2.1.1. Oxidative Enzymes Generating Reactive Oxygen Species

Reactive oxygen species (ROS) include several free radicals, such as superoxide (O2 −•) and hydroxyl radical (OH•) and nonradical molecules such as hydrogen peroxide (H2O2) and organic hydroperoxide (ROOH) (Table 2). ROS are produced in endogenously in the cytosol, the plasma membrane, the membranes of mitochondria and endoplasmic reticulum, peroxisomes, and phagocytic cells [42,43] (Figure 2). ROS is difficult for direct measurement in biological tissues due to its highly reactivity and short life.

**Figure 2.** Reactive chemical species. Reactive chemical species comprise of not only reactive oxygen species and reactive nitrogen species, but also reactive sulfur, carbonyl, halogen, and selenium species. Sulfur reacts with oxygen or nitrogen to form reactive thiol or nitrosothiol species, respectively. All reactive chemical species react in concert during regular cellular activity but may cause oxidative stress to damage cellular components such as proteins, lipids, and nucleic acids.

**Table 2.** Reactive chemical species. During regular cellular activity living cells generate numerous reactive chemical species containing oxygen, nitrogen, sulfur, carbonyl, halogen, or selenium. Reactive sulfur species can contain thiols or nitrothiols. Free radicals possess at least one unpaired electron that makes highly reactive and short-lived. Nonradicals are oxidizing chemicals or easily converted to free radicals. Superoxide, reactive sulfur species, or reactive selenium species can be reducing agents as reactive nucleophilic species.


In the cytosol, ROS can be generated by soluble intercellular components, such as catecholamines, hydroquinones, flavins, and thiols (RSHs) that undergo reduction reactions [44]. The cytosolic enzyme XDH normally catalyzes xanthine, NAD<sup>+</sup>, and water (H2O) to urate, reduced form of NAD<sup>+</sup>, NADH, and hydrogen ion (H+). Reversible oxidation of cysteine residues or irreversible Ca2+-stimulated proteolysis converts XDH to xanthine oxidase (XO) that transfers electrons to molecular oxygen (O2), producing superoxide (O2 −•) during xanthine or hypoxanthine oxidation [45]. The serum levels of uric acid, a major endogenous antioxidant was measured in patients with PPMM, RRMM, and SPMM. The uric acid levels were significantly lower in active MS than inactive MS, and the uric acid levels were independently correlated with gender, disease activity and duration of the disease [46] (Table 3). **Table 3.** Oxidative stress biomarkers of multiple sclerosis. The redox status can be monitored by the activities of oxidative and antioxidative enzymes and the presence of degradation products derived from cellular components. ↑: increase, ↓: decrease, **-**: unknown.


The plasma membrane is a network of phospholipid bilayer and integral proteins which protects the cellular organelles from the outer environment and is responsible for several cellular functions, such as cell adhesion, ion transport, cell signaling, and phagocytosis. The main ROS of the plasma membrane is a superoxide (O2 −•) produced by the membrane-bound enzyme nicotinamide adenine dinucleotide phosphate oxidase (NOX), which is composed of two membrane proteins, three cytosolic proteins, and a small GTP-binding protein [89,90]. The expression of NOX isoform NOX5 was significantly increased, but the expression NOX4 was significantly decreased in serum of RRMS patients, suggesting that differential NOX isoform expression contributes to OS-associated vascular changes in MS [48]. ROS is also produced by COX and LOX, which convert arachidonic acid to prostaglandins, thromboxanes, and leukotrienes. Phospholipase A2 generates ROS during arachidonic acid oxidation [91]. In the presence of transition metal ions, such as Fe2<sup>+</sup> and Cu+, hydrogen peroxide (H2O2), organic hydroperoxide (ROOH), and organic peroxide (ROOR) produce hydroxyl (OH•), alkoxyl (RO•), and peroxyl radical (ROO•), respectively [92] (Table 3).

Superoxide dismutase (SOD) catalyzes the disproportionation of two superoxide (O2 −•) into molecular oxygen (O2) and hydrogen peroxide (H2O2). These enzymes are present in almost all aerobic cells and in extracellular fluids. SODs contain metal ion cofactors that, depending on the isozyme, can be copper, zinc, manganese, or iron [93,94]. There are three isozymes in humans. Dimeric copper- and zinc-coordinated SOD1 is in the cytoplasm; tetrameric manganese-coordinated SOD2 is confined to the mitochondria; tetrameric copper- and zinc-coordinated SOD3 is extracellular [95]. The mitochondrial ROS production takes place at four protein complexes, ubiquinone, and cytochrome c of the ETC, embedded in the inner membrane of the mitochondria [96]. The Complexes I/III/IV utilize NADH as the substrate, while Complexes II/III/IV use succinic acid. The complex II is also glycerol 3-phosphate dependent. The primary mitochondrial ROS is superoxide (O2 −•) that is converted by

mitochondrial SOD into hydrogen peroxide (H2O2), which can be turned into hydroxyl (OH•) via the Fenton reaction [97]. The Complex I and III release superoxide (O2 −•) into the mitochondrial matrix where it can damage the mitochondrial DNA, while the Complex III also releases superoxide (O2 −•) into the intermembrane space where it is accessible to the cytosol [98]. Reduced levels of antioxidant α-tocopherol was observed in the blood of patients with Leber's hereditary optic neuropathy, which is caused by mitochondrial mutation of the Complex I, suggesting that oxidative load was elevated and antioxidant capacity was compromised [99]. Other mitochondrial enzymes that contribute to hydrogen peroxide (H2O2) production are monoamine oxidases, dihydroorotate dehydrogenase, α-glycerophosphate dehydrogenase, and α-ketoglutarate dehydrogenase (α-KGDH) complex. Succinate dehydrogenase also generates ROS [100].

Significantly higher mean activity of SOD in erythrocyte lysates was reported in RRMS than controls. Interestingly, the SOD activity of CIS was higher than that of RRMS [101]. The erythrocyte SOD activity can be a diagnostic biomarker of CIS and RRMS. The SOD activity was significantly lower in the erythrocyte lysates of RRSM patients upon relapse than controls but increased following the intravenous administration of corticosteroid methylprednisolone and remained higher during remission period than controls. The SOD is a potent predictive and therapeutic biomarker. The serum/plasma samples also showed significantly higher mean SOD activity in RRMS compared to control groups [82]. However, platelet SOD1 and SOD2 activity was unchanged in MS patients [49]. Intriguingly, SOD activity was significantly low in CSF of CIS and RRMS patients, despite the significantly high activity of plasma SOD. Furthermore, there were negative correlations between the erythrocyte SOD activity and disease duration and expanded disability status scale (EDSS) in CIS and RRMS, between the erythrocyte SOD activity and Gd<sup>+</sup> enhancement lesion volume in CIS patients [50]. These findings suggest the SOD activity as a possible diagnostic and prognostic marker (Tables 3 and 4).

The peripheral blood mononuclear cells (PBMCs) SOD1 proteins and mRNA expression were significantly lower in RRMS patients than controls and became significantly more elevated following IFN-β1b treatment than the baseline [51]. These studies suggest SOD as a potential therapeutic biomarker (Table 4). However, the erythrocyte SOD activity remained unchanged following the treatment of natalizumab, a humanized monoclonal antibody against the cell adhesion molecule α4-integrin. But levels of carbonylated protein and oxidized guanosine were reduced [52].

In the inner membrane of mitochondria and the endoplasmic reticulum, a heme-containing monooxygenase cytochrome P450 (CYP) enzymes are responsible for oxidizing steroids, cholesterols, and fatty acids. The CYPs forms ROS superoxide (O2 −•) and hydrogen peroxide (ROOH) by substrate cycling [102]. Protonation of hydrogen peroxide (ROOH) forms hydrogen peroxide (H2O2) which, furthermore, cleaves into hydroxy radicals (OH•). The redox cycling produces free radical semiquinone from quinoid substrates [103]. In the mitochondrial transport chain, flavoprotein reductase forms ROS by direct reduction of O2 and via the mediation of quinones. [104]. Superoxide (O2 −•) is produced by XO in the reperfusion phase of ischemia, LOX, COX, and NADPH-dependent oxidase [105]. In the endoplasmic reticulum NADH cytochrome b5 reductase can leak electrons to molecular oxygen (O2) to generate superoxide (O2 −•) during the NADPH-dependent oxidation of xenobiotics [106].

Most enzymes in the peroxisomes produce ROS during the catalysis of fatty acid α- and β-oxidation, amino acid and glyoxylate metabolism, and synthesis of lipidic compounds. A large fraction of hydrogen peroxide (H2O2) generated inside peroxisomes was observed to penetrate the peroxisomal membrane and diffuse to the surrounding media [107]. The peroxide can diffuse through the channel formed by the peroxisomal membrane protein Pxmp2 and hydrogen peroxide (H2O2) generated by the peroxisomal urate oxidase can release through crystalloid core tubules into the cytosol [108]. Meanwhile, peroxisomes also possess protective mechanisms to counteract oxidative stress and maintain redox balance. Reduction in peroxisomal gene and protein expression was observed in MS gray matter [109].

**Table 4.** Possible redox biomarkers in multiple sclerosis. Reactive chemical species, oxidative enzymes, antioxidants, antioxidative enzymes, degradation products, and end products are potential biomarkers for multiple sclerosis (MS). Diagnostic biomarkers allow early detection and secondary prevention; prognostic biomarkers suggest the likely clinical course; predictive biomarkers predict the response of MS patients to a specific therapy; and therapeutic biomarkers indicate a target for therapy. CIS: clinically isolated syndrome, PPMS: primary progressive MS; RRMS: relapsing-remitting MS, SPMS: secondary progressive MS, mixedMM: mixed population of MS.


The lysosomal ETC plays a central role to support the positive proton gradient to maintain an optimal pH of the acid hydrolases [110]. The ETC is made up of a flavin-adenine dinucleotide, a b-type cytochrome and ubiquinone with the donor NADH and ending to acceptor molecular oxygen (O2), transferring three electrons. Superoxide (O2 −•) is possibly produced in the acidic environment which favors dismutation of hydrogen peroxide (H2O2) into hydroxy radical (OH•) by ferrous iron [111]. Furthermore, ozone (O3) and ozone-like oxidants are generated from singlet oxygen (O2 <sup>1</sup>Δg) catalyzed by antibody or amino acid. Ozone (O3) reacts with superoxide anion (O2 −•) to form hydrogen peroxide (H2O2) in the presence of Fe<sup>+</sup><sup>2</sup> [112].

2.1.2. Oxidative Enzymes Generating Reactive Nitrogen Species

Reactive nitrogen species (RNS) are a group of nitrogen-congaing molecules including free radicals, nitric oxide (NO), and nitrogen dioxide (NO2). Free radicals are nitric oxide (NO•) and nitrogen dioxide (NO2 •) radicals, while nonradicals are nitrite (NO2 <sup>−</sup>) and nitrate (NO3 −), among others (Table 2). RNS are derived from nitric oxide (NO) and superoxide anion (O2 −•) produced by nitric oxide

synthetase 2 (NOS2), NADPH oxidase, XO, LOX, and COX, among others [113,114]. At physiological concentrations, a gaseous molecule nitric oxide (NO) is a second messenger involved in blood pressure regulation, smooth muscle relaxation, defense mechanisms, immune regulation, and neurotransmission contributing the function of memory and learning [47].

Cross-sectional studies show that the levels of nitric oxide metabolites nitrite (NO2 −) and nitrite (NO3 −), measured as a total value (tNOx), are significantly higher in plasma or serum of patients with RRMS [83,115]. A longitudinal study revealed that higher serum tNOx is significantly correlated with relapsing rate, suggesting prognostic biomarker of NOS [116]. Many studies of CSF samples reported significantly higher levels of tNOx in RRMS and PPMS, compared to healthy controls [117,118]. A study observed significantly higher levels of CSF tNOx in RRMS than SPMS, suggesting an inflammatory role of RNS [119]. Significantly higher levels of CSF tNOx were reported in patients with acute relapsing phase of RRMS than those with stable remitting phase of RRMS [120,121] (Tables 3 and 4).

In cGMP-dependent pathways, nitric oxide radical (NO•) generated by endothelial NOS in endothelium, brain, and heart relaxes blood vessels and maintains normal blood pressure, while nitric oxide radicals (NO•) produced by neuronal NOS serve as a neurotransmitter to regulate blood pressure in the brain. Inducible NOS (*i*NOS) in macrophages and smooth muscle cells gives rise to nitric oxide radicals (NO•) as in reaction to bacterial lipopolysaccharides and/or cytokines [122].

Nitric oxide radical (NO•) is produced from the metabolism of L-arginine by NOS that converts L-arginine into L-citrulline and nitric oxide radical (NO•) by a 5-electron oxidation of a guanidine nitrogen of L-arginine [123]. In mitochondria nitric oxide radicals (NO•) react with respiratory Complex III to inhibit electron transfer and facilitate superoxide anion (O2 •−) production. The nitric oxide radicals (NO•) also compete with molecular oxygen (O2) for the binding site at the binuclear center of cytochrome *c* oxidoreductase, inducing a reversible inhibition of cytochrome *c* oxidase. Nitric oxide (NO) neutralizes ROS [45]. However, RNS react with oxygen molecules (O2) and ROS, giving rise to a variety of nitrogen oxides (NOs), such as nitrogen dioxide radical (NO2 •), nitrogen dioxide (NO2), dinitrogen trioxide (N2O3), peroxynitrite (ONOO<sup>−</sup>), nitrite (NO2 −), and nitrate (NO3 −). Higher concentrations of nitric oxide (NO) become toxic by forming nitrosothiols which oxidize tyrosine, cysteine, methionine, and GSH. In mitochondria, nitric oxide radicals (NO•) inhibit Complex I by *S*-nitrosation [124]. Together with other RNS, this contributes the damage of cell membranes, proteins, and lipid membrane leading to the degradation of mitochondria, lysosomes, and DNA. The chain of events culminates in the inhibition of immune response and production of carcinogenic nitrosamines [125]. Nitric oxide (NO) is also involved in metal homeostasis including Fe, Cu, and Zn [126].

Highly toxic peroxynitrite (ONOO−) is formed by the reaction of nitric oxide (NO) and superoxide anions (O2 −•), leading to the production of more reactive compounds that oxidize methionine and tyrosine residues of proteins, lipids, and DNA. Reacting with superoxide anion (O2 −•), nitric oxide radicals (NO•) form peroxynitrite which causes reversible inhibition of cellular respiration in the mitochondria [127]. In peroxisomes nitric oxide radicals (NO•) react with superoxide anions (O2 −•) produced by XO to form peroxynitrite and hydrogen peroxides [111]. In addition, insulin resistance favors peroxintrite formation [128].

In response to bacterial lipopolysaccharides and inflammatory stimuli, *i*NOS generates nitric oxide (NO) that protects tissue hypoxia and serves as a neurotransmitter. However, overexpression *i*NOS, increase of nitric oxide (NO), and subsequent inflammation have been implicated in pathophysiology of neurodegenerative diseases including MS [47]. Calmodulin mediates the oxidative stress and inflammasome activation involving calmodulin-binding proteins, calcineurin, and calcium/calmodulin-dependent kinase II. Calmodulin inhibitors improved cognitive functions in an animal model of vascular dementia [129]. The *i*NOS activity is upregulated in acute MS plaques [19,53]. Increased activity and expression of *i*NOS in lymphocytes were found in active relapsing phase of RRMS [54]. CSF *i*NOS expression was shown in MS patients and mean CSF NOS activity was significantly higher, compared to controls [55] (Table 3).
