Biomarkers of Oxidative Stress and its Role in Atherosclerosis Development

Oxidative stress is known by excess production of reactive species, especially (ROS) and deleterious modifications in biomolecules such as proteins, and lipid mainly. It is a common mediator in pathogenesis of multiple diseases. Because of its potential to act as center in the cause of many diseases particularly cardiovascular disease, identifying biomarkers of oxidative stress has become the highly interesting and focus of many research. Measurement of reactive species (ROS) in the circulation of complex biological systems remains a challenge due to the short half-life of these reactive species and the need of special equipment to detect it. A widely used and reliable approach is the measurement of stable by-products modified under conditions of oxidative stress that have entered the circulation. The stable oxidation product that used as biomarker of oxidative stress include lipid oxidation product such as Isoprostanes (isops), MDA, and protein oxidation product such as nitrotyrosine ,Protein Glutathionilation and oxd LDL are the major one. The oxidative stress play pivotal role in the pathogenesis of the major cardiovascular risk factors termed as atherosclerosis. This is caused by excess production of reactive species (ROS) in the vessel mainly by enzymes such as NAD (P)H oxidase, xanthine oxidase, myeloperoxidase and uncoupled nitrc oxide synthase which oxidize lipoprotein (LDL). The oxidize LDL (oxLDL) lipoprotein progress the atherosclerosis process from the initially to the end stage development. Deoxyribonucleic acid: Enzyme-linked Immunosorbance Assays; eNOS: Endothelial Nitric Oxide Synthase; Chromatography; ICAM-1: Intercellular Adhesion Liquid Chromatography; Low-Density Lipoproteins; MCP-1: Monocyte Chemotactic Protein-1; M-CSF: Macrophage Colony-Stimulating Factor; MDA: Malondialdehyde; MS: Mass Spectrometry; nNOS: Neuronal Nitric Oxide Synthase cells; OxLDL: Oxidized Low Density Lipoprotein; Phox: Phagocyte Oxidase; PUFAs: Polyunsaturated Fatty acids; RNS: Reactive Nitrogen Species; ROS: Reactive Oxygen Species; SMC: Smooth Muscle Cells; SRs: Scavenger Receptors; TBA: Thiobarbitoric acid; VCAM-1: Cell Adhesion Molecule-1; XOR: Xanthine Oxidoreductase; XO: Xanthine Oxidase

and causing deleterious effects in several organs [1,2]. Oxidants removed through antioxidant defense mechanism under normal physiological condition. The incomplete removals of oxidants by antioxidants lead to its accumulation and generate oxidative stress condition. This is the result of biochemical processes leading to the production of excess reactive species and limited capabilities of endogenous antioxidant systems. Oxidizing agents (reactive species) can be produced by both endogenous source such as inflammatory cells, fibroblast, epithelial cells, endothelial cells, respiratory chain, xanthine and NADPH oxidase and exogenous source such as cigarette smoke, unhealthy diet, exogenous toxins, pollution, radiation [3,4]. Research has revealed a widespread involvement of oxidative stress in a number of disease processes such as cancer, Cardiovascular Disease (CVD), atherosclerosis, diabetes, arthritis, neurodegenerative disorders, and pulmonary, renal, and hepatic diseases during the past decade, [5][6][7][8][9]. Therefore, identifying biomarkers of oxidative stress are becoming important area of research in the recent years.
Biomarker is characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Biomarkers of oxidative stress are also important to assess the biological redox status, disease state and progression, and the health enhancing effects of antioxidants in humans. Measurement of reactive species such as ROS, RNS in the circulation of complex biological systems as biomarker of oxidative stress remains a challenge due to the short half-life of these reactive species and the need of special equipment to detect.
A popular approach for assessing biomarker of oxidative stress is the measurement of stable by-products modified under conditions of oxidative stress by excess reactive species and that have entered the circulation. These can be classified by molecules that are modified by interactions with ROS in the microenvironment.
Molecules that can be modified by excessive ROS include DNA, lipids (including phospholipids), proteins and carbohydrates. Some of these modifications have a direct effect on the function of target molecules, such as the inhibition of an enzymatic function, but other modifications just reflect the local degree of oxidative stress [6,7].

Lipid Peroxidation Product
Lipid peroxidation is reaction of oxygen with unsaturated lipids and produces a wide variety of oxidation products. A main lipid peroxidation product includes isoprostanes and aldehydes like Malondialdehyde (MDA). Determination of these lipid peroxidation products used as oxidative stress biomarker

Isoprostanes
The Isoprostanes (IsoPs) are bioactive prostaglandin-like compounds formed via a nonenzymatic mechanism through the free radical-initiated peroxidation of arachidonic acid [3,4]. Sources of free radicals for IsoPs formation include mitochondrial electron transport chain, P450 enzymes, lipoxygenase, and transition-metal catalyzed formation of free radicals. The F2-IsoPs are the most chemically stable and reliable marker for assessing oxidative stress status from the IsoPs family. It is called F2-IsoPs because they contain F-type prostane rings. Initially formed in lipid membranes as a consequence of oxidative stress and then released in free form by phospholipase action into circulation [10,11]. Elevated F2-IsoPs are found in a wide range of human clinical conditions. Elevated concentrations of F2-IsoPs are found in CVD, correlate with extent of disease, and predict the outcome [12,13]. IsoPs measured using most reliable methods such as combined Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography-Mass Spectrometry (LC-MS) in plasma and urine samples. Because of these methods are labor-intensive, require specialized and expensive instrumentation, it becomes challenge to widespread adaptation of F2-IsoPs in clinical trials [14].

Malondialdehyde
Malondialdehyde (MDA) is one of the aldehydes products of enzyme and free radical which can be formed as secondary lipid peroxidation products during lipid peroxidation. MDA is generated by decomposition of arachidonic acid and larger Polyunsaturated Fatty Acids (PUFAs). It is chemically more stable and membranepermeable than ROS and less toxic than other secondary lipid peroxidation products [15]. MDA is the most frequently used oxidative stress biomarker in many health problems such as cancer, psychiatry, chronic obstructive pulmonary disease, asthma, or cardiovascular diseases. Thiobarbitoric Acid (TBA) assay is the most commonly used method for determination of the MDA in biological fluids. Free and total MDA also determined using several technologies such as Gas Chromatography (GC), Mass Spectrometry (MS), Liquid Chromatography (LC) [16,17].

Protein Oxidation Products
Proteins represent a wide target for ROS and RNS generated under normal or oxidative stress conditions and can be considered as general scavengers of these species. Several amino acidic residues can undergo oxidative modification the major one include oxidation of sulphur-containing residues, nitration of tyrosine residues, and glutathionylation of cysteine residues [18].
Nitrotyrosine: Nitrotyrosine (Tyr-NO 2 ) is a stable oxidative stress biomarker in inflammatory diseases. It occurs by several pathways but all involves RNS and a two-step tyrosine nitration process. One widely studied pathway for nitration is mediated by reactive nitrogen species such as peroxynitrite (ONOO) and nitrogen dioxide (NO 2 ). Thus, Tyr-NO 2 was initially believed to be a specific marker of peroxynitrite-mediated damage [19]. The twostep process are a) Tyrosine is oxidized resulting in a tyrosine radical and b) A radical-radical reaction occurs between the tyrosine radical and nitrogen dioxide (NO 2 ).
Finally, Tyr-NO 2 , result from the replacement of C3 hydrogen atom of the tyrosine aromatic ring with a nitro group (R-NO 2 ) as shown in (Figure 2). This modification can occur within a polypeptide sequence (protein-associated Tyr-NO 2 ) or to free tyrosine amino acids (free Tyr-NO 2 ). Studies showed that 3-NO-Tyr plasma levels are increased in several conditions such as asthma, diabetes, and cardiovascular diseases, and reduced following therapeutic treatments [20]. to phosphorylation [24]. It has been shown to mediate redox regulation of a number of key cellular proteins including Endothelial Nitric Oxide Synthase (eNOS), SERCA and Na + -K + pump [25] as shown in (Figure 3). The impact of glutathionylation of each of these membrane proteins has been reported in either the myocardium or vascular tissue with altered function resulting in alterations in intracellular Na + and Ca 2+ . However, the direct usefulness of measuring glutathionylation of these proteins as biomarkers is hampered by difficulty in accessing the tissue in which these functionally relevant modifications occur. S-glutathionylation of susceptible proteins is commonly measured using Western Blotting [24,26].

Nitric Oxide Synthase
There are three known isoforms of NOS with different activities.
Two of the NOS isoforms are expressed constitutively these are in neuronal cells (nNOS) and endothelial cells (eNOS). Another isoform of NOS which is expressed in macrophages when needed termed as induced (iNOS) [30].  [36]. ROS and reactive halogenating species modify various biomolecules such as nucleic acids, prozteins, and lipids. MPO also plays an essential role in host immune defenses because of its unique ability to generate HOCl, which has potent antimicrobial activity as shown in (Figure 6).   [47].

Role of Myeloperoxidase in Atherosclerosis Development:
Many studies implicated MPO in the pathogenesis of atherosclerosis, showing that it is enriched within atheromatous

plaques. Inflammatory cells recruited into the vascular wall release
MPO-derived ROS that can in turn generate atherogenic OxLDL and modify High Density Lipoprotein (HDL), impairing its function in cholesterol efflux [38]. MPO high levels in the circulations are associated with cardiovascular disease [39]. MPO has emerged as one enzymatic catalyst for LDL oxidation via several chemical processes.
MPO forms the highly reactive HOCl, which preferentially reacts with amino acids rather than lipids. HOCl chlorinates electron-rich substrates on apolipoprotein B-100 such as Lys residues and Tyr forming MPO-specific 3-chlorotyrosine. Enrichment of LDL with markers of chlorination such as 3-chlorotyrosine, served to identify MPO as the first enzymatic catalyst of a specific oxidative pathway [40,41]. In addition, one MPO's role to atherosclerosis involves site specific oxidative modification of HDL, thereby attenuating its antiatherogenic properties. MPO binds to helix 8 on apolipoprotein A-I, the major protein associated with HDL. In the presence of H 2 O 2 , Cl-and/or NO 2 -, MPO may decrease the ability of HDL to facilitate cholesterol efflux from lipid-laden cells [42][43][44]. Oxidized HDL competes with native HDL as a ligand for the scavenger receptor, and this may further interfere with mobilization of cholesterol from peripheral tissues to the liver. It also resulted in proinflammatory particle that induced the expression of vascular cell adhesion molecule 1 in endothelial cells, and hence may promote the entry of circulating monocytes into the vessel wall [45,46] (Figures 7 & 8).

The Role of Oxidized Ldl in Atherosclerosis Development
The oxidation of lipoproteins is an initial phase in the development of atherosclerosis with deleterious and toxic effects on endothelial cells [48]. One of lipoproteins oxidation product ,Ox-LDL, induce endothelial dysfunction, the expression of adhesion molecules, the migration and proliferation of smooth muscle cells, and foam cell formation [49]. Atherosclerosis has three important stages heralded by a fatty streak formation, the induction of atheroma, and atherosclerotic plaques which eventually lead to atherothrombosis [50]. The lysis of the extracellular matrix leads to the destruction of the fibrous cap, and the thrombogenic contents are exposed to the blood stream initiating the coagulation process, blood clot formation, the adhesion of platelets, and thrombus formation, which may completely block the arteries [55][56][57] (Figure 9).