**6.2 Glutathione**

Glutathione (GSH) is present in all plant and animal cells and comprises three amino acids: glycine, cysteine, and glutamic acid. It is mainly synthesized in the liver [72] and exists in several redox forms, among which the most predominant is the reduced glutathione. GSH is a hydrosoluble antioxidant present in high cellular concentrations (1–10 mM) in the nucleus, mitochondria, and cytoplasm. GSH is involved in several lines of defense against ROS. First, the thiol group confers GSH with the ability to protect other thiol functions in proteins against oxidative damage [73]. Thiol groups (-SH) are widespread and highly reactive chemical entities in cells. They complex with metal ions, participate in oxidation reactions by getting oxidized themselves to sulfonic acids, and form thiol radicals and disulfides [74]. As an antioxidant, GSH reduces ROS during the enzymatic and nonenzymatic reactions. It regenerates other oxidized antioxidants like vitamin C and vitamin E [75] and is involved in the repair of lipids damaged in peroxidation processes and in the maintenance of sulfhydryl moieties of proteins in the reduced form [76, 77]. GSH functions in conjunction with three groups of enzymes to maintain an intracellular reducing environment and combat excessive formation of harmful ROS. These enzymes are glutathione peroxidase (GSHPx), glutathione reductase (GR), and glutathione oxidase (GOx). Glutathione peroxidase (GSHPx) is a selenium-containing enzyme that mediates catalytic reduction of peroxides using GSH as a sacrificial reductant [78]. The enzyme is a tetramer featuring a selenocysteine residue in each subunit [11]. The oxidation-reduction chemistry of the selenol functional group found in each selenocysteine is responsible for the activity of GSHPx, and the catalytic cycle is displayed in **Figure 14** [79]. In the first step, the selenol functional group (EnzSeH) gets oxidized by the peroxide to the corresponding selenenic acid (EnzSeOH). The thiophilic acid reacts with GSH to generate a selenenyl sulfide

**107**

**Figure 15.**

*Structure and biosynthesis of melatonin.*

*Nonenzymatic Exogenous and Endogenous Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.87778*

peroxide and GSSG.

**6.3 Melatonin**

*reductase (GR), and glutathione oxidase (GOx).*

**Figure 14.**

intermediate (EnzSeSG) which is highly reactive and is susceptible to nucleophilic displacement at the sulfur atom. Thus, attack by a second molecule of GSH at the sulfur atom regenerates the original selenol and eliminates oxidized glutathione (GSSG) as a byproduct. The latter is recycled back to GSH in an NADPH-dependent reduction process mediated by glutathione reductase (GR). GSH is also a substrate for glutathione oxidase (GOx) which catalyzes the reduction of oxygen to hydrogen

Since its discovery in 1993, melatonin's ability to reduce oxidative stress induced in all cells and organs by both oxygen- and nitrogen-based radicals has been reported in over one thousand publications. The structure of this endogenous antioxidant features an indoleamine and is biosynthesized in animals from L-tryptophan, an intermediate product of the shikimate pathway [80]. The biosynthetic process includes hydroxylation, decarboxylation, acetylation, and a methylation (**Figure 15**). Melatonin, which is produced mainly by the pineal gland in the brain [81], indirectly reduces free radical formation primarily through a process known as radical avoidance by stimulating the expression of endogenous antioxidant enzymes that metabolize reactive species and maintain redox homeostasis within cells [82]. These include superoxide dismutase (SOD), glutathione peroxidase (GSHPx), glutathione reductase, and catalase. In addition, it induces the synthesis of the antioxidant glutathione and inhibits certain enzymes that normally

*Structure and role of glutathione (GSH) in the catalytic cycle of glutathione peroxidase (GSHPx), glutathione* 

directly scavenge free radicals along with several of its metabolites that are formed during radical neutralization [83, 84]. For example, it is a very effective scavenger of the hydroxyl radical, singlet oxygen, peroxynitrite anion, and nitric oxide. Interestingly, melatonin has been shown to exhibit double the activity of vitamin E

). Melatonin can also

produce free radicals like nitric oxide synthase (generates NO•

and ranks among as the most effective lipophilic antioxidant.

#### *Nonenzymatic Exogenous and Endogenous Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.87778*

intermediate (EnzSeSG) which is highly reactive and is susceptible to nucleophilic displacement at the sulfur atom. Thus, attack by a second molecule of GSH at the sulfur atom regenerates the original selenol and eliminates oxidized glutathione (GSSG) as a byproduct. The latter is recycled back to GSH in an NADPH-dependent reduction process mediated by glutathione reductase (GR). GSH is also a substrate for glutathione oxidase (GOx) which catalyzes the reduction of oxygen to hydrogen peroxide and GSSG.

#### **Figure 14.**

*Free Radical Medicine and Biology*

free radical with ascorbate (AscH−

*Chemical structure and radical scavenging mechanism of uric acid.*

**6.2 Glutathione**

**Figure 13.**

does not directly scavenge peroxynitrite since UA cannot compete for the reaction of peroxynitrite with CO2. The antioxidant effect of uric acid may thus be related to the scavenging of the radicals CO3**·−** and NO2**.** which are formed from the reaction of peroxynitrite with CO2 [67]. As shown in **Figure 13**, UA displays a keto-enol tautomerism where the enol form predominantly exists as the monobasic urate anion at physiological pH [70]. The complete scavenging of peroxynitrite requires the presence of ascorbic acid and thiols whereby the urate anion is regenerated following reduction of the urate

atom abstraction provided evidence that the unpaired electron resides primarily on the five-membered ring of the purine structure. The radical was described as a delocalized π radical as the odd electron showed spin density on all four nitrogen atoms [71].

Glutathione (GSH) is present in all plant and animal cells and comprises three amino acids: glycine, cysteine, and glutamic acid. It is mainly synthesized in the liver [72] and exists in several redox forms, among which the most predominant is the reduced glutathione. GSH is a hydrosoluble antioxidant present in high cellular concentrations (1–10 mM) in the nucleus, mitochondria, and cytoplasm. GSH is involved in several lines of defense against ROS. First, the thiol group confers GSH with the ability to protect other thiol functions in proteins against oxidative damage [73]. Thiol groups (-SH) are widespread and highly reactive chemical entities in cells. They complex with metal ions, participate in oxidation reactions by getting oxidized themselves to sulfonic acids, and form thiol radicals and disulfides [74]. As an antioxidant, GSH reduces ROS during the enzymatic and nonenzymatic reactions. It regenerates other oxidized antioxidants like vitamin C and vitamin E [75] and is involved in the repair of lipids damaged in peroxidation processes and in the maintenance of sulfhydryl moieties of proteins in the reduced form [76, 77]. GSH functions in conjunction with three groups of enzymes to maintain an intracellular reducing environment and combat excessive formation of harmful ROS. These enzymes are glutathione peroxidase (GSHPx), glutathione reductase (GR), and glutathione oxidase (GOx). Glutathione peroxidase (GSHPx) is a selenium-containing enzyme that mediates catalytic reduction of peroxides using GSH as a sacrificial reductant [78]. The enzyme is a tetramer featuring a selenocysteine residue in each subunit [11]. The oxidation-reduction chemistry of the selenol functional group found in each selenocysteine is responsible for the activity of GSHPx, and the catalytic cycle is displayed in **Figure 14** [79]. In the first step, the selenol functional group (EnzSeH) gets oxidized by the peroxide to the corresponding selenenic acid (EnzSeOH). The thiophilic acid reacts with GSH to generate a selenenyl sulfide

). ESR studies on UA radical production by hydrogen

**106**

*Structure and role of glutathione (GSH) in the catalytic cycle of glutathione peroxidase (GSHPx), glutathione reductase (GR), and glutathione oxidase (GOx).*

### **6.3 Melatonin**

Since its discovery in 1993, melatonin's ability to reduce oxidative stress induced in all cells and organs by both oxygen- and nitrogen-based radicals has been reported in over one thousand publications. The structure of this endogenous antioxidant features an indoleamine and is biosynthesized in animals from L-tryptophan, an intermediate product of the shikimate pathway [80]. The biosynthetic process includes hydroxylation, decarboxylation, acetylation, and a methylation (**Figure 15**). Melatonin, which is produced mainly by the pineal gland in the brain [81], indirectly reduces free radical formation primarily through a process known as radical avoidance by stimulating the expression of endogenous antioxidant enzymes that metabolize reactive species and maintain redox homeostasis within cells [82]. These include superoxide dismutase (SOD), glutathione peroxidase (GSHPx), glutathione reductase, and catalase. In addition, it induces the synthesis of the antioxidant glutathione and inhibits certain enzymes that normally produce free radicals like nitric oxide synthase (generates NO• ). Melatonin can also directly scavenge free radicals along with several of its metabolites that are formed during radical neutralization [83, 84]. For example, it is a very effective scavenger of the hydroxyl radical, singlet oxygen, peroxynitrite anion, and nitric oxide. Interestingly, melatonin has been shown to exhibit double the activity of vitamin E and ranks among as the most effective lipophilic antioxidant.

#### **Figure 15.**

*Structure and biosynthesis of melatonin.*

### **6.4 Bilirubin**

Bilirubin (BIL) is an endogenous antioxidant produced from the enzymatic degradation of hemoglobin and other heme proteins (**Figure 16**). The process involves oxidative cleavage, catalyzed by the enzyme heme oxygenase, of one porphyrin exocyclic double bond of a heme residue of hemoglobin to generate biliverdin. Subsequent enzymatic reduction of biliverdin by biliverdin reductase yields bilirubin. This process is reversible and the oxidation of bilirubin by lipophilic ROS results in the formation of biliverdin. Notable structural features of bilirubin include an open chain of four connected pyrrole rings and a *Z*,*Z*-double bond geometry. In biological systems, bilirubin shows potent antioxidant properties [85, 86] especially against peroxyl radicals [87].

**Figure 16.** *Enzymatic degradation of hemoglobin heme to bilirubin.*

#### **6.5 Polyamines**

Putrescine (H2N-(CH2)4-NH2), spermidine ([H2N-(CH2)3]2-NH), and spermine (H2N-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH2) are biogenic unbranched polyamines (PAs) that exhibit antioxidant activities [88–90]. These amines are present in minute quantities in virtually all living species. While putrescine (1,4-diaminobutane) bears two primary amine groups at both terminal carbons, spermidine (triamine) and spermine (tetraamine) contain one and two additional secondary amine moieties, respectively. As antioxidants, PAs mediate protection of DNA against oxidative damage induced by hydrogen peroxide [90], scavenge free radicals [88], and reduce oxidative haemolysis of erythrocytes [90]. The amines also function as positive modulators of antioxidant genes under conditions of strong oxidative stress [88]. The protective effect of PAs is related to the stabilization of polyunsaturated phospholipids in cell membranes from peroxyl radicals, superoxides, and hydrogen peroxide [89]. In regard to their role in DNA protection against ROS, PAs are positively charged at physiological pH, enabling them to remain in proximity to negatively charged macromolecules, thus protecting them against oxidative damage [90]. Biosynthetically, the three polyamines are biosynthesized from L-ornithine, known to supply C4N building block, and L-methionine [91]. In animals, L-ornithine undergoes a pyridoxal phosphate (PLP)-dependent decarboxylation to generate putrescine. Thereafter, aminopropylation of putrescine by the enzyme spermidine synthase and decarboxy-S-adenosyl methionine produces spermidine. Repetition of the same sequence of reactions in the presence of the enzyme spermine synthase generates spermine.

### **7. Conclusions**

In addition to the oxidative damage that reactive oxygen and nitrogen species inflict on macromolecules, they also participate in damage caused by microbial

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*Nonenzymatic Exogenous and Endogenous Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.87778*

promotion of health span.

**Acknowledgements**

laboratory (Project 852).

AscH− ascorbate BIL bilirubin CAT catalase

GSH glutathione

GSSG glutathione disulfide GPx or GSHPx glutathione peroxidase GR glutathione reductase GOx glutathione oxidase GSR glutathione reductase H2O2 hydrogen peroxide HO**·** hydroxyl radical HClO hypochlorous acid IPP isopentyl diphosphate LDL low-density lipoprotein LOOH lipid hydroperoxides LOO**·** lipid peroxyl radical MPO myeloperoxidase NOS nitric oxide synthase

NO**·** nitric oxide radical ONOO**−** peroxynitrite

DHA dehydroascorbate DNA deoxyribonucleic acid DMAPP dimethylallyl diphosphate ESR electron spin resonance

EnzSeSG glutathione peroxidase selenenyl sulfide EnzSeOH glutathione peroxidase selenenic acid EnzSeH glutathione peroxidase selenol

NADPH nicotinamide adenine dinucleotide phosphate

**Abbreviations**

infections, tumor progression, and neurodegenerative diseases. In response to such oxidative injuries, tissues protect themselves by expressing genes encoding antioxidant enzymes and endogenous antioxidants to maintain oxidants at harmless levels. Oxidants themselves mediate certain cellular functions and cannot be eliminated completely. This fact emphasizes the significance of the antioxidant defense system in maintaining homeostasis and normal physiological processes, and in combating diseases and promoting immunity. The regulation of gene expression by employing oxidants and antioxidants represents a novel approach with promising therapeutic implications. Exogenous antioxidants are also critical for maintaining healthy living and longevity and must be obtained through dietary means. However, excessive dietary supplementation may disrupt the activation of the endogenous antioxidant defense system. Consequently, further research is required to fully elucidate the importance of antioxidants in the therapy of several human disease states and

Dr. Ziad Moussa is grateful to the United Arab Emirates University (UAEU) of Al-Ain and to the Research Office for supporting the research developed in his *Nonenzymatic Exogenous and Endogenous Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.87778*

infections, tumor progression, and neurodegenerative diseases. In response to such oxidative injuries, tissues protect themselves by expressing genes encoding antioxidant enzymes and endogenous antioxidants to maintain oxidants at harmless levels. Oxidants themselves mediate certain cellular functions and cannot be eliminated completely. This fact emphasizes the significance of the antioxidant defense system in maintaining homeostasis and normal physiological processes, and in combating diseases and promoting immunity. The regulation of gene expression by employing oxidants and antioxidants represents a novel approach with promising therapeutic implications. Exogenous antioxidants are also critical for maintaining healthy living and longevity and must be obtained through dietary means. However, excessive dietary supplementation may disrupt the activation of the endogenous antioxidant defense system. Consequently, further research is required to fully elucidate the importance of antioxidants in the therapy of several human disease states and promotion of health span.
