in body fluids [115]. They are stable and specific such as miR‐499 miRNA for the heart. Another advantage of miRNA as a biomarker is that they are not affected by posttranslational modifications. They can also be easily assessed by methods like polymerase chain reaction (PCR) and microarrays. PCR is an expensive method which detects small quantities of miRNA, but the results are affected by the primer used. Microarray measurements require the development of probes and can thus be useful in that many RNAs can be detected at the same time [116]. Other less used methods are direct sequencing by next‐generation sequencing [117], which eliminates the influence of primers as in the case of PCR but is still not used widely because of expense. Stem loop probe ligation [118] and Northern blotting are other methods which may be used to measure concentration of miRNA. The miRNA found most abundantly in the heart is mR‐1 which is heart specific and can be used as a sensitive and specific marker for diagnosis of acute myocardial infarction [119, 120].

Elevated miRNAs specifically miR423‐5p has also been observed in heart failure patients making them important clinical biomarkers in the diagnosis of heart failure [121]. Although miRNA measurement has shown promise, there are still various issues that need to be addressed. The concentration of miRNA in body fluids is low making its isolation rather difficult. The values obtained also tend to be different in different body fluids which need to be normalized. Therefore, it is necessary to develop a method to obtain accurate results with miRNA measurement across the various samples [116]. The product of DNA damage, 8 dydroxy‐2'‐deoxyguanosine urinary levels, seems to be elevated in dilated cardiomyopathy [122]. It is also evidently a predictor of future events following myocardial infarction [123]. Other specific biomarkers have also been studied but are not yet studied as extensively as the above biomarkers. Ascorbic acid is an endogenous antioxidant which has been linked to unstable coronary syndrome where it is thought to have an effect on the lesion [124]. Gluta‐ thione peroxidase‐I is evidently decreased in coronary artery disease patients [125] which is an antioxidant enzyme. Low levels of bilirubin have been linked to cigarette smoking and increased levels of triglycerides and cholesterol making it a potential biomarker of cardiovas‐ cular disease [126]. Oxidative bilirubin metabolites called biopyrrins are elevated in the urine in patients with heart failure [127] and are thought to be predictors of future cardiac events in acute myocardial infarction [128].

Several biomarkers of oxidative stress have been studied over the years in an effort to under‐ stand the mechanism of cardiovascular generation with the intention to use the information by targeting oxidative stress with cardio‐protective drugs. Further research into understand‐ ing the mechanism of ROS generation and their role in therapeutic intervention will be beneficial for the management of cardiovascular diseases.

## **Author details**

[97]. Out of these methods, HPLC is more specific and can measure carboxymethyl lysine CML [98] and pentosidine [99]. They mainly serve as indicators of chronic diseases [100].

Reduced glutathione (GSH) is present in large quantities in the cells and acts as an inhibitor of lipid peroxidase. Glutathione disulfide (GSSG) is the oxidized form of glutathione. Pro‐ tein glutathionylation regulates cardiovascular function [101]. Its values have been shown to increase in ischemia reperfusion injury [102], atherosclerosis [103], and cardiac hypertrophy [104]. In patients with atherosclerosis obliterans, increased glutathionylation is shown to be related to the progression of the disease proving to be a biomarker at early stage [105]. The ratio of GSH and GSSG is used as an indicator of ROS due to the fact that there occurs a decrease in GSH and increase in GSSG concentration in oxidative stress [106]. There are a number of methods to detect protein s‐glutathionylation. Quantifying the total amount of s‐ glutathionylated proteins is by measuring fluorescence [107]. Labeling glutathione is a method for glutathionylation analysis such as 35s radiolabeling [108], though it is not very sensitive and can only be used in cell culture; furthermore, it cannot detect proteins which have already undergone glutathionylation. Biotinylated glutathione either reduced or oxi‐ dized is superior to the 35s labeling methods, it detects only glutathionylated proteins thus is specific plus it can be analyzed by multiple methods such as fluorescence microscopy [109] or immunoblotting using biotin antibodies [110]. One drawback of this method is that the presence of biotin tag on glutathione may have an effect on the protein function. Anti‐ glutathione antibodies allow the detection of glutathionylated proteins in physiological con‐ ditions. Studies done with antibodies are by using mouse monoclonal antibodies [111–113]. Drawback of antibody method is the lack of specificity, and it can only detect a few proteins in total extract [109] limiting its utility in detecting glutathionylated proteins on a large scale. Recently, the use of liquid chromatography‐couple mass spectrometry using whole

proteins is found to be a good method to identify proteins in larger numbers [114].

Recently, the role of micro‐RNA (miRNA) in the generation of ROS and its consequences such as inflammation, angiogenesis, cell proliferation, and apoptosis has been a subject of research. They are found intracellularly and outside cells in body fluids [115]. They are stable and specific such as miR‐499 miRNA for the heart. Another advantage of miRNA as a biomarker is that they are not affected by posttranslational modifications. They can also be easily assessed by methods like polymerase chain reaction (PCR) and microarrays. PCR is an expensive method which detects small quantities of miRNA, but the results are affected by the primer used. Microarray measurements require the development of probes and can thus be useful in that many RNAs can be detected at the same time [116]. Other less used methods are direct sequencing by next‐generation sequencing [117], which eliminates the influence of primers as in the case of PCR but is still not used widely because of expense. Stem loop probe ligation [118] and Northern blotting are other methods which may be used to measure concentration of miRNA. The miRNA found most abundantly in the heart is mR‐1 which is heart specific

**14. Glutathione and glutathione disulfide**

106 Free Radicals and Diseases

Mini Chandra, Manikandan Panchatcharam and Sumitra Miriyala\*

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Cellular Biology and Anatomy, Louisiana Health Sciences‐Shreveport, Shreveport, LA, USA

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#### **Redoxomics and Oxidative Stress: From the Basic Research to the Clinical Practice Redoxomics and Oxidative Stress: From the Basic Research to the Clinical Practice**

Simona Tafuri, Natascia Cocchia, Francesco Landolfi, Eugenio Luigi Iorio and Francesca Ciani Simona Tafuri, Natascia Cocchia, Francesco Landolfi, Eugenio Luigi Iorio and Francesca Ciani

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64577

#### **Abstract**

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Potentially oxidant chemical species, which include not only free radicals but also other oxidizing chemical species such as reactive oxygen species (ROS), for example, hydroxyl radical and hydrogen peroxide, and nitrogen reactive species (RNS), for example, nitric oxide, play a relevant role in all biological processes and especially in cell defenses and molecular signaling. Their action is finely modulated by the antioxidant network that is composed either by endogenous or exogenous compounds (e.g., enzymes, peptides, lipids, and vitamins). An impaired modulation of oxidant species can lead to the socalled oxidative stress that is now considered an emerging health risk factor in almost all living organisms including plants, animals, and humans. Indeed, oxidative stress is related to a reduced lifespan and many diseases (e.g., cardiovascular diseases, neurodegenerative disorders, and metabolic diseases) both in humans and in animals. Unfortunately, oxidative stress does not show any clinical picture, but it can be detected only by means of specific laboratory tests. The recent recognition of a specific "redox code" and the definition of a redoxomics as a new "omics" are now enlarging the horizon of the traditional oxidative stress field leading to the definition of the so-called electrophilic stress. The aim of this chapter is to review the basic principles of redox reaction starting from the concept of free radicals and antioxidant in order to define the "electrophilic stress" as an emerging health risk factor for early aging and almost 1000 illness from infectious diseases to cancer. A paragraph is dedicated to the tests to measure oxidative stress in clinical practice either in humans or in animals in order to prevent, to treat and to monitor electrophilic-related diseases.

**Keywords:** ROS, RNS, antioxidants, redoxomics

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Reactive species, free radicals, and oxidative processes**

Free radicals play a fundamentalrole in the metabolic activity and function of different organs. Interactions between prooxidants (free radicals) and antioxidants lead to the maintenance of the intracellular homeostasis. A state of oxidative stress begins when there is an imbalance between the prooxidants and antioxidants, in favor of free radicals. Oxidative stress is a health risk factor involved in aging and in several diseases, in humans and/or in animals. In normal conditions, paired electrons create stable bonds in biomolecules; a free radical is defined as any independent species that contains one or more unpaired electrons in external orbital. Free radicals have a greater or lesser reactivity for the spontaneous tendency to exist as molecules with all electrons arranged in couples; this state is equivalent to the chemical stability.

The radicals do not show the same reactivity. Their increase of charge and the volumetric ratio is directly proportional to their reactivity. They will only reach their stability stripping electrons to other chemical species with which they are in contact and oxidize them [1].

Free radicals are classified according to the nature of atom that owns the orbital with unpaired electron. Reactive species include either radical or nonradical chemical species with oxidant potential. There are, therefore, free radicals centered on oxygen, carbon, nitrogen, or chlorine, and so on. Free radicals and other reactive species act as signaling molecules. Reactive species modulate transcription and epigenetics.

Free radicals/reactive species can be produced either by a "nonenzymatic" or an "enzymatic" way (**Figure 1**).

**Figure 1.** The "origin" of free radicals and other reactive species in living organisms.

The cell is the first target of oxidative damage. The destructive action of free radicals on cells is addressed mainly through the following reactions: membrane lipid peroxidation, oxidative modification of proteins and amino acids, nucleic acids' damage, and sugar oxidation (**Figure 2**).

**1. Reactive species, free radicals, and oxidative processes**

Free radicals play a fundamentalrole in the metabolic activity and function of different organs. Interactions between prooxidants (free radicals) and antioxidants lead to the maintenance of the intracellular homeostasis. A state of oxidative stress begins when there is an imbalance between the prooxidants and antioxidants, in favor of free radicals. Oxidative stress is a health risk factor involved in aging and in several diseases, in humans and/or in animals. In normal conditions, paired electrons create stable bonds in biomolecules; a free radical is defined as any independent species that contains one or more unpaired electrons in external orbital. Free radicals have a greater or lesser reactivity for the spontaneous tendency to exist as molecules with all electrons arranged in couples; this state is equivalent to the chemical stability.

The radicals do not show the same reactivity. Their increase of charge and the volumetric ratio is directly proportional to their reactivity. They will only reach their stability stripping electrons

Free radicals are classified according to the nature of atom that owns the orbital with unpaired electron. Reactive species include either radical or nonradical chemical species with oxidant potential. There are, therefore, free radicals centered on oxygen, carbon, nitrogen, or chlorine, and so on. Free radicals and other reactive species act as signaling molecules. Reactive species

Free radicals/reactive species can be produced either by a "nonenzymatic" or an "enzymatic"

The cell is the first target of oxidative damage. The destructive action of free radicals on cells is addressed mainly through the following reactions: membrane lipid peroxidation, oxidative modification of proteins and amino acids, nucleic acids' damage, and sugar oxi-

to other chemical species with which they are in contact and oxidize them [1].

**Figure 1.** The "origin" of free radicals and other reactive species in living organisms.

modulate transcription and epigenetics.

way (**Figure 1**).

118 Free Radicals and Diseases

dation (**Figure 2**).

**Figure 2.** The cell: the first target of oxidative damage. Reactive species can hit not only lipids but also proteins and nucleic acids.

The damage, initially cellular, if prolonged through time, spreads to the tissues, organs and then it becomes a systemic damage. Oxidative stress is responsible, then, of cardiovascular diseases, dementia, Parkinson, early senescence, inflammation, cancers, and other diseases (**Figure 3**).

**Figure 3.** Consequences of the imbalance between pro-oxidants and antioxidants.

## **2. The antioxidants and biochemical classification**

Antioxidants are substances that, when they are present at a low concentration compared to those of an oxidizable substrate, retard or prevent the oxidation of the same substrate. The keyword "oxidized substrate" includes every kind of molecule that is located *in vivo*.

In nature, there are no universal best antioxidants, but there are different antioxidants that are required to protect several molecules *in vivo* [2].

Antioxidants can be classified into enzymatic and nonenzymatic. The enzymatic antioxidants include glutathione reductase (GSH), superoxide dismutase (SOD), and catalase (CAT). Among the nonenzymatic antioxidants are vitamins (C, E, and B), carotenoids, carnitine, cysteine, some metals, taurine, and albumin [3]. Reductase and peroxidase glutathione are the main reducing endogenous agents and act as scavenger antioxidants especially in the epididymis and testes [4].

SOD is an enzyme that catalyzes the dismutation reactions of the superoxide anion (O2 − ). It can be found in intra- and extracellular forms. The intracellular forms are copper-zinc SOD present in the cytoplasm and contain copper and zinc in the active site (Cu, ZnSOD, SOD1); and manganese SOD localized mainly in the mitochondrial matrix and contains manganese in the active site (MnSOD, SOD2). Instead, SOD extracellular form (EC-SOD, SOD3), working into the extracellular space, is correlated to the polysaccharides of surface or in a free form [5].

CAT catalyzes the conversion of H2O2 to O2 and H2O and presents a heme system with a central iron atom. It acts mainly in endoplasmic reticulum, peroxisomes, mitochondria, and the cytosol of many cell types [6].

Glutathione peroxidase (GPX) catalyzes the reduction of H2O2 and organic peroxides [5].GPX contains selenium in the form of selenocysteine in its active site. It is located in the sperm in the mitochondrial matrix.

The nonenzymatic exogen antioxidants are vitamins. Vitamin E encompasses a group of potent, lipid-soluble, chain-breaking antioxidants. Structural analyses have revealed that molecules having vitamin E antioxidant activity include four tocopherols and four tocotrienols. Vitamin E (α-tocopherol) neutralizes H2O2 and quenches free radicals, therefore, stopping chain reactions that develop lipid peroxides and protecting the membrane from the oxidative damage.

Vitamin C (L-ascorbic acid or ascorbate), a pivotal nutrient for organisms, is present in the extracellular fluid. It is a principal chain-breaking antioxidant neutralizing superoxide, hydroxyl, and hydrogen peroxide radicals. Also, it has an important action to recycle vitamin E [7].

A class of natural pigments, carotenoids, is synthesized from plants and microorganisms, but not animals. They, present as microcomponents in fruits and vegetables, are responsible for their colors (yellow, orange, and red). Carotenoids are held liable for the beneficial effects of fruits and vegetables to prevent illnesses such as cardiovascular disease, cancer, and different chronic disease [7].

Cysteines, intracellular GSH precursors, enhancement the quantity of GSH synthesized, which avoids oxidative damage to the cell membrane and DNA.

In addition, albumin, taurine/hypotaurine, inositol and any metal are other minor antioxidants which help to reduce oxidative stress.

One of the plasma proteins, the albumin, reacts with peroxyl radicals and prevents the chain reactions that produce ROS (Reactive Oxygen Species) formation.

Taurine, nonenzymatic antioxidant, scavenges ROS, inositol enhances GSH activity.

Selenium is an important component in the regular development and maturation of cells and contributes to the protection of DNA and cell membranes, particularly when used as an adjunct to vitamin E.

**Figure 4.** The antioxidant pyramid. Vertical stratification (power hierarchy).

In nature, there are no universal best antioxidants, but there are different antioxidants that are

Antioxidants can be classified into enzymatic and nonenzymatic. The enzymatic antioxidants include glutathione reductase (GSH), superoxide dismutase (SOD), and catalase (CAT). Among the nonenzymatic antioxidants are vitamins (C, E, and B), carotenoids, carnitine, cysteine, some metals, taurine, and albumin [3]. Reductase and peroxidase glutathione are the main reducing endogenous agents and act as scavenger antioxidants especially in the epidi-

> − ). It can

SOD is an enzyme that catalyzes the dismutation reactions of the superoxide anion (O2

be found in intra- and extracellular forms. The intracellular forms are copper-zinc SOD present in the cytoplasm and contain copper and zinc in the active site (Cu, ZnSOD, SOD1); and manganese SOD localized mainly in the mitochondrial matrix and contains manganese in the active site (MnSOD, SOD2). Instead, SOD extracellular form (EC-SOD, SOD3), working into the extracellular space, is correlated to the polysaccharides of surface or in a free form [5].

CAT catalyzes the conversion of H2O2 to O2 and H2O and presents a heme system with a central iron atom. It acts mainly in endoplasmic reticulum, peroxisomes, mitochondria, and the

Glutathione peroxidase (GPX) catalyzes the reduction of H2O2 and organic peroxides [5].GPX contains selenium in the form of selenocysteine in its active site. It is located in the sperm in

The nonenzymatic exogen antioxidants are vitamins. Vitamin E encompasses a group of potent, lipid-soluble, chain-breaking antioxidants. Structural analyses have revealed that molecules having vitamin E antioxidant activity include four tocopherols and four tocotrienols. Vitamin E (α-tocopherol) neutralizes H2O2 and quenches free radicals, therefore, stopping chain reactions that develop lipid peroxides and protecting the membrane from the oxidative

Vitamin C (L-ascorbic acid or ascorbate), a pivotal nutrient for organisms, is present in the extracellular fluid. It is a principal chain-breaking antioxidant neutralizing superoxide, hydroxyl, and hydrogen peroxide radicals. Also, it has an important action to recycle vitamin

A class of natural pigments, carotenoids, is synthesized from plants and microorganisms, but not animals. They, present as microcomponents in fruits and vegetables, are responsible for their colors (yellow, orange, and red). Carotenoids are held liable for the beneficial effects of fruits and vegetables to prevent illnesses such as cardiovascular disease, cancer, and different

Cysteines, intracellular GSH precursors, enhancement the quantity of GSH synthesized, which

In addition, albumin, taurine/hypotaurine, inositol and any metal are other minor antioxidants

avoids oxidative damage to the cell membrane and DNA.

which help to reduce oxidative stress.

required to protect several molecules *in vivo* [2].

dymis and testes [4].

120 Free Radicals and Diseases

cytosol of many cell types [6].

the mitochondrial matrix.

damage.

E [7].

chronic disease [7].

Zinc acts as a chelator and binds ROS [8]. Chrome, another essential micronutrient, is a component of enzymes involved in carbohydrate metabolism. Its supplementation reduces fat deposition in rats, preventing obesity, initial phase of inflammation and oxidative stress [9].

**Figure 4** shows the power hierarchy of antioxidant pyramid. Endogenous enzyme antioxidants have a higher antioxidant capacity and are located at the top of the pyramid.

**Figure 5.** Antioxidant defense mechanisms.

The mechanisms of antioxidant action are shown in **Figure 5**. According to their function, they can be classified into preventive, scavenger, and repair antioxidants. Preventive antioxidants stop radical generation; scavenger antioxidants stop initiation and propagation; and repair and *de novo* antioxidants repair oxidated damaged molecules.

Polyphenols are abundant micronutrients in our diet, and there is evidence for their role in the prevention of degenerative diseases. Their bioavailability differs greatly among the polyphenol groups, depending on their composition, dietary sources, forms, and their containing so that the most abundant polyphenols in our diet are not necessarily those leading to the highest concentrations of active metabolites in target tissues. The plasma concentrations of total metabolites range from 0 to 4 mol/L with an intake of 50-mg aglycone equivalents. Among the polyphenols, isoflavones and gallic acid are the ones that are absorbed most by humans following, with different kinetics, flavonoids, catechins, and quercetin glycosides. Less absorbed polyphenols are proanthocyanidins, catechins galloylated tea, and anthocyanins. The data for other polyphenols remain poorly understood. Other studies would be necessary for the investigations on the health effects of polyphenols (**Figure 6**) [10].

**Figure 6.** Antioxidant: polyphenol classes.

## **3. Oxidative stress: from the biochemistry to the clinics**

Oxidative stress is a particular kind of chemical stress, which is induced—locally and/or systemically—by an excess of potentially oxidant reactive species, mostly centered on the oxygen (reactive oxygen species, ROS). It can be due to an increased production of reactive species and/or to a reduced efficiency of antioxidant defense system. The effects of oxidative stress can range from the impairment of cell signaling to the apoptosis or necrosis.

The systematic assessment in biological samples of primary oxidizing chemical species, such as free radicals and their derivatives, such as hydroperoxides, as well as the dosage of antioxidant compounds and/or antioxidant activities (selenium and/or glutathione peroxidase), are not a "ring terminal" in the diagnostic chain of the information flows in biological systems (DNA > RNA > protein > metabolites > oxidants), but should make a "central place" compared to genomics, transcriptomics, proteomics, and metabolomics [11]. Precisely for this reason it has been newly introduced the new concept of "redoxomics" [12], a word previously used to detect only a few oxidized byproducts in the area of proteomics [13].

Redoxomics is a new field of "applied biochemistry" and "molecular diagnostic" with the following objectives: to examine the structure, the physiological role, and the deploying of oxidant and antioxidant systems into a living organism; to identify the mutual interactions of oxidant and antioxidant systems in a biological system (e.g., cell, tissue, organ, apparatus, and whole organism) in a defined phase of its development, under basic conditions as well as after stimulation potentially stressful; to assess the implications of these results from the point of view of epidemiology, pathophysiology, clinic, pharmacology, and so on [14].

The aspiring objective of redoxomics (as well as for another "-omics" in other areas) is "mapping" dynamically—through all the analytical and sophisticated techniques, from electron spin resonance to imaging—the whole oxidative-antioxidant repertoire [15].

This "integrated" approach allows us to track any qualitative/quantitative changes of oxidative balance and can support clinicians to give an optimum and "customized" solution for fixing any anomalies of redox status related to human diseases, in particular, in the area of aesthetic and antiaging medicine [16].

## **4. The breakdown of oxidant/antioxidant balance**

The mechanisms of antioxidant action are shown in **Figure 5**. According to their function, they can be classified into preventive, scavenger, and repair antioxidants. Preventive antioxidants stop radical generation; scavenger antioxidants stop initiation and propagation; and repair

Polyphenols are abundant micronutrients in our diet, and there is evidence for their role in the prevention of degenerative diseases. Their bioavailability differs greatly among the polyphenol groups, depending on their composition, dietary sources, forms, and their containing so that the most abundant polyphenols in our diet are not necessarily those leading to the highest concentrations of active metabolites in target tissues. The plasma concentrations of total metabolites range from 0 to 4 mol/L with an intake of 50-mg aglycone equivalents. Among the polyphenols, isoflavones and gallic acid are the ones that are absorbed most by humans following, with different kinetics, flavonoids, catechins, and quercetin glycosides. Less absorbed polyphenols are proanthocyanidins, catechins galloylated tea, and anthocyanins. The data for other polyphenols remain poorly understood. Other studies would be necessary

and *de novo* antioxidants repair oxidated damaged molecules.

122 Free Radicals and Diseases

for the investigations on the health effects of polyphenols (**Figure 6**) [10].

**3. Oxidative stress: from the biochemistry to the clinics**

Oxidative stress is a particular kind of chemical stress, which is induced—locally and/or systemically—by an excess of potentially oxidant reactive species, mostly centered on the oxygen (reactive oxygen species, ROS). It can be due to an increased production of reactive species and/or to a reduced efficiency of antioxidant defense system. The effects of oxidative

stress can range from the impairment of cell signaling to the apoptosis or necrosis.

**Figure 6.** Antioxidant: polyphenol classes.

The biological concept of stress: the word "stress", as it is currently in use, was first coined by Hans Selye (1907–1982) in 1936, he defined "stress" as "the nonspecific response of the body to any demand for change."

Selye had observed in many experiments that laboratory animals underwent sharp stimuli but different physical and emotional harmful (e.g., high beam, loud noise, extreme heat or cold, and constant frustration) all shown the identical pathologic changes of gastric ulcers narrowing of the lymphoid tissue and widening of the adrenal glands. Following they showed that persistent stress could be the cause of the development of different diseases in these animals similar to those observed in humans, like heart attacks, strokes, kidney diseases, and rheumatoid arthritis. The Selye's concept and dynamics of stress overlaps with that of oxidative stress (**Figure 7**) [17]. Most of oxidative stress-related diseases are related to life style [13].

**Figure 7.** Classical stress and oxidative stress: two overlapping concepts.

Oxidative distress from oxidative eustress can happen for several reasons: formation of reactive species from stimulated polymorphonuclear leukocytes that can hit not only bacteria but also tissues; an overload of oxygen (e.g., by strong aerobic exercise) into the mitochondria and consequent increase ROS production; detoxification from acetaminophen (paracetamol) into the microsomes may increase ROS liver production; reperfusion after ischemia may lead to reactive oxygen species production due to xanthine oxidase activation.

In any case, an amount of oxidants is synthesized and by the old Greek language, *oxys* means *acids*, it is possible to establish a new paradigma: oxidative stress + acidosis = electrophilic stress.

## **5. Measurement of oxidative stress**

The focus is now put on oxidative stress biomarkers that are objectively measured and assessed as markers of normal biological processes, pathogenic processes, or pharmacological responses to therapy performed. A biomarker, to be used as a predictor of illness, must first be endorsed. The validation criteria are intrinsic qualities like the specificity, sensitivity, the degree of interand intraindividual variability, and understanding of factors that can change.

In particular, features of the sample and analytical procedures are significant, therefore, noninvasiveness of the sampling, biomarker stability, sensitivity, specificity, velocity and simplicity of the analytical method are important. Below, the most commonly used biomarkers for the evaluation of oxidative/nitrosative damage are listed.

Oxidative stress depends on an imbalance that is created between the production of ROS (prooxidants) and the action of the antioxidants. Direct assays, which measure the oxidation of the cell membrane of many cell types, are available.

The most widespread assay assesses the concentration of malondialdehyde (MDA), one of the end products of lipid peroxidation [18, 19]. The increased levels of MDA may be related, for example, to the decrease of sperm parameters. To quantify the damage of sperm DNA, another assay is also used; it measures the concentration of a specific product of oxidative DNA damage, 8-oxo-7,8, dihydro 2′deoxyguanosine (8-OHdG). This product is particularly employed as a specific marker of oxidative damage to sperm DNA [20].

The assay of the indirect chemiluminescence is one of the most popular methods for the determination of ROS in the spermatic semen. Luminol (5-amino-2,3, dihydro 1,4, phthalazinedione) and lucigen are substances used to determine the redox activity in the cells [20]. Lucigen quantizes only extracellular superoxide radicals, while the luminol is able to determine the extracellular and intracellular levels of ROS.

In order to use the nitrobluetetrazolium assay an optical microscope is needed: it allows us to determine the differentiation of ROS in different cell types. This reagent, nitrobluetetrazolium, reacts with superoxide radicals, for example, in spermatozoa and in leukocytes, changing to diformazan, a blue pigment. The concentration of diformazan is directly proportional to the intracellular concentration of ROS [21].

**Figure 7.** Classical stress and oxidative stress: two overlapping concepts.

**5. Measurement of oxidative stress**

stress.

124 Free Radicals and Diseases

reactive oxygen species production due to xanthine oxidase activation.

Oxidative distress from oxidative eustress can happen for several reasons: formation of reactive species from stimulated polymorphonuclear leukocytes that can hit not only bacteria but also tissues; an overload of oxygen (e.g., by strong aerobic exercise) into the mitochondria and consequent increase ROS production; detoxification from acetaminophen (paracetamol) into the microsomes may increase ROS liver production; reperfusion after ischemia may lead to

In any case, an amount of oxidants is synthesized and by the old Greek language, *oxys* means *acids*, it is possible to establish a new paradigma: oxidative stress + acidosis = electrophilic

The focus is now put on oxidative stress biomarkers that are objectively measured and assessed as markers of normal biological processes, pathogenic processes, or pharmacological responses to therapy performed. A biomarker, to be used as a predictor of illness, must first be endorsed. The validation criteria are intrinsic qualities like the specificity, sensitivity, the degree of inter-

In particular, features of the sample and analytical procedures are significant, therefore, noninvasiveness of the sampling, biomarker stability, sensitivity, specificity, velocity and simplicity of the analytical method are important. Below, the most commonly used biomarkers

Oxidative stress depends on an imbalance that is created between the production of ROS (prooxidants) and the action of the antioxidants. Direct assays, which measure the oxidation of the

and intraindividual variability, and understanding of factors that can change.

for the evaluation of oxidative/nitrosative damage are listed.

cell membrane of many cell types, are available.

To assess serum total oxidant and antioxidant levels, commercially available d-ROMs and anti-ROM (Reactive Oxygen Metabolites) tests (Diacron International, Grosseto, Italy) are utilized. These tests are performed using Free Carpe Diem, a dedicated spectrophotometer (Diacron International, Grosseto, Italy).

Oxidative status is evaluated by measuring hydroperoxides in the serum using d-ROMs' test. The d-ROMs test measures the oxidant ability of a serum sample toward a particular substance (modified aromatic amine) used as an indicator (chromogen, N,N-diethyl-paraphenylendiamine) (DEPPD). The phenomenon is associated with the progressive and gradual color change to pink reaction mixture (serum + chromogen), which was initially colorless. In the d-ROMs test, the metabolites of reactive oxygen species (ROMs), particularly hydroperoxides (ROOH), of a biological sample, in the presence of iron, issued by the serum proteins by an acid buffer, can produce alkoxyl and peroxyl radicals, in accordance with Fenton's reaction.

Such radicals are able to oxidize an alkyl-substituted aromatic amine that is solubilized in a chromogenic mixture, thus producing a pink-colored derivative which is photometrically quantified at 505 nm [22]. The intensity of developed color is directly proportional to the concentration of ROMs, according to the Lambert–Beer's law and is expressed as Carratelli units (1 CARR U = 0.08 mg hydrogen peroxide/dl). The method is linear up to 1000 CARR U.

The measurement of antioxidant capacity in serum samples can be performed by the anti-ROM test. The anti-ROM test measures the antioxidant capacity of serum in terms of iron-reducing capacity; in fact, it is based on the ability of serum antioxidants to reduce ferric iron to ferrous iron, which reacting with αα-dipyridyl, gives rise to a reddish purple. The intensity of color increases in proportion to the amount of iron reduced by antioxidants present in the sample.

In the BAP (Biological Antioxidant Potential) test, adding a sample of plasma to a dye solution, obtained as a mixture of ferric chloride with a derived thiocyanate solution, a discoloration is caused. The intensity of this discoloration is determined photometrically by using a wavelength of 505 nm and it results proportional to the ability of plasma to reduce ferric ions [23]. The results obtained are evaluated as μmol/L or reduced ferric ions.

The antioxidant levels can be evaluated in the semen too, both with a chemiluminescence and through a colorimetric assay. The antioxidant amounts are determined by the addition of a well-known concentration of ROS to semen, developing the chemiluminescent signal or the color changes. The antioxidants present in the sperm behave toward ROS as a scavenger, so it is possible to measure the residual levels of ROS. Then, the intensity of the developed signal is inversely proportional to the total antioxidant activity of the sample [24].

The total oxidant capacity/potential of a blood plasma/serum sample can be evaluated by exploiting the ability of N,N-diethyl-paraphenylendiamine to give electrons (oxidation) after the reaction with a biological sample. The newly generated radical cation can be detected thus providing a suitable measure of oxidant capacity—either by evaluating the absorbance change at 505 nm (the solution becomes pink to red, depending on the concentration) or the specific spin resonance signal [22].

## **6. The oxidative stress evaluation in clinical practice**

Oxidative stress does not show any clinical picture; thus, the study of basic mechanisms of oxidative stress can lead to the identification of suitable biomarkers. Searching for the "ideal" biomarker of oxidative stress is not an easy matter; it should be validated by means of the golden standard technique (e.g., electron spin resonance); acceptably high levels of sensitivity, specificity, and precision; chemically stable over the time; able to measure suitably oxidative stress level; able to provide reliable information even in an early stage of the disease; able to anticipate the progression of disease during a systemic monitoring; modifiable with adequate sensitivity after medical/surgery/antioxidant treatments; minimally invasive, highly compliant, fast; and optimal cost/benefit ratio. Unfortunately, an accomplished biomarker of oxidative stress with all these features is not yet available.

The blood plasma/serum of apparently healthy people (and that one of many animal species) is able to oxidize the DEPPD (as described above) in a precise range of absorbance change as a function of either genetic or environment factors (age, gender, race, physiological conditions like pregnancy, and so on) and according to a Gauss-like curve of distribution (**Figure 8**) [25].

Redoxomics and Oxidative Stress: From the Basic Research to the Clinical Practice http://dx.doi.org/10.5772/64577 127

**Figure 8.** Causes, mechanisms, and clinical pictures of oxidative stress.

In the BAP (Biological Antioxidant Potential) test, adding a sample of plasma to a dye solution, obtained as a mixture of ferric chloride with a derived thiocyanate solution, a discoloration is caused. The intensity of this discoloration is determined photometrically by using a wavelength of 505 nm and it results proportional to the ability of plasma to reduce ferric ions [23].

The antioxidant levels can be evaluated in the semen too, both with a chemiluminescence and through a colorimetric assay. The antioxidant amounts are determined by the addition of a well-known concentration of ROS to semen, developing the chemiluminescent signal or the color changes. The antioxidants present in the sperm behave toward ROS as a scavenger, so it is possible to measure the residual levels of ROS. Then, the intensity of the developed

The total oxidant capacity/potential of a blood plasma/serum sample can be evaluated by exploiting the ability of N,N-diethyl-paraphenylendiamine to give electrons (oxidation) after the reaction with a biological sample. The newly generated radical cation can be detected thus providing a suitable measure of oxidant capacity—either by evaluating the absorbance change at 505 nm (the solution becomes pink to red, depending on the concentration) or the

Oxidative stress does not show any clinical picture; thus, the study of basic mechanisms of oxidative stress can lead to the identification of suitable biomarkers. Searching for the "ideal" biomarker of oxidative stress is not an easy matter; it should be validated by means of the golden standard technique (e.g., electron spin resonance); acceptably high levels of sensitivity, specificity, and precision; chemically stable over the time; able to measure suitably oxidative stress level; able to provide reliable information even in an early stage of the disease; able to anticipate the progression of disease during a systemic monitoring; modifiable with adequate sensitivity after medical/surgery/antioxidant treatments; minimally invasive, highly compliant, fast; and optimal cost/benefit ratio. Unfortunately, an accomplished bio-

The blood plasma/serum of apparently healthy people (and that one of many animal species) is able to oxidize the DEPPD (as described above) in a precise range of absorbance change as a function of either genetic or environment factors (age, gender, race, physiological conditions like pregnancy, and so on) and according to a Gauss-like curve of distribution

signal is inversely proportional to the total antioxidant activity of the sample [24].

The results obtained are evaluated as μmol/L or reduced ferric ions.

**6. The oxidative stress evaluation in clinical practice**

marker of oxidative stress with all these features is not yet available.

specific spin resonance signal [22].

126 Free Radicals and Diseases

(**Figure 8**) [25].

The blood plasma/serum of apparently healthy people, which are exposed to factors that are classically able to induce a condition of oxidative stress, shows a total oxidant capacity constantly and significantly higher than that found in apparently healthy nonexposed peoples, and patients suffering from diseases classically related to oxidative stress show significantly higher levels of blood plasma/serum total oxidant capacity compared to those found in apparently healthy controls (**Figure 9**).

**Figure 9.** Oxidative stress-related diseases. Pathophysiological and clinical pictures.

Some studies have shown that between the work done on standing and chronic venous insufficiency of the lower limbs, there is a statistically significant correlation. This correlation has been associated with an oxidative stress in an advanced stage that, according to several studies, would represent a risk factor for cardiovascular systemic disorders; in fact, standing workers exhibit significantly higher mean levels of ROS after work [26].

It is known that elevated levels of blood reactive oxygen species are correlated with the severity of periodontitis. An improvement of clinical parameters in chronic periodontitis patients, through a nonsurgical periodontal treatment, is able to determine a decrease in blood reactive oxygen species [27].