Vitamin E and Oxidative Stress

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

damage and disease: induction, repair and significance. Mutat Res

[252] Dizardaroglu M, Kirkali G, Jaruge P. Formamidopyrimidines in DNA: mechanisms of formation, repair, and biological effects. Free Radic Biol

Med 2008; 45(12):1610-1621.

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Med Sci 2014; 3(1):15.

44(5):479-496.

[253] Vineis P, Pursianinen KP. Air pollution and cancer: biomarker studies in human populations. Carcinogenesis

[254] Valko M, Izakovic M, Mazur M, Christopher J, Rhodes C, Telser J. Role of oxygen radicals in DNA damage and cancer incidence. Mol Cell Biochem

[255] Valluru L, Dasari S, Wudayagiri R. Role of free radicals and antioxidants in gynaecological cancer: Current status and future prospects. Oxid Antioxid

[256] Liou GY, Storz P. Reactive oxygen species in cancer. Free Radic Res 2010;

[257] Miranda-Vilela AL, Portillo FA, de-Araujo VG, Estevanato LLC, Mezzomo BP, de-Almeida MFM, Lacava LGM. The protective effects of nutritional antioxidant therapy on Ehrlich solid tumor-bearing mice depend on the type of antioxidant therapy chosen: histology, genotoxicity and haematology evaluations. J Nutr Biochem 2011; 22(11):1091-1098.

[258] Block K, Koch A, Mead M, Tothy PK, Newman PA, Gyllenhaal C. Impact of antioxidant supplementation on chemotherapeutic efficacy: A systematic review of the evidence from randomized controlled trials. Cancer Treatment Rev 2007; 33(5):407-418.

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[244] Husain K, Centeno BA, Chen DT, Hingorani SR, Sebti SM, Malafa MP. Vitamin E δ-tocotrienol prolongs survival in the LSLKrasG12D/1;LSL-Trp53R172H/1;Pdx=1-Cre(KPC) transgenic mouse model of pancreatic

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[245] Hayes JD, Dinkova-Kostova AT, Tew KD. Oxidative stress in cancer. Cancer Cell 2020; 38(2):167-197.

[246] Seis H. Oxidative stress: A concept of redox biology and medicine. Redox

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[247] Ziech D, Franco R,

Georgkalikas AG, Georgakila S, Malamou-Mitsi V, Schoneveld O, Pappa A, Panayiotidis MI. The role of reactive oxygen species and oxidative stress in environmental carcinogenesis and biomarker development. Chem Biol

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[250] Cooke MS, Evans MD,

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[248] Marnette LJ. Oxyradicals and DNA damage. Carcinogenesis 2000;

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Dizardaroglu M, Lunec J. Oxidative

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**236**

**239**

**Chapter 12**

**Abstract**

oxidative process in some diseases.

**1. Introduction**

Vitamin E in Human Health and

Oxidative Stress Related Diseases

*Israel Ehizuelen Ebhohimen, Taiwo Stephen Okanlawon,* 

of radical species has been implicated in the onset and progression of several diseases. The efficacy of antioxidants acting via the inhibition of radical chain reactions, scavenging of free radicals, direct donation of electrons to radical species and chelation of metal ions have been reported to attenuate the oxidative process. Vitamin E is an effective antioxidant and its hydrophobic nature and membrane permeability offer some benefits to application and bioavailability. This chapter highlights the following; structural differences in the vitamin family, biosynthesis in plants and the native biological role, antioxidant mechanisms of vitamin E, an overview of the prophylactic action of vitamin E as well as the effect on the

**Keywords:** vitamin E, antioxidant, bioactivity, tocopherol, tocotrienol

synthetic PC-8, a γ-tocotrienol homolog but had longer side chains [1].

The tocochromanols generally called the vitamin E family are amphipathic organic molecules with antioxidant capacity. They are categorized as tocopherols, tocotrienols and plastochromanol-8 (PC-8) [1]. All groups have a similar chemical structure comprising a polar chromanol head linked with a hydrophobic prenyl tail. The positive effect of tocopherols and tocotrienols on reproduction in animals called research attention to this family of organic compounds. Vitamin E was first described by Evans and Bishop in 1922 [2] as substance 'X' [3, 4]. The class of compounds was later called 'tocopherol' coined from the Greek words 'to'kos' meaning 'child birth', and 'phe'rein' meaning 'to bring forth'. The suffix '-ol' was included due to the presence of an alcohol functional group. The tocopherols have a saturated prenyl tail while the tocotrienols have unsaturated tails with carbon–carbon double bonds at 3′,7′ and 11′ positions. Each group comprises four molecular forms (α, β, γ and δ) that are differentiated by the methyl group substitutions in the chromanol head group (**Figures 1** and **2**) which strongly influence their antioxidant activity in various systems [3–5]. Plastochromanol-8 (PC-8), is also a natural component of plant tissues and was first discovered in the leaves of the rubber tree (*Hevea brasiliensis*), where its concentration exceeded that of α-tocopherol and plastoquinone [6]. Structural studies revealed that the compounds were identical to those of

Oxidative stress characterized by an imbalance in the production and degradation

*Augustine Ododo Osagie and Owen Norma Izevbigie*

#### **Chapter 12**

## Vitamin E in Human Health and Oxidative Stress Related Diseases

*Israel Ehizuelen Ebhohimen, Taiwo Stephen Okanlawon, Augustine Ododo Osagie and Owen Norma Izevbigie*

#### **Abstract**

Oxidative stress characterized by an imbalance in the production and degradation of radical species has been implicated in the onset and progression of several diseases. The efficacy of antioxidants acting via the inhibition of radical chain reactions, scavenging of free radicals, direct donation of electrons to radical species and chelation of metal ions have been reported to attenuate the oxidative process. Vitamin E is an effective antioxidant and its hydrophobic nature and membrane permeability offer some benefits to application and bioavailability. This chapter highlights the following; structural differences in the vitamin family, biosynthesis in plants and the native biological role, antioxidant mechanisms of vitamin E, an overview of the prophylactic action of vitamin E as well as the effect on the oxidative process in some diseases.

**Keywords:** vitamin E, antioxidant, bioactivity, tocopherol, tocotrienol

#### **1. Introduction**

The tocochromanols generally called the vitamin E family are amphipathic organic molecules with antioxidant capacity. They are categorized as tocopherols, tocotrienols and plastochromanol-8 (PC-8) [1]. All groups have a similar chemical structure comprising a polar chromanol head linked with a hydrophobic prenyl tail. The positive effect of tocopherols and tocotrienols on reproduction in animals called research attention to this family of organic compounds. Vitamin E was first described by Evans and Bishop in 1922 [2] as substance 'X' [3, 4]. The class of compounds was later called 'tocopherol' coined from the Greek words 'to'kos' meaning 'child birth', and 'phe'rein' meaning 'to bring forth'. The suffix '-ol' was included due to the presence of an alcohol functional group. The tocopherols have a saturated prenyl tail while the tocotrienols have unsaturated tails with carbon–carbon double bonds at 3′,7′ and 11′ positions. Each group comprises four molecular forms (α, β, γ and δ) that are differentiated by the methyl group substitutions in the chromanol head group (**Figures 1** and **2**) which strongly influence their antioxidant activity in various systems [3–5]. Plastochromanol-8 (PC-8), is also a natural component of plant tissues and was first discovered in the leaves of the rubber tree (*Hevea brasiliensis*), where its concentration exceeded that of α-tocopherol and plastoquinone [6]. Structural studies revealed that the compounds were identical to those of synthetic PC-8, a γ-tocotrienol homolog but had longer side chains [1].


**Figure 1.** *Tocopherol.*

**Figure 2.** *Tocotrienol.*

The human body tends to accumulate α-tocopherol due to the activity of the liver α-tocopherol transfer protein (α-TTP), which enriches plasma with a-tocopherol [7]. Besides α-TTP, which resides only in the liver, a system of tocopherol-binding proteins (TBPs) cause the localization of tocopherols in various human tissues where they are required [8].

The main dietary sources of vitamin E compounds include vegetable oils, nuts and seeds. The vitamin E family has been studied extensively due to their diverse biological functions and α-tocopherol is reported to have the highest biological activity [3]. Although α-tocopherol is universally distributed in the plant kingdom and is the predominant vitamin E form in photosynthetic tissues, γ-tocopherol and tocotrienols predominate in the seeds of several dicots and monocots [5].

#### **1.1 Biological functions of vitamin E**

The most notable biological function of this lipid-soluble is their antioxidant capacity. All vitamin E compounds meet the definition of an antioxidant moiety with the capacity to inhibit oxidative reactions *in vitro* [3]. Vitamin E is widely accepted as one of the most potent antioxidant in nature and the antioxidant property is based on the capacity to rapidly transfer its phenolic hydrogen atom to

**241**

*Vitamin E in Human Health and Oxidative Stress Related Diseases*

neutralize free radicals (**Figure 3**). The regeneration mechanism, mostly by vitamin

The antioxidant capacity of α, β and γ isoforms of both tocopherol and tocotrienol are similar and the δ isoforms have weaker activity [11, 12]. As the main chain-breaking antioxidant in body tissues, the vitamin E isoforms inhibit lipid peroxidation especially in polyunsaturated fatty acid component of cell membranes. The in vivo antioxidant capacity of vitamin E is not completely clear. The suggested in vivo activity is based on the reported in vitro activity [3]. Based on the capacity to inhibit oxidation, vitamin E may help ameliorate or suppress the progression of

Other reported biological functions of vitamin E include; regulation of inflam-

Tocochromanols are only synthesized by photosynthetic organisms. In plants, tocochromanol biosynthesis utilizes cytosolic aromatic amino acid pathway for head group synthesis while the tail is synthesized by the plastidic deoxyxylulose-

The formation of homogentisic acid (HGA) from p-hydroxyphenylpyruvic acid (HPP) by p-hydroxyphenylpyruvic acid dioxygenase (HPPD) is the rate-limiting

The biosynthesis of tocopherols and the tocotrienols follow the same pathway, each class requiring specific substrates and enzymes. HGA is prenylated with either phytyl-diphosphate (PDP) or geranylgeranyl diphosphate (GGDP) to yield the committed intermediates 2-methyl-6-phytylplastoquinol (MPBQ ) and 2-methyl-6-geranylgeranylplastoquinol (MGGBQ ) in tocopherol and tocotrienol synthesis

MPBQ methyltransferase (MPBQ MT) transfers a second methyl group to MPBQ to form 2,3-dimethyl-5-phytyl-1,4-benzoquinone (DMPBQ ) and to MGGBQ to form 2,3-dimethyl-5-geranylgeranyl-1,4-benzoquinone (DMGGBQ ) respctively. Tocopherol cyclase converts MPBQ and DMPBQ to δ- and γ-tocopherols, respectively, and the corresponding geranylgeranylated intermediates to δ- and γ-tocotrienols. Finally, γ-tocopherol methyltransferase (γ-TMT) adds a methyl group to C-6 of the chromanol ring, converting δ- and γ-tocopherols and tocotri-

Among the vitamin E family present in foods, α-tocopherol is the most important to human health [15]. Although all tocopherols are absorbed equally during digestion, only α-tocopherol is preferentially retained and distributed throughout the body [16]. The concentration of γ-tocopherol is far higher than that of α-tocopherol in oil seeds though the former is the biosynthetic precursor of the

enols to β- and α-tocopherols and tocotrienols, respectively [14].

matory response, gene expression, cell proliferation, as well as modulation of

cellular signaling and activity of membrane bound enzymes.

step in the synthesis of the head group (**Figure 4**).

*DOI: http://dx.doi.org/10.5772/intechopen.99169*

C is essential for maintaining viability [9, 10].

*Reaction of alpha-tocopherol with hydroxyl radical.*

oxidative stress related diseases [13].

**1.2 Biosynthesis of vitamin E**

5-phosphate pathway.

**Figure 3.**

respectively.

*Vitamin E in Human Health and Oxidative Stress Related Diseases DOI: http://dx.doi.org/10.5772/intechopen.99169*

#### **Figure 3.**

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

The human body tends to accumulate α-tocopherol due to the activity of the liver α-tocopherol transfer protein (α-TTP), which enriches plasma with a-tocopherol [7]. Besides α-TTP, which resides only in the liver, a system of tocopherol-binding proteins (TBPs) cause the localization of tocopherols in various human tissues

The main dietary sources of vitamin E compounds include vegetable oils, nuts and seeds. The vitamin E family has been studied extensively due to their diverse biological functions and α-tocopherol is reported to have the highest biological activity [3]. Although α-tocopherol is universally distributed in the plant kingdom and is the predominant vitamin E form in photosynthetic tissues, γ-tocopherol and

The most notable biological function of this lipid-soluble is their antioxidant capacity. All vitamin E compounds meet the definition of an antioxidant moiety with the capacity to inhibit oxidative reactions *in vitro* [3]. Vitamin E is widely accepted as one of the most potent antioxidant in nature and the antioxidant property is based on the capacity to rapidly transfer its phenolic hydrogen atom to

tocotrienols predominate in the seeds of several dicots and monocots [5].

**240**

where they are required [8].

**Figure 1.** *Tocopherol.*

**Figure 2.** *Tocotrienol.*

**1.1 Biological functions of vitamin E**

*Reaction of alpha-tocopherol with hydroxyl radical.*

neutralize free radicals (**Figure 3**). The regeneration mechanism, mostly by vitamin C is essential for maintaining viability [9, 10].

The antioxidant capacity of α, β and γ isoforms of both tocopherol and tocotrienol are similar and the δ isoforms have weaker activity [11, 12]. As the main chain-breaking antioxidant in body tissues, the vitamin E isoforms inhibit lipid peroxidation especially in polyunsaturated fatty acid component of cell membranes. The in vivo antioxidant capacity of vitamin E is not completely clear. The suggested in vivo activity is based on the reported in vitro activity [3]. Based on the capacity to inhibit oxidation, vitamin E may help ameliorate or suppress the progression of oxidative stress related diseases [13].

Other reported biological functions of vitamin E include; regulation of inflammatory response, gene expression, cell proliferation, as well as modulation of cellular signaling and activity of membrane bound enzymes.

#### **1.2 Biosynthesis of vitamin E**

Tocochromanols are only synthesized by photosynthetic organisms. In plants, tocochromanol biosynthesis utilizes cytosolic aromatic amino acid pathway for head group synthesis while the tail is synthesized by the plastidic deoxyxylulose-5-phosphate pathway.

The formation of homogentisic acid (HGA) from p-hydroxyphenylpyruvic acid (HPP) by p-hydroxyphenylpyruvic acid dioxygenase (HPPD) is the rate-limiting step in the synthesis of the head group (**Figure 4**).

The biosynthesis of tocopherols and the tocotrienols follow the same pathway, each class requiring specific substrates and enzymes. HGA is prenylated with either phytyl-diphosphate (PDP) or geranylgeranyl diphosphate (GGDP) to yield the committed intermediates 2-methyl-6-phytylplastoquinol (MPBQ ) and 2-methyl-6-geranylgeranylplastoquinol (MGGBQ ) in tocopherol and tocotrienol synthesis respectively.

MPBQ methyltransferase (MPBQ MT) transfers a second methyl group to MPBQ to form 2,3-dimethyl-5-phytyl-1,4-benzoquinone (DMPBQ ) and to MGGBQ to form 2,3-dimethyl-5-geranylgeranyl-1,4-benzoquinone (DMGGBQ ) respctively. Tocopherol cyclase converts MPBQ and DMPBQ to δ- and γ-tocopherols, respectively, and the corresponding geranylgeranylated intermediates to δ- and γ-tocotrienols. Finally, γ-tocopherol methyltransferase (γ-TMT) adds a methyl group to C-6 of the chromanol ring, converting δ- and γ-tocopherols and tocotrienols to β- and α-tocopherols and tocotrienols, respectively [14].

Among the vitamin E family present in foods, α-tocopherol is the most important to human health [15]. Although all tocopherols are absorbed equally during digestion, only α-tocopherol is preferentially retained and distributed throughout the body [16]. The concentration of γ-tocopherol is far higher than that of α-tocopherol in oil seeds though the former is the biosynthetic precursor of the

latter. This suggests that the γ-tocopherol methyltansferase reaction is limited. One approach to increase α-tocopherol yield in these seeds is to increase the expression of γ-tocopherol methyltansferase gene [17].

#### **Figure 4.**

*Tocopherol biosynthesis [14]. Abbreviations: PAT, prephenate amino transferase; AdeH, arogenate dehydrogenase; TAT, tyrosine amino transferase; HPPD, p-hydroxyphenylpyruvate dioxygenase; HPT, homogentisate phytyltransferase; phytyl-DP, phytyl-diphosphate; MPBQ, 2-methyl-6-phytyl-1,4-benzoquinone; DMPBQ, 2,3-dimethyl-5-phytyl-1,4-benzoquinone; MT, methyltransferase; SAM, S-adenosyl methionine; TC, tocopherol cyclase; MPBQ MT, MPBQ methyltransferase; gamma-TMT, gamma-tocopherol methyltransferase.*

**243**

*Vitamin E in Human Health and Oxidative Stress Related Diseases*

The reactive oxygen species (ROS) represents the most important class of radical species generated in living systems formed from the partial reduction of molecular oxygen. Notable members of this family of radicals include superoxide anion (O2

The superoxide radical is readily dismutated to hydrogen peroxide. The reactivity of hydrogen peroxide as a molecule is low but it can penetrate cell membranes

> – 3 2

2 3 Fe H O Fe HO HO 2 2

HO H O H O O H 22 2 2

O H O O HO HO 2 22 2

2 3 Fe HO H Fe H O2

The hydroxyl radical is regarded as the most reactive oxygen radical and can cause oxidative damage to cells by attacking biomolecules located a few nanometers

Low levels of ROS production are required for important physiological functions, including proliferation, host defense, signal transduction, and gene expression [20]. There is a cellular balance between ROS generation and clearance in eukaryotic cells. This is achieved by the activity of several antioxidative defense mechanisms that comprise enzymes and antioxidants. The five main types of primary intracellular antioxidant enzymes are Cu/Zn-superoxide dismutase (Cu/Zn-SOD, SOD1) in the cytosol, manganese superoxide dismutase (Mn-SOD, SOD2) in the mitochondrial matrix, catalase, glutathione peroxidase (GPx), and glutathione reductase (GR). Small molecular weight and nonenzymatic antioxidants are also involved in the protection of the intracellular

<sup>2</sup> <sup>2</sup> Fe O Fe O •

+ + +→ + (1)

<sup>+</sup> +−• + →+ + (2)

• • <sup>+</sup> + → ++ (3)

• − • + →+ + (4)

+ •+ + + +→ + (5)

–

hydroxyl radical (OH∙), hydrogen peroxide (H2O2), and singlet oxygen (1

are generated by the respiratory chain in mitochondria, enzymatic reactions, exposure to UV light, ionizing radiation and heavy metal ions. The mitochondrial electron transport chain generates superoxide radicals through the single-electron leak at respiratory complexes I and III of the oxidative phosphorylation pathway. The flavin-dependent enzymes in the mitochondrial matrix also produce a consid-

− ),

O2) which

*DOI: http://dx.doi.org/10.5772/intechopen.99169*

erable amount of reactive oxygen species.

The second step is the Fenton reaction:

Haber and Weiss reaction:

The chain termination reaction:

from the site of generation [19].

and generate hydroxyl radical via the Fenton's reaction [18]. The first step involves the reduction of ferric to ferrous ion:

–

**2. Oxidative stress**

#### **2. Oxidative stress**

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

of γ-tocopherol methyltansferase gene [17].

latter. This suggests that the γ-tocopherol methyltansferase reaction is limited. One approach to increase α-tocopherol yield in these seeds is to increase the expression

*Tocopherol biosynthesis [14]. Abbreviations: PAT, prephenate amino transferase; AdeH, arogenate dehydrogenase; TAT, tyrosine amino transferase; HPPD, p-hydroxyphenylpyruvate dioxygenase; HPT, homogentisate phytyltransferase; phytyl-DP, phytyl-diphosphate; MPBQ, 2-methyl-6-phytyl-1,4-benzoquinone; DMPBQ, 2,3-dimethyl-5-phytyl-1,4-benzoquinone; MT, methyltransferase; SAM, S-adenosyl methionine; TC, tocopherol cyclase; MPBQ MT, MPBQ methyltransferase; gamma-TMT, gamma-tocopherol methyltransferase.*

**242**

**Figure 4.**

The reactive oxygen species (ROS) represents the most important class of radical species generated in living systems formed from the partial reduction of molecular oxygen. Notable members of this family of radicals include superoxide anion (O2 − ), hydroxyl radical (OH∙), hydrogen peroxide (H2O2), and singlet oxygen (1 O2) which are generated by the respiratory chain in mitochondria, enzymatic reactions, exposure to UV light, ionizing radiation and heavy metal ions. The mitochondrial electron transport chain generates superoxide radicals through the single-electron leak at respiratory complexes I and III of the oxidative phosphorylation pathway. The flavin-dependent enzymes in the mitochondrial matrix also produce a considerable amount of reactive oxygen species.

The superoxide radical is readily dismutated to hydrogen peroxide. The reactivity of hydrogen peroxide as a molecule is low but it can penetrate cell membranes and generate hydroxyl radical via the Fenton's reaction [18].

The first step involves the reduction of ferric to ferrous ion:

$$\text{Fe}^{3+} + \text{O}\_{\text{g}^{\text{-}}} \rightarrow \text{Fe}^{2+} + \text{O}\_2 \tag{1}$$

The second step is the Fenton reaction:

$$\text{Fe}^{2+} + \text{H}\_2\text{O}\_2 \rightarrow \text{Fe}^{3+} + \text{HO}^- + \text{HO}^\bullet \tag{2}$$

Haber and Weiss reaction:

$$\text{H}\text{O}^{\bullet} + \text{H}\_{2}\text{O}\_{2} \rightarrow \text{H}\_{2}\text{O} + \text{O}\_{2}^{\bullet^{-}} + \text{H}^{\*} \tag{3}$$

$$\text{O}\_2^{\bullet^-} + \text{H}\_2\text{O}\_2 \rightarrow \text{O}\_2 + \text{HO}^- + \text{HO}^\bullet \tag{4}$$

The chain termination reaction:

$$\text{Fe}^{2+} + \text{HO}^{\bullet} + \text{H}^{\bullet} \rightarrow \text{Fe}^{3+} + \text{H}\_2\text{O} \tag{5}$$

The hydroxyl radical is regarded as the most reactive oxygen radical and can cause oxidative damage to cells by attacking biomolecules located a few nanometers from the site of generation [19].

Low levels of ROS production are required for important physiological functions, including proliferation, host defense, signal transduction, and gene expression [20]. There is a cellular balance between ROS generation and clearance in eukaryotic cells. This is achieved by the activity of several antioxidative defense mechanisms that comprise enzymes and antioxidants. The five main types of primary intracellular antioxidant enzymes are Cu/Zn-superoxide dismutase (Cu/Zn-SOD, SOD1) in the cytosol, manganese superoxide dismutase (Mn-SOD, SOD2) in the mitochondrial matrix, catalase, glutathione peroxidase (GPx), and glutathione reductase (GR). Small molecular weight and nonenzymatic antioxidants are also involved in the protection of the intracellular

components against the reactive oxygen species. However, when cellular production of ROS overwhelms these antioxidative mechanisms, oxidative stress occurs [18].

The use of the term 'oxidative stress' became frequent in the 1970s, but its origin dates back to the 1950s when researchers were studying the toxic effects of ionizing radiation and free radicals. Oxidative stress refers to a pathological state that arises from an imbalance between the production of free radicals and the ability to neutralize them by antioxidants. When the antioxidant capacity is reduced, prooxidants can react with surrounding biomolecules and the extent of the reaction is dependent on the susceptibility of the biomolecules [20–22].

#### **2.1 Free radical reaction with biomolecules**

Biological molecules, notably DNA, proteins and lipids, can be affected by free radicals. The reaction of reactive oxygen species (ROS) with these macromolecules if not checked generates additional free radicals thereby causing more damage. The incorporation of modified bases into a growing DNA molecule has serious phenotypic consequences [23]. Mitochondrial DNA is mainly vulnerable, because of its closeness to the site of metabolic ROS generation [24]. Telomeres are also vulnerable to ROS attack. ROS-accelerated reduction in telomere length hasten cell senescence [25].

Oxidation of proteins induce the formation of irreversible disulphide bridges, changes in secondary and tertiary structure and ultimately impaired function. The degree of the damage depends on the location of the proteins, their composition and structure [26]. Some amino acids (tryptophan, tyrosine, histidine and cysteine) are more susceptible to oxidation than others [24].

Damage to lipids is also of great significance because of the negative impact on membrane structure and function. The composition of biological membranes is very important to the membrane function but also influence susceptibility to oxidative damage. Polyunsaturated fatty acids (PUFAs) are much more prone to peroxidation than monounsaturated or saturated fatty acid acids. Oxidation of lipids can generate a wide range of reactive intermediates which can trigger complex chain reactions with widespread effects.

#### *2.1.1 DNA oxidation*

Technological advancement in analytical chemistry have provided sensitive and specific methods for identifying and quantifying DNA adducts. Application of these techniques to the analysis of nuclear DNA from human tissues has made it clear that the notion "human genome is pristine if there is no exposure to environmental carcinogens" is incorrect. Much damage is done to DNA molecules endogenously by intermediates of oxygen reduction that either attack the nitrogenous bases or the deoxyribosyl backbone of DNA (**Figure 5**) [28].

Hydroxyl radical (HO• ) is a provable candidate in DNA oxidation because it is extremely reactive. Hydroxyl radicals cannot diffuse beyond two molecular diameters because of their high reactivity [29, 30]. It can add to DNA bases or abstract hydrogen atoms to produce DNA adducts in no specific order [28]. The effect on nuclear DNA can only be possible if H2O2 generate HO• on reaction with a metal ion in the vicinity of a DNA molecule [31, 32].

Peroxinitrite, a product of the coupling of nitric oxide and superoxide ion (O2−) has also been identified as an extremely strong DNA oxidant. Apart from its ability to generate HO• , its protonated form (peroxinitrous acid, ONOOH) is an extremely reactive oxidant [28].

**245**

diseases.

*2.1.2 Proteins*

**Figure 5.**

*Vitamin E in Human Health and Oxidative Stress Related Diseases*

Secondary products of lipid peroxidation reactions are also culpably involved in DNA oxidation. Malondialdehyde and 4-hydroxylnonenal can oxidize DNA thereby

When a H-atom is abstracted from C5' carbon atom in the sugar moiety, the C5′ centered radical generated binds to the C8-position of the purine base in the same nucleoside. The products of this intramolecular cyclization are 8,5'-cyclopurine-2′ deoxynucleosides (**Figure 5**). The reactions of carbon-centered sugar radicals result

of the purine base which can be converted to the 2,6-diamino-4-hydroxy-5-formamidopyrimidine by reduction that ultimately lead to ring opening. The oxidation of

with the heterocyclic part of the pyrimidine bases to yield several base adducts. For

yields C5–OH and C6–OH adducts respectively. Further oxidation of these adducts by water and concomitant deprotonation results in the formation of the respective glycols.

The interest in the study of protein oxidation started in the early 1990s with the aim to explain oxidative damage to specific purified enzymes or in oxidative stress processes involving proteins. The process of protein oxidation by free radicals is an important biochemical event in living cells and is implicated in a number of human

the C-8-hydroxy-adduct radical of purines yield 8-hydroxypurine HO•

with purines in DNA produces a C-8-hydroxy-adduct radical

with cytosine and thymine at C5- and C6-positions,

can react also

*DOI: http://dx.doi.org/10.5772/intechopen.99169*

generating several DNA adducts [30].

*2.1.1.1 Mechanism of DNA oxidation*

*Oxidation of deoxyribose in purines [27].*

in the DNA strand breaks [27]. The reaction of HO•

example, the reaction of HO•

*Vitamin E in Human Health and Oxidative Stress Related Diseases DOI: http://dx.doi.org/10.5772/intechopen.99169*

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

dependent on the susceptibility of the biomolecules [20–22].

ine) are more susceptible to oxidation than others [24].

bases or the deoxyribosyl backbone of DNA (**Figure 5**) [28].

nuclear DNA can only be possible if H2O2 generate HO•

in the vicinity of a DNA molecule [31, 32].

chain reactions with widespread effects.

**2.1 Free radical reaction with biomolecules**

occurs [18].

senescence [25].

*2.1.1 DNA oxidation*

Hydroxyl radical (HO•

components against the reactive oxygen species. However, when cellular production of ROS overwhelms these antioxidative mechanisms, oxidative stress

dates back to the 1950s when researchers were studying the toxic effects of ionizing radiation and free radicals. Oxidative stress refers to a pathological state that arises from an imbalance between the production of free radicals and the ability to neutralize them by antioxidants. When the antioxidant capacity is reduced, prooxidants can react with surrounding biomolecules and the extent of the reaction is

The use of the term 'oxidative stress' became frequent in the 1970s, but its origin

Biological molecules, notably DNA, proteins and lipids, can be affected by free radicals. The reaction of reactive oxygen species (ROS) with these macromolecules if not checked generates additional free radicals thereby causing more damage. The incorporation of modified bases into a growing DNA molecule has serious phenotypic consequences [23]. Mitochondrial DNA is mainly vulnerable, because of its closeness to the site of metabolic ROS generation [24]. Telomeres are also vulnerable to ROS attack. ROS-accelerated reduction in telomere length hasten cell

Oxidation of proteins induce the formation of irreversible disulphide bridges, changes in secondary and tertiary structure and ultimately impaired function. The degree of the damage depends on the location of the proteins, their composition and structure [26]. Some amino acids (tryptophan, tyrosine, histidine and cyste-

Damage to lipids is also of great significance because of the negative impact on membrane structure and function. The composition of biological membranes is very important to the membrane function but also influence susceptibility to oxidative damage. Polyunsaturated fatty acids (PUFAs) are much more prone to peroxidation than monounsaturated or saturated fatty acid acids. Oxidation of lipids can generate a wide range of reactive intermediates which can trigger complex

Technological advancement in analytical chemistry have provided sensitive and specific methods for identifying and quantifying DNA adducts. Application of these techniques to the analysis of nuclear DNA from human tissues has made it clear that the notion "human genome is pristine if there is no exposure to environmental carcinogens" is incorrect. Much damage is done to DNA molecules endogenously by intermediates of oxygen reduction that either attack the nitrogenous

extremely reactive. Hydroxyl radicals cannot diffuse beyond two molecular diameters because of their high reactivity [29, 30]. It can add to DNA bases or abstract hydrogen atoms to produce DNA adducts in no specific order [28]. The effect on

Peroxinitrite, a product of the coupling of nitric oxide and superoxide ion (O2−) has also been identified as an extremely strong DNA oxidant. Apart from its ability

, its protonated form (peroxinitrous acid, ONOOH) is an extremely

) is a provable candidate in DNA oxidation because it is

on reaction with a metal ion

**244**

to generate HO•

reactive oxidant [28].

**Figure 5.** *Oxidation of deoxyribose in purines [27].*

Secondary products of lipid peroxidation reactions are also culpably involved in DNA oxidation. Malondialdehyde and 4-hydroxylnonenal can oxidize DNA thereby generating several DNA adducts [30].

#### *2.1.1.1 Mechanism of DNA oxidation*

When a H-atom is abstracted from C5' carbon atom in the sugar moiety, the C5′ centered radical generated binds to the C8-position of the purine base in the same nucleoside. The products of this intramolecular cyclization are 8,5'-cyclopurine-2′ deoxynucleosides (**Figure 5**). The reactions of carbon-centered sugar radicals result in the DNA strand breaks [27].

The reaction of HO• with purines in DNA produces a C-8-hydroxy-adduct radical of the purine base which can be converted to the 2,6-diamino-4-hydroxy-5-formamidopyrimidine by reduction that ultimately lead to ring opening. The oxidation of the C-8-hydroxy-adduct radical of purines yield 8-hydroxypurine HO• can react also with the heterocyclic part of the pyrimidine bases to yield several base adducts. For example, the reaction of HO• with cytosine and thymine at C5- and C6-positions, yields C5–OH and C6–OH adducts respectively. Further oxidation of these adducts by water and concomitant deprotonation results in the formation of the respective glycols.

#### *2.1.2 Proteins*

The interest in the study of protein oxidation started in the early 1990s with the aim to explain oxidative damage to specific purified enzymes or in oxidative stress processes involving proteins. The process of protein oxidation by free radicals is an important biochemical event in living cells and is implicated in a number of human diseases.

#### *2.1.2.1 Oxidation of the protein backbone*

The removal of one hydrogen atom from an amino acid residue in a polypeptide molecule by a free radical generates a carbon-centered radical. The reaction of the carbon-centered radical with O2 forms an alkylperoxyl radical intermediate which can be converted to an alkylperoxide. The resulting alkoxyl radical may be converted to a hydroxyl protein. Steps in this pathway are mediated by interactions with HO2 • and are catalyzed by Fe2+. The radical intermediates generated can further react with other amino acid residues within the same or in different protein molecules thereby generating a new carbon-centered radical that can undergo similar reactions. However, when oxygen is absent, two carboncentered radicals may react with each other to form a protein–protein cross-link (**Figure 6**) [33].

Peptide bond cleavage occurs by either the diamide or α-amidation pathways. ROS attack on glutamyl, aspartyl and prolyl side chains of polypeptide residues lead to peptide bond cleavage via a different pathway. Oxalic acid is formed and the N-terminal amino acid of the peptide derived from the C-terminal portion of the protein will exist as an N-pyruvyl derivative. The involvement of proline oxidation is linked to the observation that the number of peptides formed during radiolysis of proteins is approximately equal to the number of prolyl residues. It was thus proposed that oxidation of prolyl residues would lead to peptide bond cleavage. This was verified by Uchida et al. [34] who showed that the oxidation of proline residues leads to the formation of 2-pyrrolidone and concomitant peptide bond cleavage. The hydrolysis of 2-pyrrolidone by an acid yields 4-aminobutyric acid. The observation of 4-aminobutyric acid in protein hydrolysates serve as reasonable evidence of the involvement of proline oxidation in peptide bond cleavage.

#### *2.1.3 Lipid peroxidation*

The adverse effects of lipid peroxidation in biological systems gained attention in the 1960's and it is now known that this reaction is a relevant event in biology and medicine [35].

Lipid peroxidation is a chain reaction, catalyzed by transition metals ultimately resulting in the breakdown of membrane phospholipids that contain polyunsaturated fatty acids (PUFAs). The severity of the resulting damage depends on the nature and concentration of the oxidant and it may range from reductions in membrane fluidity to full disruption of bilayer integrity [30]. The two most dominant ROS that can affect lipids profoundly are; hydroxyl radical (• OH) and hydroperoxyl radical (HO2•), a protonated form of O2 − . The hydroxyl radical (• OH) attack biomolecules located a few nanometers from the site of generation [36]. H2O2 generated from HO2• can react with redox active metals to generate • OH through Fenton or Haber-Weiss reactions. The HO2• can also directly initiate the oxidation of polyunsaturated phospholipids in cell membrane [37].

**247**

**Figure 7.**

*Lipid peroxidation process [30].*

*Vitamin E in Human Health and Oxidative Stress Related Diseases*

Lipid peroxidation reactions involve the abstraction of hydrogen from a carbon in a lipid molecule followed by the insertion of oxygen to form lipid peroxyl radicals and hydroperoxides. Glycolipids, phospholipids, and cholesterol are susceptible to these damaging and possibly lethal peroxidative alterations. The enzymes; lipoxy-

Lipid peroxidation reactions are categorized into three phases: initiation, propagation, and termination. In the initiation step, pro-oxidants like hydroxyl radical removes an allylic hydrogen forming a carbon-centered lipid radical (L•). During the propagation phase, the lipid radical (L•) rapidly reacts with oxygen to form a lipid peroxyl radical (LOO•). The LOO• can react with neighboring lipid molecules

The reaction process can be terminated by antioxidants that donate hydrogen atom(s) to the lipid peroxyl radical species resulting in the formation of non-radical products. For example, vitamin E donate hydrogen atom to the LOO• species. The resulting 'oxidized' vitamin E radical reacts with another LOO• forming non-radical

Lipid peroxidation produces a number of oxidation products categorized as primary and secondary products. Lipid hydroperoxides (LOOH) are the main primary products of lipid peroxidation. Several aldehydes are formed as secondary products from the hydroperoxides including; malondialdehyde (MDA), propanal, hexanal, and 4-hydroxynonenal (4-HNE) [39, 40]. 4-HNE and MDA have been reported to be the most toxic and most mutagenic product of lipid peroxidation

The decomposition of arachidonic acid (AA) and larger PUFAs as well as enzymatic processes during the biosynthesis of thromboxane A2 (TXA2) and 12-l-hydroxy-5,8,10-heptadecatrienoic acid (HHT), or the non-enzymatic processes

products. The chain reaction continues in the absence of antioxidants [38].

genase, cyclooxygenase and cytochrome P450 can also oxidize lipids.

form a new L• and lipid hydroperoxide (LOOH) (**Figure 7**).

*DOI: http://dx.doi.org/10.5772/intechopen.99169*

*2.1.3.1 Lipid peroxidation process*

*2.1.3.2 Lipid peroxidation products*

respectively [41].

**Figure 6.** *Formation of carbon-centered radicals during protein oxidation [33].*

#### *2.1.3.1 Lipid peroxidation process*

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

The removal of one hydrogen atom from an amino acid residue in a polypeptide molecule by a free radical generates a carbon-centered radical. The reaction of the carbon-centered radical with O2 forms an alkylperoxyl radical intermediate which can be converted to an alkylperoxide. The resulting alkoxyl radical may be converted to a hydroxyl protein. Steps in this pathway are mediated

generated can further react with other amino acid residues within the same or in different protein molecules thereby generating a new carbon-centered radical that can undergo similar reactions. However, when oxygen is absent, two carboncentered radicals may react with each other to form a protein–protein cross-link

Peptide bond cleavage occurs by either the diamide or α-amidation pathways. ROS attack on glutamyl, aspartyl and prolyl side chains of polypeptide residues lead to peptide bond cleavage via a different pathway. Oxalic acid is formed and the N-terminal amino acid of the peptide derived from the C-terminal portion of the protein will exist as an N-pyruvyl derivative. The involvement of proline oxidation is linked to the observation that the number of peptides formed during radiolysis of proteins is approximately equal to the number of prolyl residues. It was thus proposed that oxidation of prolyl residues would lead to peptide bond cleavage. This was verified by Uchida et al. [34] who showed that the oxidation of proline residues leads to the formation of 2-pyrrolidone and concomitant peptide bond cleavage. The hydrolysis of 2-pyrrolidone by an acid yields 4-aminobutyric acid. The observation of 4-aminobutyric acid in protein hydrolysates serve as reasonable evidence of the involvement of proline oxidation in peptide bond

The adverse effects of lipid peroxidation in biological systems gained attention in the 1960's and it is now known that this reaction is a relevant event in biology and

Lipid peroxidation is a chain reaction, catalyzed by transition metals ultimately

attack biomolecules located a few nanometers from the site of generation [36]. H2O2

Fenton or Haber-Weiss reactions. The HO2• can also directly initiate the oxidation

−

OH) and

OH through

OH)

. The hydroxyl radical (•

resulting in the breakdown of membrane phospholipids that contain polyunsaturated fatty acids (PUFAs). The severity of the resulting damage depends on the nature and concentration of the oxidant and it may range from reductions in membrane fluidity to full disruption of bilayer integrity [30]. The two most dominant ROS that can affect lipids profoundly are; hydroxyl radical (•

hydroperoxyl radical (HO2•), a protonated form of O2

of polyunsaturated phospholipids in cell membrane [37].

*Formation of carbon-centered radicals during protein oxidation [33].*

generated from HO2• can react with redox active metals to generate •

and are catalyzed by Fe2+. The radical intermediates

*2.1.2.1 Oxidation of the protein backbone*

•

by interactions with HO2

(**Figure 6**) [33].

cleavage.

medicine [35].

*2.1.3 Lipid peroxidation*

**246**

**Figure 6.**

Lipid peroxidation reactions involve the abstraction of hydrogen from a carbon in a lipid molecule followed by the insertion of oxygen to form lipid peroxyl radicals and hydroperoxides. Glycolipids, phospholipids, and cholesterol are susceptible to these damaging and possibly lethal peroxidative alterations. The enzymes; lipoxygenase, cyclooxygenase and cytochrome P450 can also oxidize lipids.

Lipid peroxidation reactions are categorized into three phases: initiation, propagation, and termination. In the initiation step, pro-oxidants like hydroxyl radical removes an allylic hydrogen forming a carbon-centered lipid radical (L•). During the propagation phase, the lipid radical (L•) rapidly reacts with oxygen to form a lipid peroxyl radical (LOO•). The LOO• can react with neighboring lipid molecules form a new L• and lipid hydroperoxide (LOOH) (**Figure 7**).

The reaction process can be terminated by antioxidants that donate hydrogen atom(s) to the lipid peroxyl radical species resulting in the formation of non-radical products. For example, vitamin E donate hydrogen atom to the LOO• species. The resulting 'oxidized' vitamin E radical reacts with another LOO• forming non-radical products. The chain reaction continues in the absence of antioxidants [38].

#### *2.1.3.2 Lipid peroxidation products*

Lipid peroxidation produces a number of oxidation products categorized as primary and secondary products. Lipid hydroperoxides (LOOH) are the main primary products of lipid peroxidation. Several aldehydes are formed as secondary products from the hydroperoxides including; malondialdehyde (MDA), propanal, hexanal, and 4-hydroxynonenal (4-HNE) [39, 40]. 4-HNE and MDA have been reported to be the most toxic and most mutagenic product of lipid peroxidation respectively [41].

The decomposition of arachidonic acid (AA) and larger PUFAs as well as enzymatic processes during the biosynthesis of thromboxane A2 (TXA2) and 12-l-hydroxy-5,8,10-heptadecatrienoic acid (HHT), or the non-enzymatic processes

**Figure 7.** *Lipid peroxidation process [30].* by bicyclic endoperoxides produced during lipid peroxidation, can generate MDA in vivo. MDA generated by these processes can be enzymatically metabolized or form adducts with biomolecules [30].

#### **2.2 Oxidative stress and human diseases**

Oxidative stress has been implicated in the onset and progression of pathological conditions such as cancer, cardiovascular disease, neurological disorders, diabetes as well as aging [42–44]. The negative impact of oxidative stress is due to the damaging consequences of free radicals on important biological molecules [45]. Free radical mediated oxidative stress increases with age and may overwhelm natural repair systems [46]. A review of the mechanism of human diseases resulting from oxidative stress was published by Rahman [47]. The influence of oxidative stress on aging is now established and the associated diseases include; cardiovascular diseases, Huntington's disease, Alzheimer's disease, stroke, Parkinson's disease and cancer [47, 48].

The use of oxidative stress biomarkers for the diagnosis of acute and chronic diseases indicate the involvement of oxidative stress in such pathological conditions. This is confirmed by the difference in the concentration of these biomarkers in healthy and ill subjects evaluated for long periods. Representative biomarkers of oxidative damage associated with some human diseases were summarized by Valko et al. [20] and are presented in **Table 1**.

#### **3. Roles of vitamin E in human health and disease**

Natural antioxidants including vitamin E have gained relevance in combating oxidative stress [49]. The vitamin E required by humans is solely acquired from diet. The lipohilic nature of vitamin E support the theory of high antioxidant capacity in cell membranes [47]. The capacity to be replenished by other antioxidants such as ascorbic acid is another important factor [9, 10]. Vitamin E has been reported to slow down the progression of oxidative assaults on biomolecules thus suppressing diseases [13]. The remaining sections of this chapter shall focus on the roles of vitamin E in the management of some illnesses and human wellbeing.


*Abbreviations: MDA, malondialdehyde; HNE, 4-hydroxy-2-nonenal; AGE, advanced glycation end products; 8-OH-dG, 8-hydroxy-20-deoxyguanosine; GSH, reduced glutathione; GSSG, oxidized glutathione; NO2-Tyr, 3-nitro-tyrosine.*

**249**

*Vitamin E in Human Health and Oxidative Stress Related Diseases*

react with another tocopheroxyl radical to form stable products [50].

**3.2 The role of vitamin E in cardiovascular disease**

(CHF), vascular injury and organ dysfunction [55, 56].

aggregation, increased NO production and arterial dilation.

prophylactic action. The vitamin E family have been reported to regulate cell growth and induce apoptosis in tumor cells. Several other anticarcinogenic mechanisms have been reported for the vitamin E family including the stimulation of the migration of macrophages and lymphocytes that contain tumor necrosis factor to tumor sites [51] and modulation of the expression of oncogenes. Additional studies have revealed that vitamin E succinate, a modified product can specifically induce apoptosis in tumor and cancer cells but not normal epithelial cells in mammary and

The hypothesis that atherosclerosis may be prevented by blocking the oxidative modification of LDL cholesterol, a key process in the onset and progression of atherosclerosis renewed interest in vitamin E. Research outcomes have documented beneficial effects of vitamin E on several stages of the atherosclerotic process [53].

The development of atherosclerosis depends on the pro- and anti-inflammatory as well as pro- and anti-oxidant balance [54]. The oxidation of LDL is a principal component of atherosclerosis and is implicated in the onset of cardiovascular diseases via several mechanisms. At low concentrations, oxidized LDL can stimulate the production of inflammatory markers such as cell adhesion molecules and macrophage colony stimulating factor by endothelial cells. The resulting endothelial dysfunction can result in either cell growth or apoptotic cell death that can cause

At high concentrations, oxidized LDL are recognized by scavenger receptors and they are phagocytosed by macrophages resulting in the formation of lipid-laden foam cells. The cytotoxic property of oxidized LDL in cultured endothelial cells, the ability to inhibit macrophage motility and the inhibitory effect on nitric oxideinduced vasodilation are other potential atherogenic possibilities. Experimental evidence also back the involvement of free radicals in congestive heart failure

The vitamin E family has received considerable attention in atherosclerosis research based on the capacity to inhibit LDL oxidation and decrease uptake of oxidized LDL by macrophages in human arterial lesions. The vitamin E compounds are reported to be favorable modulators of the atherogenic process at the molecular and cellular levels [57, 58]. Other potential mechanisms of action include; reduction of endothelial injury, reduction in the expression of adhesion molecules, reduction in endothelial cell adhesion, inhibition of inflammatory cytokines and chemokines synthesis, inhibition of smooth muscle cell proliferation, inhibition of platelet

An in vitro study on the effect of vitamin E on LDL oxidation in the blood plasma of healthy volunteers revealed that enrichment with vitamin E increased resistance to LDL-oxidation in a dose-dependent manner [59] and decreased uptake

Epidemiological studies and observational surveys link populations that consume a high amount of vitamin E to a reduced incidence of chronic diseases. This disease preventing capacity is largely linked to the antioxidant capacity of the vitamin E family. The antioxidant property is based on the capacity of vitamin E to donate hydrogen to free radicals and its lipid membrane solubility. The resulting tocopheroxyl radical is far less reactive compared to free radicals so does not propagate the oxidative chain reaction. The tocopheroxyl radical can be reduced by ascorbic acid or

Vitamin E is an immune booster which may play an essential role in the observed

*DOI: http://dx.doi.org/10.5772/intechopen.99169*

**3.1 Vitamin E in disease prophylaxis**

prostatic glands [50, 52].

vasoconstriction.

#### **Table 1.**

*Oxidative stress biomarkers associated with some human diseases [20].*

#### **3.1 Vitamin E in disease prophylaxis**

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

adducts with biomolecules [30].

cancer [47, 48].

**2.2 Oxidative stress and human diseases**

et al. [20] and are presented in **Table 1**.

**3. Roles of vitamin E in human health and disease**

by bicyclic endoperoxides produced during lipid peroxidation, can generate MDA in vivo. MDA generated by these processes can be enzymatically metabolized or form

Oxidative stress has been implicated in the onset and progression of pathological conditions such as cancer, cardiovascular disease, neurological disorders, diabetes as well as aging [42–44]. The negative impact of oxidative stress is due to the damaging consequences of free radicals on important biological molecules [45]. Free radical mediated oxidative stress increases with age and may overwhelm natural repair systems [46]. A review of the mechanism of human diseases resulting from oxidative stress was published by Rahman [47]. The influence of oxidative stress on aging is now established and the associated diseases include; cardiovascular diseases, Huntington's disease, Alzheimer's disease, stroke, Parkinson's disease and

The use of oxidative stress biomarkers for the diagnosis of acute and chronic diseases indicate the involvement of oxidative stress in such pathological conditions. This is confirmed by the difference in the concentration of these biomarkers in healthy and ill subjects evaluated for long periods. Representative biomarkers of oxidative damage associated with some human diseases were summarized by Valko

Natural antioxidants including vitamin E have gained relevance in combating oxidative stress [49]. The vitamin E required by humans is solely acquired from diet. The lipohilic nature of vitamin E support the theory of high antioxidant capacity in cell membranes [47]. The capacity to be replenished by other antioxidants such as ascorbic acid is another important factor [9, 10]. Vitamin E has been reported to slow down the progression of oxidative assaults on biomolecules thus suppressing diseases [13]. The remaining sections of this chapter shall focus on the roles of vitamin E in the management of some illnesses and human wellbeing.

**Oxidation products used as biomarkers for oxidative stress Disease** MDA, GSH/GSSG ratio, NO2-Tyr, 8-OH-dG Cancer

HNE, GSH/GSSG ratio, acrolein, NO2-Tyr, F2-isoprostanes Cardiovascular disease F2-isoprostanes, GSH/GSSG ratio Rheumatoid arthritis MDA, HNE, GSH/GSSG ratio, F2-isoprostanes, NO2-Tyr, AGE Alzheimer's disease HNE, GSH/GSSG ratio, carbonylated proteins, Fe-level Parkinson's disease F2-isoprostanes, GSH/GSSG ratio Ischemia and reperfusion

MDA, HNE, Acrolein, NO2-Tyr, F2-isoprostanes Atherosclerosis

*Abbreviations: MDA, malondialdehyde; HNE, 4-hydroxy-2-nonenal; AGE, advanced glycation end products; 8-OH-dG, 8-hydroxy-20-deoxyguanosine; GSH, reduced glutathione; GSSG, oxidized glutathione; NO2-Tyr,* 

Diabetes mellitus

MDA, GSH/GSSG ratio, F2-isoprostanes, NO2-Tyr, AGE, S-gluathionylated

*Oxidative stress biomarkers associated with some human diseases [20].*

**248**

**Table 1.**

proteins

*3-nitro-tyrosine.*

Epidemiological studies and observational surveys link populations that consume a high amount of vitamin E to a reduced incidence of chronic diseases. This disease preventing capacity is largely linked to the antioxidant capacity of the vitamin E family. The antioxidant property is based on the capacity of vitamin E to donate hydrogen to free radicals and its lipid membrane solubility. The resulting tocopheroxyl radical is far less reactive compared to free radicals so does not propagate the oxidative chain reaction. The tocopheroxyl radical can be reduced by ascorbic acid or react with another tocopheroxyl radical to form stable products [50].

Vitamin E is an immune booster which may play an essential role in the observed prophylactic action. The vitamin E family have been reported to regulate cell growth and induce apoptosis in tumor cells. Several other anticarcinogenic mechanisms have been reported for the vitamin E family including the stimulation of the migration of macrophages and lymphocytes that contain tumor necrosis factor to tumor sites [51] and modulation of the expression of oncogenes. Additional studies have revealed that vitamin E succinate, a modified product can specifically induce apoptosis in tumor and cancer cells but not normal epithelial cells in mammary and prostatic glands [50, 52].

The hypothesis that atherosclerosis may be prevented by blocking the oxidative modification of LDL cholesterol, a key process in the onset and progression of atherosclerosis renewed interest in vitamin E. Research outcomes have documented beneficial effects of vitamin E on several stages of the atherosclerotic process [53].

#### **3.2 The role of vitamin E in cardiovascular disease**

The development of atherosclerosis depends on the pro- and anti-inflammatory as well as pro- and anti-oxidant balance [54]. The oxidation of LDL is a principal component of atherosclerosis and is implicated in the onset of cardiovascular diseases via several mechanisms. At low concentrations, oxidized LDL can stimulate the production of inflammatory markers such as cell adhesion molecules and macrophage colony stimulating factor by endothelial cells. The resulting endothelial dysfunction can result in either cell growth or apoptotic cell death that can cause vasoconstriction.

At high concentrations, oxidized LDL are recognized by scavenger receptors and they are phagocytosed by macrophages resulting in the formation of lipid-laden foam cells. The cytotoxic property of oxidized LDL in cultured endothelial cells, the ability to inhibit macrophage motility and the inhibitory effect on nitric oxideinduced vasodilation are other potential atherogenic possibilities. Experimental evidence also back the involvement of free radicals in congestive heart failure (CHF), vascular injury and organ dysfunction [55, 56].

The vitamin E family has received considerable attention in atherosclerosis research based on the capacity to inhibit LDL oxidation and decrease uptake of oxidized LDL by macrophages in human arterial lesions. The vitamin E compounds are reported to be favorable modulators of the atherogenic process at the molecular and cellular levels [57, 58]. Other potential mechanisms of action include; reduction of endothelial injury, reduction in the expression of adhesion molecules, reduction in endothelial cell adhesion, inhibition of inflammatory cytokines and chemokines synthesis, inhibition of smooth muscle cell proliferation, inhibition of platelet aggregation, increased NO production and arterial dilation.

An in vitro study on the effect of vitamin E on LDL oxidation in the blood plasma of healthy volunteers revealed that enrichment with vitamin E increased resistance to LDL-oxidation in a dose-dependent manner [59] and decreased uptake of oxidized LDL by macrophages in human arterial lesions. In another study, vitamin E enrichment increased LDL vitamin E concentration by approximately 2.5 folds and the susceptibility to oxidation was reduced by 30–40% [60].

The results obtained from in vitro studies have not been replicated exactly in clinical trials.

#### **3.3 The role of vitamin E in cancer**

Cancer is a complicated disease condition characterized by the inability to control cell growth. Carcinogenesis has been categorized into three stages; initiation, promotion, progression and the action of free radicals have been implicated in all stages due to their capacity to react with all components of DNA. The concentration of oxidized DNA adducts is directly linked to the size of benign tumors and can directly affect the transformation to malignancy [47].

The vitamin E compounds are powerful antioxidants thus can inhibit DNA oxidation. The natural forms of vitamin E have been reported as effective agents for cancer therapy [61]. The result of the selenium and vitamin E cancer prevention trial (SELECT) revealed that dietary supplementation of α-tocopherol at 400 IU/d increased the risk of prostate cancer [62]. The anticancer properties of α-tocopherol have been studied the most among the vitamin E compounds and available results reveal that the anticancer property of the compound is not very promising.

However, the vitamin E forms; γ-tocopherol, δ-tocopherol, γ-tocotrienol and δ-tocotrienol have been reported to have higher anticancer property compared to α-tocopherol. These vitamin E forms are able to inhibit multiple cancer promoting pathways by inhibiting the formation of eicosanoids. In conjunction with their metabolic product; 13′-carboxycromanol, they inhibit the cyclooxygenases (COX-1 and -2) and 5-lipoxgenase (5-LOX). Gamma- and δ-tocotrienols also suppress the activation of *nuclear factor kappa B* (NF-kB) and signal transducer and activator of transcription factor 3 (STAT3). These activities neutralize pro-inflammatory tumor microenvironments that favor cancer development, invasiveness, and resistance to treatment. These vitamin E compounds also target cancer cells and cancer stem cells by promoting apoptosis, antiangiogenesis, and antiproliferation partially via modulating epigenetic events and other signaling pathways. The modulatory effect of tocotrienols on immunity may also contribute to cancer prevention [61].

The anti-inflammatory mechanism of vitamin E compounds and their metabolites is based on their capacity to inhibit the cycloxogenases and the lipoxygenase involved in eicosanoid synthesis. Reduced synthesis of prostaglandins and leukotrienes have been reported to slow down tumorigenesis, angiogenesis and metastasis. The activity of COX-2 and 5-LOX as well as the concentration of Prostaglandin E2 (PGE2) are increased in tumor cells. These events promote angiogenesis and resistance to apoptosis via PGE2 receptor-mediated signaling in cancer cells [61, 63, 64].

The non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit inflammation via the inhibition of COX and 5-LOX have been shown to inhibit tumor development in various cancer models [65, 66]. Studies have revealed that γ-tocopherol, δ-tocopherol and γ-tocotrienol as well as their metabolites can inhibit COX- and 5-LOX at physiological concentrations [61].

Pro-inflammatory cytokines secreted by macrophages associated with tumor and cancer cells also promote tumor growth and invasiveness. The cytokines (interleukin-1 (IL-1), interleukin-6 (IL-6)) and *tumor necrosis factor alpha* (TNF-α), activate NF-kB and STAT3 in cancer cells. The activation of NF-kB and STAT3 increase the expression of genes that promote cell survival, proliferation, angiogenesis and

**251**

*Vitamin E in Human Health and Oxidative Stress Related Diseases*

invasiveness [67]. The inhibition of NF-kB and STAT3 as well as their regulatory cytokines is a potent target to suppress tumor development and progression.

Vitamin E compounds have been shown to block NF-kB and STAT3 activation and their regulated genes in macrophages and cancer cells [68]. The inability to express survival genes following the inhibition of NF-kB and STAT3 sensitizes the cancer cells to therapeutic drugs. γ- and δ-Tocotrienols have been reported to be

The immune system play an important role in the defense against cancer by detecting and killing tumor cells [68]. A combination of tocotrienols as supplements have been reported to modulate immune response and has a higher anticancer activity than α-tocopherol alone [70]. Tocotrienol supplementation enhanced lymphocyte proliferation without affecting major cytokines in old mice but not young ones, suggesting an age-dependent immune modulatory function [71]. The supplementation with the vitamin E compounds increase interferon gamma (IFN-γ) and interleukin 4 (IL-4), thus enhancing antibody production while suppressing TNF-α in stimulated splenocytes. This activity observed in response to tetanus toxoid vaccination suggest anticancer activity via

The vitamin E forms have been reported to directly target cancer cells. γ-Tocopherol, δ-tocopherol, γ-totrienol, δ-tocotrienol and 13′-carboxychromanol have been reported to induce the arrest of cancer cell growth, apoptosis and autophagy in several types of cancer cells [72]. The preferential accumulation of γ-totrienol, δ-tocotrienol and 13′-carboxychromanol in cancer cells may be responsible for the observed higher anticancer activity compared to the tocopherol

The capacity of these vitamin E forms to induce pathways associated with antiproliferation [74], elevation of mitochondria apoptotic proteins [73], autophagy marker LC3II and endoplasmic reticulum stress markers such as c-Jun N-Terminal kinase (JNK) phosphorylation and death receptor-5 (DR5) pro-apoptotic pathway [75] may contribute to the reported anticancer activity. The anticancer activity has also been linked with the capacity of the vitamin E forms and 13′-carboxylchromanol to modulate sphingolipid metabolism. At elevated concentrations, sphingolipids such as dihydroceramide, dihydroshpingosine and ceramides induce stress and apoptosis as well as inhibit cell growth [76]. This has been shown in prostate, colon, pancreatic and breast cancer cells were an elevation of the sphingolipids precede or happen simultaneously with cell death [74]. Suppressing de novo synthesis of sphingolipids reverses the anticancer activity of the vitamin E forms. Research is still ongoing to completely unravel the interactions and effect of sphingolipid

Cataracts are one of the commonest reasons for critical vision distress in adult humans. They essentially happen because of the aggregation of proteins oxidized by free radicals. A few observational examinations have uncovered a likely connection between vitamin E supplements and the danger of cataracts development. Leske et al. [77] reported that lens clarity was higher in individuals receiving

vitamin E supplements and those with higher plasma concentrations of the vitamin. In another investigation, vitamin E supplementation was related with reduced opacification of the lens. However, in a randomized Age-Related Eye Illness Study (AREDS), vitamin E had no clear impact [78]. Like in other disease conditions, the exact mechanism of the observed positive effects of vitamin in the reduction of

*DOI: http://dx.doi.org/10.5772/intechopen.99169*

active inhibitors of NF-kB and STAT3 [69].

immune modulation [68].

counterparts [73].

modulation in vivo [61].

**3.4 The role of vitamin E in cataracts**

cataract formation in vivo is in progress [79].

#### *Vitamin E in Human Health and Oxidative Stress Related Diseases DOI: http://dx.doi.org/10.5772/intechopen.99169*

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

clinical trials.

**3.3 The role of vitamin E in cancer**

to cancer prevention [61].

cancer cells [61, 63, 64].

5-LOX at physiological concentrations [61].

directly affect the transformation to malignancy [47].

of oxidized LDL by macrophages in human arterial lesions. In another study, vitamin E enrichment increased LDL vitamin E concentration by approximately 2.5

The results obtained from in vitro studies have not been replicated exactly in

Cancer is a complicated disease condition characterized by the inability to control cell growth. Carcinogenesis has been categorized into three stages; initiation, promotion, progression and the action of free radicals have been implicated in all stages due to their capacity to react with all components of DNA. The concentration of oxidized DNA adducts is directly linked to the size of benign tumors and can

The vitamin E compounds are powerful antioxidants thus can inhibit DNA oxidation. The natural forms of vitamin E have been reported as effective agents for cancer therapy [61]. The result of the selenium and vitamin E cancer prevention trial (SELECT) revealed that dietary supplementation of α-tocopherol at 400 IU/d increased the risk of prostate cancer [62]. The anticancer properties of α-tocopherol have been studied the most among the vitamin E compounds and available results reveal that the anticancer property of the compound is not very promising.

However, the vitamin E forms; γ-tocopherol, δ-tocopherol, γ-tocotrienol and δ-tocotrienol have been reported to have higher anticancer property compared to α-tocopherol. These vitamin E forms are able to inhibit multiple cancer promoting pathways by inhibiting the formation of eicosanoids. In conjunction with their metabolic product; 13′-carboxycromanol, they inhibit the cyclooxygenases (COX-1 and -2) and 5-lipoxgenase (5-LOX). Gamma- and δ-tocotrienols also suppress the activation of *nuclear factor kappa B* (NF-kB) and signal transducer and activator of transcription factor 3 (STAT3). These activities neutralize pro-inflammatory tumor microenvironments that favor cancer development, invasiveness, and resistance to treatment. These vitamin E compounds also target cancer cells and cancer stem cells by promoting apoptosis, antiangiogenesis, and antiproliferation partially via modulating epigenetic events and other signaling pathways. The modulatory effect of tocotrienols on immunity may also contribute

The anti-inflammatory mechanism of vitamin E compounds and their metabolites is based on their capacity to inhibit the cycloxogenases and the lipoxygenase involved in eicosanoid synthesis. Reduced synthesis of prostaglandins and leukotrienes have been reported to slow down tumorigenesis, angiogenesis and metastasis. The activity of COX-2 and 5-LOX as well as the concentration of Prostaglandin E2 (PGE2) are increased in tumor cells. These events promote angiogenesis and resistance to apoptosis via PGE2 receptor-mediated signaling in

The non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit inflammation via the inhibition of COX and 5-LOX have been shown to inhibit tumor development in various cancer models [65, 66]. Studies have revealed that γ-tocopherol, δ-tocopherol and γ-tocotrienol as well as their metabolites can inhibit COX- and

Pro-inflammatory cytokines secreted by macrophages associated with tumor and cancer cells also promote tumor growth and invasiveness. The cytokines (interleukin-1 (IL-1), interleukin-6 (IL-6)) and *tumor necrosis factor alpha* (TNF-α), activate NF-kB and STAT3 in cancer cells. The activation of NF-kB and STAT3 increase the expression of genes that promote cell survival, proliferation, angiogenesis and

folds and the susceptibility to oxidation was reduced by 30–40% [60].

**250**

invasiveness [67]. The inhibition of NF-kB and STAT3 as well as their regulatory cytokines is a potent target to suppress tumor development and progression.

Vitamin E compounds have been shown to block NF-kB and STAT3 activation and their regulated genes in macrophages and cancer cells [68]. The inability to express survival genes following the inhibition of NF-kB and STAT3 sensitizes the cancer cells to therapeutic drugs. γ- and δ-Tocotrienols have been reported to be active inhibitors of NF-kB and STAT3 [69].

The immune system play an important role in the defense against cancer by detecting and killing tumor cells [68]. A combination of tocotrienols as supplements have been reported to modulate immune response and has a higher anticancer activity than α-tocopherol alone [70]. Tocotrienol supplementation enhanced lymphocyte proliferation without affecting major cytokines in old mice but not young ones, suggesting an age-dependent immune modulatory function [71]. The supplementation with the vitamin E compounds increase interferon gamma (IFN-γ) and interleukin 4 (IL-4), thus enhancing antibody production while suppressing TNF-α in stimulated splenocytes. This activity observed in response to tetanus toxoid vaccination suggest anticancer activity via immune modulation [68].

The vitamin E forms have been reported to directly target cancer cells. γ-Tocopherol, δ-tocopherol, γ-totrienol, δ-tocotrienol and 13′-carboxychromanol have been reported to induce the arrest of cancer cell growth, apoptosis and autophagy in several types of cancer cells [72]. The preferential accumulation of γ-totrienol, δ-tocotrienol and 13′-carboxychromanol in cancer cells may be responsible for the observed higher anticancer activity compared to the tocopherol counterparts [73].

The capacity of these vitamin E forms to induce pathways associated with antiproliferation [74], elevation of mitochondria apoptotic proteins [73], autophagy marker LC3II and endoplasmic reticulum stress markers such as c-Jun N-Terminal kinase (JNK) phosphorylation and death receptor-5 (DR5) pro-apoptotic pathway [75] may contribute to the reported anticancer activity. The anticancer activity has also been linked with the capacity of the vitamin E forms and 13′-carboxylchromanol to modulate sphingolipid metabolism. At elevated concentrations, sphingolipids such as dihydroceramide, dihydroshpingosine and ceramides induce stress and apoptosis as well as inhibit cell growth [76]. This has been shown in prostate, colon, pancreatic and breast cancer cells were an elevation of the sphingolipids precede or happen simultaneously with cell death [74]. Suppressing de novo synthesis of sphingolipids reverses the anticancer activity of the vitamin E forms. Research is still ongoing to completely unravel the interactions and effect of sphingolipid modulation in vivo [61].

#### **3.4 The role of vitamin E in cataracts**

Cataracts are one of the commonest reasons for critical vision distress in adult humans. They essentially happen because of the aggregation of proteins oxidized by free radicals. A few observational examinations have uncovered a likely connection between vitamin E supplements and the danger of cataracts development. Leske et al. [77] reported that lens clarity was higher in individuals receiving vitamin E supplements and those with higher plasma concentrations of the vitamin. In another investigation, vitamin E supplementation was related with reduced opacification of the lens. However, in a randomized Age-Related Eye Illness Study (AREDS), vitamin E had no clear impact [78]. Like in other disease conditions, the exact mechanism of the observed positive effects of vitamin in the reduction of cataract formation in vivo is in progress [79].

#### **3.5 Roles of vitamin E in other diseases**

The role of vitamin E gas been studied in several other diseases conditions linked to oxidative stress. For example, stroke has been linked with free radical reactions arising from xanthine oxidase, cyclooxygenase and inflammation [80]. These free radical reactions can cause neuronal death [81]. The oxidative assault is increased by the biochemical processes associated with stroke.

Oxidative stress is also implicated in the onset and progression of the neurological disorders; Alzheimer's disease and Parkinson's disease. In both conditions, logarithmic age-dependent increase in the oxidation of proteins, lipids and DNA as well as decreased in vivo antioxidant activity has been reported [47]. In Alzheimer's disease, oxidation induce protein cross linking and aggregation of β-amyloid protein which in turn induce the oxidation of carbohydrate side chains of membrane lipids leading to neuronal membrane breakdown [82]. Oxidation of lipids also accompany the process and has been quantitatively assessed by increased concentration of 4-hydroxyl-2-nonenal-glutathione conjugates in the brain [83].

In diabetes, free radical-induced OS has been reported to play a significant role in the development of insulin resistance, β-cell dysfunction and impaired glucose tolerance. Hyperglycemia worsens the oxidative burden following the formation of advanced glycation end products (AGEs).

The biochemical importance of oxidative stress in the onset and progression of disease conditions underscores the relevance of vitamin E in prevention and management.

In HIV infection, it is not exactly clear if vitamin E supplementation has the same effect at all stages of the disease. However, high serum concentration of vitamins A and E have been reported to affect disease progression. Vitamin E concentrations higher than 23.5 μM reduced the risk of progression of HIV-1. Vitamin E has also been shown to normalize immune system parameters in murine acquired immunodeficiency syndrome as well as protect against bone marrow toxicity of azidothymidine. Protection against azidothymidine toxicity was confirmed in stage IV HIV patients on alpha-tocopherol supplementation. High doses have also been reported to restore delayed skin hypersensitivity, stimulate interleukin-2 production and T-helper (CD4 T-cell) proliferation [79].

#### **4. Discussion**

The in vitro antioxidant activity of vitamin E compounds are well documented. However in vivo studies on prevention and treatment of oxidative stress-related diseases have been disappointing. Till date, there is no approval for the clinical use of vitamin E as a drug even though vitamin E consumption has been reported to boost immunity, improve skin health and vision. Vitamin E remains a popular supplement and is generally regarded as safe by the FDA.

Vitamin E is the main lipid soluble antioxidant [79] in the cell membrane thus will always attract research attention considering the relevance of membrane lipid oxidation [84]. The bioactivity of vitamin E in the cell membrane may be a direct approach to limit oxidation of other biomolecules that can form conjugates with oxidized lipids.

Emerging research outputs are shedding light on the disparity between in vitro and in vivo antioxidant capacity of vitamin E [85]. In macrophages, the oxidation of lipids occur in the lysosomes. Alboaklah and Leake [85] conducted an experiment on LDL oxidation at lysosomal pH (about 4.5). In their experiment, LDL enriched with vitamin E was oxidized by Cu2+ more slowly compared to control LDL. At

**253**

in vivo.

*Vitamin E in Human Health and Oxidative Stress Related Diseases*

pH 4.5, the enriched LDL was not protected against oxidation by low concentrations of Cu2+ or Fe3+. They observed that the enriched LDL reduced the Cu2+ and Fe3+ to

pH. This may partly be responsible for the observed reduction in the bioactivity of

The poor water solubility of vitamin E has been implicated in the low oral bioavailability [87]. Recently, nanoformulations such as nanovesicles, solid-lipid nanoparticles, nanostructured lipid carriers, nanoemulsions, and polymeric nanoparticles have shown promising outcomes in improving the efficacy and

Lipid-based nanovesicles such as niosomes and liposomes are highly promising for the delivery of lipophilic drugs and active compounds. Although niosomes and liposomes have similar physicochemical properties, niosomes have a higher permeability to small solutes and ions than liposomes. The application of these nanovesicles

In a study to enhance the tumor-suppressing effect of tocotrienols in vivo, Fu et al. [88] first developed a D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)-based noisome [88, 89]. This system appeared to significantly increase tocotrienol uptake in vitro using A431, B16F10 and T98G cell lines and hence improved the therapeutic efficacy. TPGS can be functionalized as an excellent solubilizer, emulsifier, permeation and bioavailability enhancer for hydrophobic drugs [90]. TPGS has demonstrated capacity to selectively induce apoptogenic activity against many cancer types by targeting the activation of mitochondrial mediators of apoptosis [91]. Another 6-*O*-palmitoyl-ascorbic acid (PA) based niosomes (comprising AP, TPGS, cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG (2000)-carboxylic acid)) was developed by Tan et al. [92] targeting transferrin receptors for intravenous administration of γ-tocotrienol aimed at treating breast cancer. Both in vitro and in vivo studies have proven that tumor-targeted niosomes significantly improve the therapeutic efficacy of γ-tocotrienol. These studies suggests that nanovesicles can be suitable carriers for improved delivery and enhanced

Although the antioxidant and antiproliferative properties of vitamin E against oxidative stress related diseases have been reported, there is currently no approval for clinical application. A modified product, TPGS has been approved by the FDA as a safe pharmaceutical adjuvant with high biocompatibility. Despite the reported bioactivity of the tocotrienols, studies so far are inconclusive. The reported in vitro biochemical properties of the vitamin E family will continually call the attention of researchers since they are natural and can play essential roles in ameliorating the impact of oxidative assault in biological membranes. To maximize the prophylactic and curative properties of vitamin E, further research on absorption, cellular uptake, solubility, and stability is required to improve bioavailabilty and efficacy

as drug delivery vehicles is suitable because they are non-toxic and stable.

The bioavailability of vitamin E has been another complication in clinical applications. Recent data indicate that the absorption of vitamin E is far more complex than previously thought. Details about the digestion, absorption and transport of vitamin E are presented in a review by Reboul [86]. The author concluded that the process is only partly understood and suggested further studies to decipher the

and Fe2+ at a faster rate than control LDL at lysosomal

*DOI: http://dx.doi.org/10.5772/intechopen.99169*

the more pro-oxidant Cu+

vitamin E compounds in vivo.

molecular mechanisms [86].

bioavailability of vitamin E.

efficacy of vitamin E [93].

**5. Conclusion**

#### *Vitamin E in Human Health and Oxidative Stress Related Diseases DOI: http://dx.doi.org/10.5772/intechopen.99169*

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

The role of vitamin E gas been studied in several other diseases conditions linked to oxidative stress. For example, stroke has been linked with free radical reactions arising from xanthine oxidase, cyclooxygenase and inflammation [80]. These free radical reactions can cause neuronal death [81]. The oxidative assault is increased

Oxidative stress is also implicated in the onset and progression of the neurological disorders; Alzheimer's disease and Parkinson's disease. In both conditions, logarithmic age-dependent increase in the oxidation of proteins, lipids and DNA as well as decreased in vivo antioxidant activity has been reported [47]. In Alzheimer's disease, oxidation induce protein cross linking and aggregation of β-amyloid protein which in turn induce the oxidation of carbohydrate side chains of membrane lipids leading to neuronal membrane breakdown [82]. Oxidation of lipids also accompany the process and has been quantitatively assessed by increased concen-

In diabetes, free radical-induced OS has been reported to play a significant role in the development of insulin resistance, β-cell dysfunction and impaired glucose tolerance. Hyperglycemia worsens the oxidative burden following the formation of

The biochemical importance of oxidative stress in the onset and progression of disease conditions underscores the relevance of vitamin E in prevention and

In HIV infection, it is not exactly clear if vitamin E supplementation has the same effect at all stages of the disease. However, high serum concentration of vitamins A and E have been reported to affect disease progression. Vitamin E concentrations higher than 23.5 μM reduced the risk of progression of HIV-1. Vitamin E has also been shown to normalize immune system parameters in murine acquired immunodeficiency syndrome as well as protect against bone marrow toxicity of azidothymidine. Protection against azidothymidine toxicity was confirmed in stage IV HIV patients on alpha-tocopherol supplementation. High doses have also been reported to restore delayed skin hypersensitivity, stimulate interleukin-2 produc-

The in vitro antioxidant activity of vitamin E compounds are well documented. However in vivo studies on prevention and treatment of oxidative stress-related diseases have been disappointing. Till date, there is no approval for the clinical use of vitamin E as a drug even though vitamin E consumption has been reported to boost immunity, improve skin health and vision. Vitamin E remains a popular supplement

Vitamin E is the main lipid soluble antioxidant [79] in the cell membrane thus will always attract research attention considering the relevance of membrane lipid oxidation [84]. The bioactivity of vitamin E in the cell membrane may be a direct approach to limit oxidation of other biomolecules that can form conjugates with

Emerging research outputs are shedding light on the disparity between in vitro and in vivo antioxidant capacity of vitamin E [85]. In macrophages, the oxidation of lipids occur in the lysosomes. Alboaklah and Leake [85] conducted an experiment on LDL oxidation at lysosomal pH (about 4.5). In their experiment, LDL enriched with vitamin E was oxidized by Cu2+ more slowly compared to control LDL. At

tration of 4-hydroxyl-2-nonenal-glutathione conjugates in the brain [83].

**3.5 Roles of vitamin E in other diseases**

advanced glycation end products (AGEs).

tion and T-helper (CD4 T-cell) proliferation [79].

and is generally regarded as safe by the FDA.

management.

**4. Discussion**

oxidized lipids.

by the biochemical processes associated with stroke.

**252**

pH 4.5, the enriched LDL was not protected against oxidation by low concentrations of Cu2+ or Fe3+. They observed that the enriched LDL reduced the Cu2+ and Fe3+ to the more pro-oxidant Cu+ and Fe2+ at a faster rate than control LDL at lysosomal pH. This may partly be responsible for the observed reduction in the bioactivity of vitamin E compounds in vivo.

The bioavailability of vitamin E has been another complication in clinical applications. Recent data indicate that the absorption of vitamin E is far more complex than previously thought. Details about the digestion, absorption and transport of vitamin E are presented in a review by Reboul [86]. The author concluded that the process is only partly understood and suggested further studies to decipher the molecular mechanisms [86].

The poor water solubility of vitamin E has been implicated in the low oral bioavailability [87]. Recently, nanoformulations such as nanovesicles, solid-lipid nanoparticles, nanostructured lipid carriers, nanoemulsions, and polymeric nanoparticles have shown promising outcomes in improving the efficacy and bioavailability of vitamin E.

Lipid-based nanovesicles such as niosomes and liposomes are highly promising for the delivery of lipophilic drugs and active compounds. Although niosomes and liposomes have similar physicochemical properties, niosomes have a higher permeability to small solutes and ions than liposomes. The application of these nanovesicles as drug delivery vehicles is suitable because they are non-toxic and stable.

In a study to enhance the tumor-suppressing effect of tocotrienols in vivo, Fu et al. [88] first developed a D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)-based noisome [88, 89]. This system appeared to significantly increase tocotrienol uptake in vitro using A431, B16F10 and T98G cell lines and hence improved the therapeutic efficacy. TPGS can be functionalized as an excellent solubilizer, emulsifier, permeation and bioavailability enhancer for hydrophobic drugs [90]. TPGS has demonstrated capacity to selectively induce apoptogenic activity against many cancer types by targeting the activation of mitochondrial mediators of apoptosis [91]. Another 6-*O*-palmitoyl-ascorbic acid (PA) based niosomes (comprising AP, TPGS, cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG (2000)-carboxylic acid)) was developed by Tan et al. [92] targeting transferrin receptors for intravenous administration of γ-tocotrienol aimed at treating breast cancer. Both in vitro and in vivo studies have proven that tumor-targeted niosomes significantly improve the therapeutic efficacy of γ-tocotrienol. These studies suggests that nanovesicles can be suitable carriers for improved delivery and enhanced efficacy of vitamin E [93].

#### **5. Conclusion**

Although the antioxidant and antiproliferative properties of vitamin E against oxidative stress related diseases have been reported, there is currently no approval for clinical application. A modified product, TPGS has been approved by the FDA as a safe pharmaceutical adjuvant with high biocompatibility. Despite the reported bioactivity of the tocotrienols, studies so far are inconclusive. The reported in vitro biochemical properties of the vitamin E family will continually call the attention of researchers since they are natural and can play essential roles in ameliorating the impact of oxidative assault in biological membranes. To maximize the prophylactic and curative properties of vitamin E, further research on absorption, cellular uptake, solubility, and stability is required to improve bioavailabilty and efficacy in vivo.

### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Israel Ehizuelen Ebhohimen1 \*, Taiwo Stephen Okanlawon<sup>2</sup> , Augustine Ododo Osagie3 and Owen Norma Izevbigie4

1 Department of Chemical Sciences, Samuel Adegboyega University, Ogwa, Edo State, Nigeria

2 Department of Biological Sciences, Samuel Adegboyega University, Ogwa, Edo State, Nigeria

3 Department of Medical Biochemistry, University of Benin, Benin City, Nigeria

4 Department of Surgery, Lagos State University Teaching Hospital, Lagos, Nigeria

\*Address all correspondence to: israel.ebhohimen@gmail.com

© 2021 The Author(s). Licensee IntechOpen. 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.

**255**

*Vitamin E in Human Health and Oxidative Stress Related Diseases*

and model systems Journal of Cellular

[11] Brigelius-Flohe R. Vitamin E: The shrew waiting to be tamed. Free Radic

[12] Muller L, Theile K, Bohm V. In vitro antioxidant activity of tocopherols and tocotrienols and comparison of vitamin

antioxidant capacity in human plasma. Mol Nutr Food Res 2010;54:731-742.

[13] Tran K, Wong JT, Lee E, Chan AC, Choy PC. Vitamin E potentiates

arachidonate release and phospholipase A2 activity in rat heart myoblastic cells.

[15] Kamal-Eldin A, Appelqvist LA. The chemistry and antioxidant properties of tocopherols and tocotrienols. Lipids. 1996;31(7):671-701. DOI:10.1007/ BF02522884. PMID: 8827691.

[16] Traber MG, Sies H. Vitamin E in humans: Demand and delivery. Annu Rev Nutr. 1996;16:321-347. DOI:10.1146/ annurev.nu.16.070196.001541. PMID:

[17] Shintani D, DellaPenna D. Elevating the Vitamin E content of plants through

[18] Nita M, Grzybowski A. The role of the reactive oxygen species and

oxidative stress in the Pathomechanism of the age-related ocular diseases and other pathologies of the anterior and posterior eye segments in adults. Oxid Med Cell Longev. 2016;2016:3164734.

metabolic engineering. Science.

1998;282:2098-2100

doi:10.1155/2016/3164734

and Molecular Medicine.

Biol Med 2009; 46:543-554

E concentration and lipophilic

Biochem J. 1996; 319:385-391.

2005;10:574-579.

8839930.

[14] DellaPenna D. Progress in the dissection and manipulation of vitamin E synthesis. Trends in Plant Science

2010;14:840-860.

*DOI: http://dx.doi.org/10.5772/intechopen.99169*

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[2] Evans HM, Bishop KS. On the existence of a hitherto unrecognized dietary factor essential for reproduction.

Mangialasche, F., Mecocci P. Vitamin E family: Role in the pathogenesis and treatment of Alzheimer's disease. Alzheimer's and dementia (New York,

Science 1922;56:650-651.

[3] Boccardi, V, Baroni, M,

N. Y.). 2016;2(3), 182-191. DOI:10.1016/j.trci.2016.08.002

antiox7010002

tplants.2019.08.006.

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[4] Mene-Saffrane L. Vitamin E

Biosynthesis and its regulation in plants. Antioxidants. 2018;7:2. doi:10.3390/

[5] Muñoz P, Munne-Bosch S. Vitamin E in plants: Biosynthesis, transport, and function. Trends in Plant Science. 2019;24:1040-1051. DOI:10.1016/j.

[6] Whittle KJ, Dunphy PJ, Pennock, JF. Plastochromanol in the leaves of *Hevea brasiliensis*. Biochem. J. 1965;96:17-19.

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[8] Fanali, G. et al. α-Tocopherol binding to human serum albumin. Biofactors.

[9] Burton GW, Hughes L, Foster DO, Pietrzak E, Goss-Sampson MA, Muller DPR. Antioxidant mechanisms of vitamin e and β-carotene. Free radicals: From basic science to Medicine.

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*Vitamin E in Human Health and Oxidative Stress Related Diseases DOI: http://dx.doi.org/10.5772/intechopen.99169*

#### **References**

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

The authors declare no conflict of interest.

**254**

**Author details**

**Conflict of interest**

Israel Ehizuelen Ebhohimen1

Augustine Ododo Osagie3

Ogwa, Edo State, Nigeria

Ogwa, Edo State, Nigeria

\*, Taiwo Stephen Okanlawon<sup>2</sup>

and Owen Norma Izevbigie4

3 Department of Medical Biochemistry, University of Benin, Benin City, Nigeria

4 Department of Surgery, Lagos State University Teaching Hospital, Lagos, Nigeria

© 2021 The Author(s). Licensee IntechOpen. 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,

1 Department of Chemical Sciences, Samuel Adegboyega University,

2 Department of Biological Sciences, Samuel Adegboyega University,

\*Address all correspondence to: israel.ebhohimen@gmail.com

provided the original work is properly cited.

,

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its role in the prevention of

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[52] Israel K, Yu W, Sanders B, Kline K. Vitamin E succinate induces apoptosis in human prostate cancer cells: Role for Fas in vitamin E succinate-triggered apoptosis. Nutr Cancer 2000;36:90-100.

[53] Pruthi S, Allison TG, Hensrud DD. Vitamin E supplementation in the prevention of coronary heart disease. Mayo Clin Proc. 2001;76:1131-1136

[54] Scott J. Pathophysiology and biochemistry of cardiovascular disease.

Curr Opion Genet Develop.

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Cherubini A. Oxidative stress in brain aging, neurodegenerative and vascular diseases: An overview. J Chromat B.

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2005;827:65-75.

Interventions in Aging 2007:2(2) 219-236

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Acad Sci. 1993; 90:7915-7922.

2020;26:1-8.

*DOI: http://dx.doi.org/10.5772/intechopen.99169*

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Biochimica et Biophysica Acta.

Cheeseman KH, Slater FT, Lang L, Esterbauer H. Separation and characterization of the aldehydic products of lipid peroxidation stimulated by carbontetrachloride or ADP-iron in isolated rat hepatocytes and rat liver microsomal suspensions. Biochemical Journal 1985;227:629-638.

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[43] Jenner P. Oxidative stress in Parkinson's disease. Annals of Neurology. 2003;53:26-36.

[44] Dalle-Donne I, Rossi R, Colombo R, Giustarini D, Milzani A. Biomarkers of oxidative damage in human disease. Clinical Chemistry 2006;52: 601-623.

[45] Harman D. Ageing: A theory based on free radical and radiation chemistry. J Gerontol, 1956;2:298-300. DOI:10.1016/j.tplants.2019.08.006.

[46] Kowald A, Kirkwood TB. Accumulation of defective mitochondria through delayed degradation of damaged organelles and its possible role in ageing of post-mitotic and dividing cells. J Theor Biol, 2000;202:145-160.

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[51] Shklar G, Schwartz J. Tumor necrosis factor in experimental cancer: Regression with alpha tocopherol, beta-carotene, and algae extract. Eur J Cancer Clin Oncol 1988;24:839-850.

[52] Israel K, Yu W, Sanders B, Kline K. Vitamin E succinate induces apoptosis in human prostate cancer cells: Role for Fas in vitamin E succinate-triggered apoptosis. Nutr Cancer 2000;36:90-100.

[53] Pruthi S, Allison TG, Hensrud DD. Vitamin E supplementation in the prevention of coronary heart disease. Mayo Clin Proc. 2001;76:1131-1136

[54] Scott J. Pathophysiology and biochemistry of cardiovascular disease. Curr Opion Genet Develop. 2004;14:271-279.

[55] Mariani E, Polidori MC, Cherubini A. Oxidative stress in brain aging, neurodegenerative and vascular diseases: An overview. J Chromat B. 2005;827:65-75.

[56] Elahi MM, Matata BM. Free radicals in blood: Evolving concepts in the mechanism of ischemic heart disease. Arch Biochem Biophys. 2006;450:78-88.

[57] Meydani M. Vitamin E and atherosclerosis: Beyond prevention of LDL oxidation. J. Nutr. 2001;131:366S–368S

[58] Saremi A, Arora R. Vitamin E and cardiovascular disease. American Journal of Therapeutics. 2010;17:e56-e65.

[59] Esterbauer H, Dieber-Rotheneder M, Striegl G. Role of vitamin E in preventing the oxidation of low-density lipoprotein. Am J Clin Nutr. 1991;53:314S–321S.

[60] Reaven PD, Khouw A, Beltz WF. Effect of dietary antioxidant combinations in humans: Protection of LDL by vitamin E but not by betacarotene. Arterioscler Thromb. 1993;13:590-600.

[61] Jiang Q. Natural forms of vitamin E as effective agents for cancer prevention and therapy. Adv Nutr 2017;8:850-67 DOI:DOI:10.3945/an.117.016329.

[62] Klein EA, Thompson IM Jr., Tangen CM, Crowley JJ, Lucia MS, Goodman PJ, Minasian LM, Ford LG, Parnes HL, Gaziano JM, et al. Vitamin E and the risk of prostate cancer: The selenium and Vitamin E Cancer prevention trial (SELECT). JAMA 2011;306:1549-1556.

[63] Taketo, M. M. Cyclooxygenase-2 inhibitors in tumorigenesis (part I). J Natl Cancer Inst 1998;90:1529-1536

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[66] Reddy BS, Hirose Y, Lubet R, Steele V, Kelloff G, Paulson S, Seibert K, Rao CV. Chemoprevention of colon cancer by specific cyclooxygenase-2 inhibitor, celecoxib, administered during different stages of carcinogenesis. Cancer Res 2000;60:293-297.

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[68] Jiang Q. Natural forms of vitamin E: Metabolism, antioxidant, and antiinflammatory activities and their role in disease prevention and therapy. Free Radic Biol Med 2014;72:76-90.

[69] Kunnumakkara AB, Sung B, Ravindran J, Diagaradjane P, Deorukhkar A, Dey S, Koca C, Yadav VR, Tong Z, Gelovani JG, et al. G-Tocotrienol inhibits pancreatic tumors and sensitizes them to gemcitabine treatment by modulating the inflammatory microenvironment. Cancer Res 2010;70:8695-8705.

[70] Radhakrishnan AK, Mahalingam D, Selvaduray KR, Nesaretnam K. Supplementation with natural forms of vitamin E augments antigen-specific TH1-type immune response to tetanus toxoid. Biomed Res Int. 2013;2013:782067.

[71] Ren Z, Pae M, Dao MC, Smith D, Meydani SN, Wu D. Dietary supplementation with tocotrienols enhances immune function in C57BL/6 mice. J Nutr. 2010;140:1335-1341.

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**259**

*Vitamin E in Human Health and Oxidative Stress Related Diseases*

[81] Alexandrova M, Bochev P, Markova V. Dynamics of free radical processes in acute ischemic stroke: Infl uence on neurological status and outcome. J Clin Neurosci.

[82] Behl C, Davis JB, Lesley R. Hydrogen peroxide mediates amyloid protein activity. Cell. 1994;77:817-827.

[83] Völkel W, Sicilia T, Pähler A. Increased brain levels of 4-hydroxy-2 nonenal glutathione conjugates in severe Alzheimer's disease. Neurochem Intern.

[84] Lúcio M, Nunes C, Gaspar D, Ferreira H, Lima JLFC, Reis S.

DOI 10.1007/s11483-009-9129-4

Research. 2020;54:574-584

2017;6(4):95. doi:10.3390/

Biol Med. 1997; 22: 977-984.

jconrel.2011.04.01571

antiox6040095

Antioxidant activity of vitamin E and trolox: Understanding of the factors that govern lipid peroxidation studies in vitro. Food Biophysics. 2009;4:312-320.

[85] AlboaklahH KM, Leake DS. Effect of vitamin E on low density lipoprotein oxidation at lysosomal pH. Free Radical

[86] Reboul E. Vitamin E Bioavailability: Mechanisms of intestinal absorption in the spotlight. Antioxidants (Basel).

[87] Penn JS, Tolman BL, Bullard LE. Effect of a water-soluble vitamin E analog, trolox C, on retinal vascular development in an animal model of retinopathy of prematurity. Free Radic

[88] Fu JY, Zhang W, Blatchford DR, Tetley L, McConnell G, Dufès C. Novel tocotrienol-entrapping vesicles can eradicate solid tumors after intravenous administration. J Control Release. 2011;154(1):20-26. doi:10.1016/j.

[89] Zhang Z, Tan S, Feng SS. Vitamin E TPGS as a molecular biomaterial for

2004;11:501-506.

2006;48:679-686.

*DOI: http://dx.doi.org/10.5772/intechopen.99169*

Hutchinson SZ, Neuger AM, Lush R, Coppola D, Sebti S, Malafa MP. Vitamin E delta-tocotrienol levels in tumor and pancreatic tissue of mice after oral administration. Pharmacology

[74] Campbell SE, Stone WL, Lee S, Whaley S, Yang H, Qui M, Goforth P, Sherman D, McHaffie D, Krishnan K. Comparative effects of RRR alpha-and

proliferation and apoptosis in human colon cancer cell lines. BMC Cancer

Sanders BG, Kline K. Involvement of de novo ceramide synthesis in gammatocopherol and gammatocotrienolinduced apoptosis in human breast cancer cells. Mol Nutr Food Res

[76] Hannun YA, Obeid LM. Principles of bioactive lipid signalling: Lessons from sphingolipids. Nat Rev Mol Cell

[77] Leske MC, Chylack LT Jr., Wu SY. The Lens opacities case-control study. Risk factors for cataract. Archives of Ophthalmology. 1991;109:244-251

[78] Murphy TH, Schnaar RL, Coyle JT. Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cysteine uptake. FASEB J.

[79] Rizvi S, Raza, ST, Ahmed F, Ahmad A, Abbas S, Mahdi F. The role of vitamin e in human health and some diseases. Sultan Qaboos University Medical Journal. 2014;14(2):e157–e165.

[80] Piantadosi CA, Zhang J.

Mitochondrial generation of reactive oxygen species after brain ischemia in the rat. Stroke. 1996;27:327-332.

RRR-gamma-tocopherol on

[75] Gopalan A, Jiang Q, Jang Y,

[73] Husain K, Francois RA,

2009;83:157-163.

2006;6:13.

2012;56:1803-1811

Biol 2008;9:139-150.

[PubMed: 1993036]

1990;4:1624-1633.

*Vitamin E in Human Health and Oxidative Stress Related Diseases DOI: http://dx.doi.org/10.5772/intechopen.99169*

[73] Husain K, Francois RA, Hutchinson SZ, Neuger AM, Lush R, Coppola D, Sebti S, Malafa MP. Vitamin E delta-tocotrienol levels in tumor and pancreatic tissue of mice after oral administration. Pharmacology 2009;83:157-163.

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

a potent preventive and therapeutic agent in the min mouse model of adenomatous polyposis. Cancer Res

[66] Reddy BS, Hirose Y, Lubet R, Steele V, Kelloff G, Paulson S, Seibert K, Rao CV. Chemoprevention of colon cancer by specific cyclooxygenase-2 inhibitor, celecoxib, administered

2000;60:5040-5044.

during different stages of carcinogenesis. Cancer Res

[67] Grivennikov SI, Greten FR,

cancer. Cell 2010;140:883-899.

[69] Kunnumakkara AB, Sung B, Ravindran J, Diagaradjane P, Deorukhkar A, Dey S, Koca C, Yadav VR, Tong Z, Gelovani JG, et al. G-Tocotrienol inhibits pancreatic tumors and sensitizes them to

gemcitabine treatment by modulating the inflammatory microenvironment. Cancer Res 2010;70:8695-8705.

[70] Radhakrishnan AK, Mahalingam D,

Supplementation with natural forms of vitamin E augments antigen-specific TH1-type immune response to tetanus

[71] Ren Z, Pae M, Dao MC, Smith D,

[72] Shah SJ, Sylvester PW. Gamma-Tocotrienol inhibits neoplastic

mammary epithelial cell proliferation by decreasing Akt and nuclear factor kappaB activity. Exp Biol Med (Maywood) 2005;230:235-241.

Selvaduray KR, Nesaretnam K.

toxoid. Biomed Res Int. 2013;2013:782067.

Meydani SN, Wu D. Dietary supplementation with tocotrienols enhances immune function in C57BL/6 mice. J Nutr. 2010;140:1335-1341.

Karin M. Immunity, inflammation, and

[68] Jiang Q. Natural forms of vitamin E: Metabolism, antioxidant, and antiinflammatory activities and their role in disease prevention and therapy. Free Radic Biol Med 2014;72:76-90.

2000;60:293-297.

[56] Elahi MM, Matata BM. Free radicals in blood: Evolving concepts in the mechanism of ischemic heart disease. Arch Biochem Biophys. 2006;450:78-88.

[57] Meydani M. Vitamin E and atherosclerosis: Beyond prevention of

[58] Saremi A, Arora R. Vitamin E and cardiovascular disease. American

[59] Esterbauer H, Dieber-Rotheneder M, Striegl G. Role of vitamin E in preventing the oxidation of low-density

[60] Reaven PD, Khouw A, Beltz WF.

combinations in humans: Protection of LDL by vitamin E but not by betacarotene. Arterioscler Thromb.

[61] Jiang Q. Natural forms of vitamin E as effective agents for cancer prevention and therapy. Adv Nutr 2017;8:850-67 DOI:DOI:10.3945/an.117.016329.

[62] Klein EA, Thompson IM Jr., Tangen CM, Crowley JJ, Lucia MS, Goodman PJ, Minasian LM, Ford LG, Parnes HL, Gaziano JM, et al. Vitamin E and the risk of prostate cancer: The selenium and Vitamin E Cancer prevention trial (SELECT). JAMA

[63] Taketo, M. M. Cyclooxygenase-2 inhibitors in tumorigenesis (part I). J Natl Cancer Inst 1998;90:1529-1536

[64] Taketo, M. M. Cyclooxygenase-2 inhibitors in tumorigenesis (part II). J Natl Cancer Inst 1998;90:1609-1620.

[65] Jacoby RF, Seibert K, Cole CE,

cyclooxygenase-2 inhibitor celecoxib is

Kelloff G, Lubet RA. The

LDL oxidation. J. Nutr. 2001;131:366S–368S

Journal of Therapeutics.

lipoprotein. Am J Clin Nutr.

Effect of dietary antioxidant

2010;17:e56-e65.

1991;53:314S–321S.

1993;13:590-600.

2011;306:1549-1556.

**258**

[74] Campbell SE, Stone WL, Lee S, Whaley S, Yang H, Qui M, Goforth P, Sherman D, McHaffie D, Krishnan K. Comparative effects of RRR alpha-and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines. BMC Cancer 2006;6:13.

[75] Gopalan A, Jiang Q, Jang Y, Sanders BG, Kline K. Involvement of de novo ceramide synthesis in gammatocopherol and gammatocotrienolinduced apoptosis in human breast cancer cells. Mol Nutr Food Res 2012;56:1803-1811

[76] Hannun YA, Obeid LM. Principles of bioactive lipid signalling: Lessons from sphingolipids. Nat Rev Mol Cell Biol 2008;9:139-150.

[77] Leske MC, Chylack LT Jr., Wu SY. The Lens opacities case-control study. Risk factors for cataract. Archives of Ophthalmology. 1991;109:244-251 [PubMed: 1993036]

[78] Murphy TH, Schnaar RL, Coyle JT. Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cysteine uptake. FASEB J. 1990;4:1624-1633.

[79] Rizvi S, Raza, ST, Ahmed F, Ahmad A, Abbas S, Mahdi F. The role of vitamin e in human health and some diseases. Sultan Qaboos University Medical Journal. 2014;14(2):e157–e165.

[80] Piantadosi CA, Zhang J. Mitochondrial generation of reactive oxygen species after brain ischemia in the rat. Stroke. 1996;27:327-332.

[81] Alexandrova M, Bochev P, Markova V. Dynamics of free radical processes in acute ischemic stroke: Infl uence on neurological status and outcome. J Clin Neurosci. 2004;11:501-506.

[82] Behl C, Davis JB, Lesley R. Hydrogen peroxide mediates amyloid protein activity. Cell. 1994;77:817-827.

[83] Völkel W, Sicilia T, Pähler A. Increased brain levels of 4-hydroxy-2 nonenal glutathione conjugates in severe Alzheimer's disease. Neurochem Intern. 2006;48:679-686.

[84] Lúcio M, Nunes C, Gaspar D, Ferreira H, Lima JLFC, Reis S. Antioxidant activity of vitamin E and trolox: Understanding of the factors that govern lipid peroxidation studies in vitro. Food Biophysics. 2009;4:312-320. DOI 10.1007/s11483-009-9129-4

[85] AlboaklahH KM, Leake DS. Effect of vitamin E on low density lipoprotein oxidation at lysosomal pH. Free Radical Research. 2020;54:574-584

[86] Reboul E. Vitamin E Bioavailability: Mechanisms of intestinal absorption in the spotlight. Antioxidants (Basel). 2017;6(4):95. doi:10.3390/ antiox6040095

[87] Penn JS, Tolman BL, Bullard LE. Effect of a water-soluble vitamin E analog, trolox C, on retinal vascular development in an animal model of retinopathy of prematurity. Free Radic Biol Med. 1997; 22: 977-984.

[88] Fu JY, Zhang W, Blatchford DR, Tetley L, McConnell G, Dufès C. Novel tocotrienol-entrapping vesicles can eradicate solid tumors after intravenous administration. J Control Release. 2011;154(1):20-26. doi:10.1016/j. jconrel.2011.04.01571

[89] Zhang Z, Tan S, Feng SS. Vitamin E TPGS as a molecular biomaterial for

**Chapter 13**

**Abstract**

can overturn male infertility.

sperm parameters

**1. Introduction**

**261**

and Antioxidants

Male Infertility, Oxidative Stress

*Vegim Zhaku, Ashok Agarwal, Sheqibe Beadini, Ralf Henkel,*

Within the male reproductive system, oxidative stress (OS) has been identified as prevailing etiology of male infertility. The effects of reactive oxygen species (ROS) on male fertility depend on the dimensions, "modus operandi" of the ROS and the oxido-reduction potential (ORP) of the male reproductive tract. Hereupon, for an adequate response to OS, the cells of our body are endowed with a wellsophisticated system of defense in order to be protected. Various antioxidant enzymes and small molecular free radical scavengers, maintain the delicate balance between oxidants and reductants (antioxidants), crucial to cellular function and fertility. Therapeutic use of antioxidants is an optimal and coherent option in terms of mitigating OS and improving semen parameters. Therefore, recognizing and managing OS through either decreasing ROS levels or by increasing antioxidant force, appear to be a requesting approach in the management of male infertility. However, a clear defined attitude of the experts about the clinical efficacy of antioxidant therapy is still deprived. Prominently, antioxidant such as coenzyme Q10, vitamin C and E, lycopene, carnitine, zinc and selenium have been found useful in controlling the balance between ROS production and scavenging activities. In spite of that, healthy lifestyle, without smoke and alcohol, everyday exercise, reduction of psychological stress and quality well-designed meals, are habits that

**Keywords:** Male infertility, reactive oxygen species, oxidative stress, antioxidants,

The World Health Organization (WHO) defines infertility as the inability (failure) to attain clinical pregnancy after one year or more of regular unprotected sexual intercourse [1]. Since infertility presents a certain disability (impaired reproductive function), medical assessment and treatment falls under the umbrella of the United Nations Convention on the Rights of Persons with Disabilities – UNCRPD, which is formally accepted by many countries. The article 1 of this Convention summarizes the overall objective as: "to promote, protect and ensure the full and equal enjoyment of all human rights and fundamental freedoms by all persons with disabilities, and to promote respect for their inherent dignity" [2]. Due to its health, cultural and socio-economic impact, infertility is a major globally underestimated public health concern [3, 4]. Therefore, proper evaluation of male

*Renata Finelli, Nexhbedin Beadini and Sava Micic*

drug delivery. Biomaterials. 2012; 33: 4889-4906.

[90] Guo Y, Luo J, Tan S, Otieno BO, Zhang Z. The applications of Vitamin E TPGS in drug delivery. Eur J Pharm Sci. 2013; 49: 175-186.

[91] Yang C, Wu T, Qi Y, Zhang Z. Recent advances in the application of Vitamin E TPGS for Drug Delivery. 2018; 8(2): 464-485. DOI:10.7150/thno.22711

[92] Tan GR, Feng SS, Leong DT. The reduction of anti-cancer drug antagonism by the spatial protection of drugs with PLA-TPGS nanoparticles. Biomaterials. 2014;35: 3044-3051.

[93] Zaffarin ASM, Ng SF., Ng MH, Hassan H, Alias E. Pharmacology and pharmacokinetics of Vitamin E: Nanoformulations to enhance Bioavailability. International Journal of Nanomedicine. 2020;15: 9961-9974.

#### **Chapter 13**

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

drug delivery. Biomaterials. 2012; 33:

[90] Guo Y, Luo J, Tan S, Otieno BO, Zhang Z. The applications of Vitamin E TPGS in drug delivery. Eur J Pharm Sci.

[91] Yang C, Wu T, Qi Y, Zhang Z. Recent advances in the application of Vitamin E TPGS for Drug Delivery. 2018; 8(2): 464-485. DOI:10.7150/thno.22711

[92] Tan GR, Feng SS, Leong DT. The reduction of anti-cancer drug

[93] Zaffarin ASM, Ng SF., Ng MH, Hassan H, Alias E. Pharmacology and pharmacokinetics of Vitamin E: Nanoformulations to enhance

Bioavailability. International Journal of Nanomedicine. 2020;15: 9961-9974.

antagonism by the spatial protection of drugs with PLA-TPGS nanoparticles. Biomaterials. 2014;35: 3044-3051.

4889-4906.

2013; 49: 175-186.

**260**

## Male Infertility, Oxidative Stress and Antioxidants

*Vegim Zhaku, Ashok Agarwal, Sheqibe Beadini, Ralf Henkel, Renata Finelli, Nexhbedin Beadini and Sava Micic*

#### **Abstract**

Within the male reproductive system, oxidative stress (OS) has been identified as prevailing etiology of male infertility. The effects of reactive oxygen species (ROS) on male fertility depend on the dimensions, "modus operandi" of the ROS and the oxido-reduction potential (ORP) of the male reproductive tract. Hereupon, for an adequate response to OS, the cells of our body are endowed with a wellsophisticated system of defense in order to be protected. Various antioxidant enzymes and small molecular free radical scavengers, maintain the delicate balance between oxidants and reductants (antioxidants), crucial to cellular function and fertility. Therapeutic use of antioxidants is an optimal and coherent option in terms of mitigating OS and improving semen parameters. Therefore, recognizing and managing OS through either decreasing ROS levels or by increasing antioxidant force, appear to be a requesting approach in the management of male infertility. However, a clear defined attitude of the experts about the clinical efficacy of antioxidant therapy is still deprived. Prominently, antioxidant such as coenzyme Q10, vitamin C and E, lycopene, carnitine, zinc and selenium have been found useful in controlling the balance between ROS production and scavenging activities. In spite of that, healthy lifestyle, without smoke and alcohol, everyday exercise, reduction of psychological stress and quality well-designed meals, are habits that can overturn male infertility.

**Keywords:** Male infertility, reactive oxygen species, oxidative stress, antioxidants, sperm parameters

#### **1. Introduction**

The World Health Organization (WHO) defines infertility as the inability (failure) to attain clinical pregnancy after one year or more of regular unprotected sexual intercourse [1]. Since infertility presents a certain disability (impaired reproductive function), medical assessment and treatment falls under the umbrella of the United Nations Convention on the Rights of Persons with Disabilities – UNCRPD, which is formally accepted by many countries. The article 1 of this Convention summarizes the overall objective as: "to promote, protect and ensure the full and equal enjoyment of all human rights and fundamental freedoms by all persons with disabilities, and to promote respect for their inherent dignity" [2]. Due to its health, cultural and socio-economic impact, infertility is a major globally underestimated public health concern [3, 4]. Therefore, proper evaluation of male

infertility is a substantial stride in qualifying, quantifying and configuring necessary laboratory assessment, credential treatment strategies as well as policies to diminish the burden of this global sensitive health issue.

There are approximately 186 million infertile people [5] or 15% of couples globally, 50% due to male factor infertility which experience problems in conceiving [6, 7]. In male dominated societies, generally, the female partner is blamed for barrenness, even though ancient Greeks were aware that male factor is a contributor to the reproductive success [8].

In fertile couples, spontaneous conception is most likely to occur in 30% of cases during the first month, 75% after 6 months, 90% after 12 months and 95% between 18 to 24 months [9]. Also, there are studies which consider that 80% of couples having unprotected sexual intercourse will achieve pregnancy in the 6-month [10] or 12-month interval [11].

In addition, male fertility reaches its maximum potential at ages of about 25 to 30 years and declines sharply in the beginning of fifties [12], however, there are men reported to give life to offspring into their eighties [13]. Paternal age of >40 years is associated with more than 20% higher chance of congenital defects in the offspring [14]. Over the past decades, an age-related decline in semen quality resulting in declined fertility was observed [15].

Oxidative stress (OS) has been identified as one of the major contributors affecting male fertility potential [16] and has thus been extensively studied in the last three decades. Although cells of the human body have efficient mechanisms to cope with factors disturbing the normal cell homeostasis, OS may arise due to an imbalance between generation of oxidants and antioxidants mechanisms, resulting in cell damage.

Reactive oxygen species (ROS) are important mediators of OS status, because of their capacity to oxidize proteins, lipids, and DNA, resulting in cellular dysfunction [17]. ROS are oxygen-based molecules that have unpaired electrons on their most outlier spin-orbit, derived from the reaction of carbon-centered radical with oxygen (except hydrogen peroxide), which makes them highly reactive [18]. The most common ROS are hydroxyl radical (OH•), hydrogen peroxide (H2O2) and the superoxide anion (O2•-). ROS are generated not only by leukocytes (neutrophils and macrophages mostly) [19], but also by any aerobe living cell including spermatozoa [20]. Moreover, another subclass of free radicals deriving from nitrogenbased molecules are called reactive nitrogen species (RNS) [21, 22]. At physiologic amount, RNS are important for various functions within the male reproductive tract such as: (1) signal transduction, (2) regulation and assembly of tight junction within the blood-testis barrier, (3) mediation of cytotoxic and pathological events, (4) production of hormones, (5) inflammation and (6) other important physiological changes of spermatozoa [23].

Some of the most common ROS and RNS are listed in **Table 1**. Effects, consequences, mode of formation and action of these molecules are presented in details in **Table 2**.

Under physiological conditions, high levels of ROS are counterbalanced by antioxidants, which competently maintain a delicate redox balance by donating their electrons to the ROS and thus interrupting further intake of electrons from surrounding compartments [37]. The seminal antioxidant system comprises a network of enzymatic and non-enzymatic molecules, dispersed mostly within seminal plasma and spermatozoa [38]. The three major antioxidant enzymes are glutathione peroxidase (GPx), catalase (CAT) and the superoxide dismutase (SOD) [39].

without any scientific rationale, ensuing neither semen parameters improvement, nor fertilization outcomes. Contrary, some other studies even showed a worsening of semen parameters [40–42], because an excess intake of antioxidants can contribute in the establishment of reductive stress (RS), a condition which has been reported being as harmful as OS [43]. Therefore, there still lack of conclusive consensus regarding the clinical advantages of antioxidants - based therapy in male infertility.

*The mode of formation of the biologically active ROS responsible for the major consequences of oxidative stress.*

**Hydrogen peroxide (H2O2) Ref.** Hydrogen peroxide is not a free radical, because it does not contain an unpaired electron, but it is classified as ROS because it participates in the generation of highly reactive hydroxyl free radicals through interactions with iron and copper, based on the Fenton

**Reactive oxygen species Reactive nitrogen Species**

**•** *Singlet oxygen* **<sup>1</sup>**

*Lipid peroxyl* **LOO•** *Lipid hydroperoxide* **LOOH** *Nitryl chloride* **NO2Cl** *Thyl* **RS•** *Ozone* **O3** *Nitrous acid* **HNO2**

*Nitric oxide* **NO•** *Hydrogen peroxide* **H2O2** *Dinitrogen trioxide* **N2O3**

*Hydroxyl* **OH•** *Peroxynitrite* **ONOO—** *Nitroxyl cation* **NO+**

**•—** *Hypochloric acid* **HOCl** *Nitroxyl anion* **NO**

**Hydroxyl (•OH)** Ref. This represents the neutral form of the hydroxide ion, deriving from the reaction between Fe2+ and H2O2 (Fenton reaction). It is the most reactive free radical. The hydroxyl radicals and hydroxide ions can be generated also by the reaction of H2O2 and O2•- catalyzed by iron (Haber-Weiss reaction). The hydroxyl radical has the potential of reacting fast and

**Peroxynitrite (ONOO—)** Ref.

**Hypochloric acid (HOCl)** Ref. Hypochloric acid is produced by macrophages and neutrophils during respiratory burning that accompanies phagocytosis. This radical is generated in the reaction between H2O2

of structural proteins, resulting in the formation of nitrosotioles, which can disunite metal-protein interactions and result in the formation of metal-derived free radicals.

Peroxyl radicals remove electrons from lipids during the process of lipid peroxidation. During this process, intermediates are generated that participate in further reactions with oxygen to form lipid peroxyl (LOO•) and lipid hydroperoxide (LOOH) which are

It is generated by electron transport leaks from several reaction in cytosol. It does not spread easily and faraway its origin. It is responsible for cell injury, by deconstructing iron–sulphur clusters in proteins through the inactivation of iron regulatory protein-1.

**—)** Ref.

**)** Ref.

[22,24–26]

[27–29]

[30, 31]

[32]

[33–35].

[36]

—, it can react with thio groups

**O2** *Nitrogen dioxide* **NO2**

reaction.

**Table 1.**

**Superoxide (O2•**

*Peroxyl* **RO2**

*Superoxide* **O2**

*Most common ROS and RNS.*

nonspecifically.

**Peroxyl radical (ROO•**

and chloride ion (Cl).

**Table 2.**

**263**

Superoxide is insoluble for the cell membrane.

**Radicals Non-radicals**

*Male Infertility, Oxidative Stress and Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.98204*

It is generated during reaction of nitric oxide (NO) with O2

responsible for sperm DNA and protein damage.

With an increasing knowledge on the role of OS in the clinical manifestation of male infertility, antioxidant prescription and its implementation in treating male infertility may be helpful. Several antioxidant compounds are currently prescribed


#### **Table 1.**

infertility is a substantial stride in qualifying, quantifying and configuring necessary laboratory assessment, credential treatment strategies as well as policies to diminish

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

There are approximately 186 million infertile people [5] or 15% of couples globally, 50% due to male factor infertility which experience problems in conceiving [6, 7]. In male dominated societies, generally, the female partner is blamed for barrenness, even though ancient Greeks were aware that male factor is a contribu-

In fertile couples, spontaneous conception is most likely to occur in 30% of cases during the first month, 75% after 6 months, 90% after 12 months and 95% between 18 to 24 months [9]. Also, there are studies which consider that 80% of couples having unprotected sexual intercourse will achieve pregnancy in the 6-month [10]

In addition, male fertility reaches its maximum potential at ages of about 25 to 30 years and declines sharply in the beginning of fifties [12], however, there are men reported to give life to offspring into their eighties [13]. Paternal age of >40 years is associated with more than 20% higher chance of congenital defects in the offspring [14]. Over the past decades, an age-related decline in semen quality

Oxidative stress (OS) has been identified as one of the major contributors affecting male fertility potential [16] and has thus been extensively studied in the last three decades. Although cells of the human body have efficient mechanisms to cope with factors disturbing the normal cell homeostasis, OS may arise due to an imbalance between generation of oxidants and antioxidants mechanisms, resulting

Reactive oxygen species (ROS) are important mediators of OS status, because of their capacity to oxidize proteins, lipids, and DNA, resulting in cellular dysfunction [17]. ROS are oxygen-based molecules that have unpaired electrons on their most outlier spin-orbit, derived from the reaction of carbon-centered radical with oxygen (except hydrogen peroxide), which makes them highly reactive [18]. The most common ROS are hydroxyl radical (OH•), hydrogen peroxide (H2O2) and the superoxide anion (O2•-). ROS are generated not only by leukocytes (neutrophils and macrophages mostly) [19], but also by any aerobe living cell including spermatozoa [20]. Moreover, another subclass of free radicals deriving from nitrogenbased molecules are called reactive nitrogen species (RNS) [21, 22]. At physiologic amount, RNS are important for various functions within the male reproductive tract such as: (1) signal transduction, (2) regulation and assembly of tight junction within the blood-testis barrier, (3) mediation of cytotoxic and pathological events, (4) production of hormones, (5) inflammation and (6) other important physiolog-

Some of the most common ROS and RNS are listed in **Table 1**. Effects, consequences, mode of formation and action of these molecules are presented in details in

Under physiological conditions, high levels of ROS are counterbalanced by antioxidants, which competently maintain a delicate redox balance by donating their electrons to the ROS and thus interrupting further intake of electrons from surrounding compartments [37]. The seminal antioxidant system comprises a network of enzymatic and non-enzymatic molecules, dispersed mostly within seminal plasma and spermatozoa [38]. The three major antioxidant enzymes are glutathione peroxidase (GPx), catalase (CAT) and the superoxide dismutase (SOD) [39]. With an increasing knowledge on the role of OS in the clinical manifestation of male infertility, antioxidant prescription and its implementation in treating male infertility may be helpful. Several antioxidant compounds are currently prescribed

the burden of this global sensitive health issue.

resulting in declined fertility was observed [15].

tor to the reproductive success [8].

or 12-month interval [11].

in cell damage.

**Table 2**.

**262**

ical changes of spermatozoa [23].

*Most common ROS and RNS.*


#### **Table 2.**

*The mode of formation of the biologically active ROS responsible for the major consequences of oxidative stress.*

without any scientific rationale, ensuing neither semen parameters improvement, nor fertilization outcomes. Contrary, some other studies even showed a worsening of semen parameters [40–42], because an excess intake of antioxidants can contribute in the establishment of reductive stress (RS), a condition which has been reported being as harmful as OS [43]. Therefore, there still lack of conclusive consensus regarding the clinical advantages of antioxidants - based therapy in male infertility.

#### **2. Oxidative stress and male infertility**

OS is a condition characterized by an elevated generation of ROS and a reduced response of biological mechanisms to promptly neutralize the reactive intermediates or to repair the damage [44]. An increased quantity of ROS and RNS has now been established with strict evidence to be a prominent attribute of many acute and chronic pathologies [45].

Nearly eight decades after the Macleods discovery in 1943, highlighting ROS as key players in cell physiology and sperm motility [46], scientists all over the world turned their attention toward the association between free radicals and the male infertility.

#### **2.1 Sources of ROS**

Semen comprises a variety of cells including spermatozoa, germ cells, leukocytes and epithelial cells [47], whereby leukocytes produce about 1000-times more ROS than immature sperm cells [48].

ROS originate from a different countless endogenous and exogenous sources.

Endogenous sources of ROS can be generated extracellularly and intracellularly. Intracellular ROS include O2 —, H2O2 and OH, generated mainly in the mitochondria [49]. In the mitochondria, about 5% of the consumed oxygen is physiologically converted into ROS. The ROS production is increased when the electron transporting chain (ETC) derails as a result of mitochondrial dysfunction [50].

Exogenous sources of ROS include smoking, alcohol and drugs abuse, environmental pollutants, heavy metals, ionizing radiation, diets rich in energy-yielding nutrients like carbohydrates, saturated fats and proteins [51].

#### **2.2 Mechanism of ROS production within human sperm**

ROS are generated in two pathways: the extrinsic and the intrinsic pathway, described in **Figure 1**.

Leukocytes are responsible for the extrinsic pathway of generating ROS, while spermatozoa for the intrinsic pathway of ROS generation [52]. Granulocytes are the white blood cells (WBC) in seminal fluid which are predominantly responsible for demolishing pathogens by ROS production [53, 54].

increased ROS levels by activating (1) the NADPH - nicotinamide adenine dinucleotide phosphate located in the plasma membrane of spermatozoa, and (2) NADPH – dependent oxide-reductase, known as diphorase, detected in the middle piece of mitochondrial level [64–66]. In a study by Sabeur et al., calcium-dependent NADPH oxidase 5 (NOX5) of spermatozoa plays a considerable role in ROS generation [67]. However, there is a difference between NOX5 found in spermatozoa, which does not require protein kinase C for expressing its activity, and in leuko-

*Mechanism of free radical production within semen. (a) The intrinsic and extrinsic pathway contribute in the formation of O2•-. (b) Superoxide is transformed directly and indirectly to secondary (e, f, g) ROS. Adapted*

*from reference [28]. (mathematical symbols + and - stand for positive and negative feedback).*

ROS are very important molecules as they act as cellular mediators essential for (1) normal spermatogenesis, (2) activation of steroidogenic pathway, (3) modulation of mitochondrial and death receptor-apoptotic pathways. These fundamental cascades are required for the process of: maturation, hyperactivation, capacitation, acrosome reaction as well as sperm-oocyte fusion, crucial for the fertilization pro-

After spermiation, spermatozoa are transported into the epididymis where they undergo a maturation process, leading to significant chemical and physiological modifications including recombination of cell-surface proteins, and enzymatic and

cytes, where protein kinase C is essential [68].

*Male Infertility, Oxidative Stress and Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.98204*

**2.3 Physiological role of ROS**

cess, all presented in **Figure 2**.

*2.3.1 Maturation*

**265**

**Figure 1.**

An association between OS and the elevated leukocyte numbers has been found [19]. On the other hand, the relationship between the seminal leukocyte concentration and male infertility is not clear. In fact, leukocytospermia, i.e. the presence of more than 1<sup>10</sup><sup>6</sup> WBC/mL, is not predictive of male infertility [55, 56]. However, the significance of WBC activation in ROS generation and its impact on elevated OS levels cannot be left unnoticed. Various studies reported high levels of proinflammatory chemokines in human semen along with high ROS quantity [57, 58]. Recently, in the seminal plasma of oligozoospermic and azoospermic men it was observed a negative correlation between levels of interleukin-6 (IL-6), interferon alpha (IFN-α) and interferon gamma (IFN-γ) and sperm parameters such as concentration, motility and morphology [59, 60].

Among spermatozoa, it has been shown that morphologically abnormal spermatozoa are the main source of ROS generation [61]. Excess residual cytoplasm (ERC) around the mid-piece of spermatozoa (observed in teratozoospermic sperm) contains high levels of cytoplasmic enzymes responsible for generating ROS [62].

ERC has a considerable amount of enzymes to regulate glucose metabolism, specifically glucose-6-phosphate dehydrogenase (G6PD) [63], which induces

*Male Infertility, Oxidative Stress and Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.98204*

#### **Figure 1.**

**2. Oxidative stress and male infertility**

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

chronic pathologies [45].

**2.1 Sources of ROS**

than immature sperm cells [48].

Intracellular ROS include O2

described in **Figure 1**.

**264**

infertility.

OS is a condition characterized by an elevated generation of ROS and a reduced response of biological mechanisms to promptly neutralize the reactive intermediates or to repair the damage [44]. An increased quantity of ROS and RNS has now been established with strict evidence to be a prominent attribute of many acute and

Nearly eight decades after the Macleods discovery in 1943, highlighting ROS as key players in cell physiology and sperm motility [46], scientists all over the world turned their attention toward the association between free radicals and the male

Semen comprises a variety of cells including spermatozoa, germ cells, leukocytes and epithelial cells [47], whereby leukocytes produce about 1000-times more ROS

ROS originate from a different countless endogenous and exogenous sources. Endogenous sources of ROS can be generated extracellularly and intracellularly.

dria [49]. In the mitochondria, about 5% of the consumed oxygen is physiologically

ROS are generated in two pathways: the extrinsic and the intrinsic pathway,

Leukocytes are responsible for the extrinsic pathway of generating ROS, while spermatozoa for the intrinsic pathway of ROS generation [52]. Granulocytes are the white blood cells (WBC) in seminal fluid which are predominantly responsible for

An association between OS and the elevated leukocyte numbers has been found [19]. On the other hand, the relationship between the seminal leukocyte concentration and male infertility is not clear. In fact, leukocytospermia, i.e. the presence of more than 1<sup>10</sup><sup>6</sup> WBC/mL, is not predictive of male infertility [55, 56]. However, the significance of WBC activation in ROS generation and its impact on elevated OS

Among spermatozoa, it has been shown that morphologically abnormal spermatozoa are the main source of ROS generation [61]. Excess residual cytoplasm (ERC) around the mid-piece of spermatozoa (observed in teratozoospermic sperm) contains high levels of cytoplasmic enzymes responsible for generating ROS [62]. ERC has a considerable amount of enzymes to regulate glucose metabolism, specifically glucose-6-phosphate dehydrogenase (G6PD) [63], which induces

levels cannot be left unnoticed. Various studies reported high levels of proinflammatory chemokines in human semen along with high ROS quantity [57, 58]. Recently, in the seminal plasma of oligozoospermic and azoospermic men it was observed a negative correlation between levels of interleukin-6 (IL-6), interferon alpha (IFN-α) and interferon gamma (IFN-γ) and sperm parameters such as

converted into ROS. The ROS production is increased when the electron transporting chain (ETC) derails as a result of mitochondrial dysfunction [50]. Exogenous sources of ROS include smoking, alcohol and drugs abuse, environmental pollutants, heavy metals, ionizing radiation, diets rich in energy-yielding

nutrients like carbohydrates, saturated fats and proteins [51].

**2.2 Mechanism of ROS production within human sperm**

demolishing pathogens by ROS production [53, 54].

concentration, motility and morphology [59, 60].

—, H2O2 and OH, generated mainly in the mitochon-

*Mechanism of free radical production within semen. (a) The intrinsic and extrinsic pathway contribute in the formation of O2•-. (b) Superoxide is transformed directly and indirectly to secondary (e, f, g) ROS. Adapted from reference [28]. (mathematical symbols + and - stand for positive and negative feedback).*

increased ROS levels by activating (1) the NADPH - nicotinamide adenine dinucleotide phosphate located in the plasma membrane of spermatozoa, and (2) NADPH – dependent oxide-reductase, known as diphorase, detected in the middle piece of mitochondrial level [64–66]. In a study by Sabeur et al., calcium-dependent NADPH oxidase 5 (NOX5) of spermatozoa plays a considerable role in ROS generation [67]. However, there is a difference between NOX5 found in spermatozoa, which does not require protein kinase C for expressing its activity, and in leukocytes, where protein kinase C is essential [68].

#### **2.3 Physiological role of ROS**

ROS are very important molecules as they act as cellular mediators essential for (1) normal spermatogenesis, (2) activation of steroidogenic pathway, (3) modulation of mitochondrial and death receptor-apoptotic pathways. These fundamental cascades are required for the process of: maturation, hyperactivation, capacitation, acrosome reaction as well as sperm-oocyte fusion, crucial for the fertilization process, all presented in **Figure 2**.

#### *2.3.1 Maturation*

After spermiation, spermatozoa are transported into the epididymis where they undergo a maturation process, leading to significant chemical and physiological modifications including recombination of cell-surface proteins, and enzymatic and

limited capability to repair DNA damage [76]. Protamination occurs when sperma-

Another important event is the formation of "mitochondrial capsule" made

Hyperactivation is a particular state of sperm motility characterized by vigorous,

Undoubtedly, ROS play an inclusive role in the regulation of these processes, by triggering hyperactivation and capacitation. This occurs by induction of Ca2+ and

 influx, probably through the deactivation of the enzyme Ca2+-ATPase and further basification of the cytosol [80]. ROS (especially O2•-) upregulate the Ca2+ mediate adenylate cyclase (AC) enzymatic activity, increasing cAMP (cyclic adenosine monophosphate) generation by activating protein kinase A (PKA). Further, this triggers NADPH oxidase activation and thereby promotes the upregulation of ROS production [81]. PKA-mediated phosphorylation leads to protein tyrosine kinase (PTK) activation, phosphorylating consecutive tyrosine residues in the

large asymmetric flagellar (whiplash-type) beat and head sperm shifting (large lateral head displacement) [79]. Hyperactivation is reported to facilitate the capacitation process and is indispensable for successful accomplishment of acrosomal

tozoa pass through the caput and caudal part of the epididymis [77].

axonemal fibrous sheath and the cytoskeleton of sperm tail [69, 82].

Capacitation has been documented in 1951 by Austin and Chang [83, 84]. Capacitation involves cholesterol outflow from the sperm membrane and a global intensification of tyrosine phosphorylation [85]. The signal transduction pathway is guided by the cAMP and modulated by the oxido-reductive state [86]. During capacitation, spermatozoa undergo molecular modifications such as alkalization of inner cell pH, activation of cAMP-dependent pathways, cholesterol efflux from cell-membrane and phosphorylation of surface proteins by cAMP-dependent kinase [87]. Researchers have emphasized the impact of free radicals in modulating the cAMP pathway, which involves PKA activation and phosphorylation of its substrates [88]. A correlation between elevated protein phosphorylation rate, increased presence of the second messengers and ROS synthesis have been observed during capacitation [69]. The cholesterol oxidation and its consequent discharge from the sperm membrane is necessary in tuning-up spermatozoa for the next step, resulting in greater bicarbonate and Ca2+ ion permeability via activation of sodium/bicar-

The hyperactivated spermatozoon tends to penetrate over the cumulus-oocytecomplex and attach to the zona pellucida of the egg, whereas acrosome reaction (AR) is a well-regulated exocytotic reaction in response to coordinated stimuli [90]. These changes are triggered by tyrosine phosphorylation of sperm-membrane proteins regulated by ROS signaling [63, 88]. NO is implicated in AR by activating the second messenger cyclic guanosine-mono-phosphate (cGMP), PKC and protein

[88]. In the oocyte, the release of Ca2+ is followed by cleavage of phosphatydilinositol-4,5-bisphosphonate (PIP2) into inositol tri-phosphate (IP3) and

and NO are needed for AR

reaction, sperm-egg fusion, and fecundation [74].

*Male Infertility, Oxidative Stress and Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.98204*

bonate cotransporter (NBC) and ion channels [89].

kinase G (PKG) [91]. Physiological levels of H2O2, O2

*2.3.4 Acrosome reaction (AR)*

**267**

degradation [78].

HCO3

*2.3.3 Capacitation*

*2.3.2 Hyperactivation*

by a complex protein material, which is necessary to abolish proteolytic

#### **Figure 2.**

*Physiological and pathological consequences of ROS. ROS dose is a critical parameter in determining the ultimate cellular response, low (necessary) dose for physiological processes and high (toxicity) dose expressing their pathological effects.*

nuclear modifications [69, 70]. These result in the assembly of the signal transduction machinery that is crucial for the sperm capacity to undergo hyperactivation and capacitation [69, 71]. The nuclear DNA of mammalian spermatozoa is densely packed, as histones are substituted by smaller-sized (arginine-rich) protamine [72]. Protamines substitute histones during spermiogenesis [73] and compact DNA tightly through inter/intramolecular disulphide bonds between cysteine residues [74]. The oxidizing process of thiol groups on protamines and the formation of disulfide bonds increase chromatin stability and DNA protection from any physical or chemical damage [75], which is fundamental because human spermatozoa have

#### *Male Infertility, Oxidative Stress and Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.98204*

limited capability to repair DNA damage [76]. Protamination occurs when spermatozoa pass through the caput and caudal part of the epididymis [77].

Another important event is the formation of "mitochondrial capsule" made by a complex protein material, which is necessary to abolish proteolytic degradation [78].

#### *2.3.2 Hyperactivation*

Hyperactivation is a particular state of sperm motility characterized by vigorous, large asymmetric flagellar (whiplash-type) beat and head sperm shifting (large lateral head displacement) [79]. Hyperactivation is reported to facilitate the capacitation process and is indispensable for successful accomplishment of acrosomal reaction, sperm-egg fusion, and fecundation [74].

Undoubtedly, ROS play an inclusive role in the regulation of these processes, by triggering hyperactivation and capacitation. This occurs by induction of Ca2+ and HCO3 influx, probably through the deactivation of the enzyme Ca2+-ATPase and further basification of the cytosol [80]. ROS (especially O2•-) upregulate the Ca2+ mediate adenylate cyclase (AC) enzymatic activity, increasing cAMP (cyclic adenosine monophosphate) generation by activating protein kinase A (PKA). Further, this triggers NADPH oxidase activation and thereby promotes the upregulation of ROS production [81]. PKA-mediated phosphorylation leads to protein tyrosine kinase (PTK) activation, phosphorylating consecutive tyrosine residues in the axonemal fibrous sheath and the cytoskeleton of sperm tail [69, 82].

#### *2.3.3 Capacitation*

Capacitation has been documented in 1951 by Austin and Chang [83, 84]. Capacitation involves cholesterol outflow from the sperm membrane and a global intensification of tyrosine phosphorylation [85]. The signal transduction pathway is guided by the cAMP and modulated by the oxido-reductive state [86]. During capacitation, spermatozoa undergo molecular modifications such as alkalization of inner cell pH, activation of cAMP-dependent pathways, cholesterol efflux from cell-membrane and phosphorylation of surface proteins by cAMP-dependent kinase [87]. Researchers have emphasized the impact of free radicals in modulating the cAMP pathway, which involves PKA activation and phosphorylation of its substrates [88]. A correlation between elevated protein phosphorylation rate, increased presence of the second messengers and ROS synthesis have been observed during capacitation [69]. The cholesterol oxidation and its consequent discharge from the sperm membrane is necessary in tuning-up spermatozoa for the next step, resulting in greater bicarbonate and Ca2+ ion permeability via activation of sodium/bicarbonate cotransporter (NBC) and ion channels [89].

#### *2.3.4 Acrosome reaction (AR)*

The hyperactivated spermatozoon tends to penetrate over the cumulus-oocytecomplex and attach to the zona pellucida of the egg, whereas acrosome reaction (AR) is a well-regulated exocytotic reaction in response to coordinated stimuli [90]. These changes are triggered by tyrosine phosphorylation of sperm-membrane proteins regulated by ROS signaling [63, 88]. NO is implicated in AR by activating the second messenger cyclic guanosine-mono-phosphate (cGMP), PKC and protein kinase G (PKG) [91]. Physiological levels of H2O2, O2 and NO are needed for AR [88]. In the oocyte, the release of Ca2+ is followed by cleavage of phosphatydilinositol-4,5-bisphosphonate (PIP2) into inositol tri-phosphate (IP3) and

nuclear modifications [69, 70]. These result in the assembly of the signal transduction machinery that is crucial for the sperm capacity to undergo hyperactivation and capacitation [69, 71]. The nuclear DNA of mammalian spermatozoa is densely packed, as histones are substituted by smaller-sized (arginine-rich) protamine [72]. Protamines substitute histones during spermiogenesis [73] and compact DNA tightly through inter/intramolecular disulphide bonds between cysteine residues [74]. The oxidizing process of thiol groups on protamines and the formation of disulfide bonds increase chromatin stability and DNA protection from any physical or chemical damage [75], which is fundamental because human spermatozoa have

*Physiological and pathological consequences of ROS. ROS dose is a critical parameter in determining the ultimate cellular response, low (necessary) dose for physiological processes and high (toxicity) dose expressing*

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

**Figure 2.**

**266**

*their pathological effects.*

diacylglycerol (DAG), which are responsible for acrosomal exocytosis and activation of PKC. This further results in Ca2+ inflow and activation of PLA2 (phospholipase A2), which play a key role in the cleavage of secondary fatty and consequently increasing the membrane fluidity, necessary sperm-oocyte fusion [92].

generation by mitochondria, followed by the release of cytochrome C, which in turn activates the apoptotic caspases, triggering the apoptosis [74, 82, 105]. High levels of cytochrome C have been found in seminal plasma of infertile men [82, 106].

It is reported that infertile males with high seminal OS levels present high fragmentation of sperm DNA [107]. Numerous contributors can include lifestyle factors, radiation, advanced male age, varicocele, infection and idiopathic causes [108, 109]. Guanine base (G) is the most common DNA's organic base exposed to OS assault and converts into 8-hydroxy-deoxyguanosine (8-OHdG) by free radicals [110]. Mechanisms by which OS cause DNA damage involve warping single and double-stranded DNA crosslinks, direct oxidation of DNA bases and DNA mutations [111]. Comparing to nuclear DNA, mitochondrial DNA is more susceptible to DNA damage, due to the lack of histones and protamines, and nucleotide excision

In addition, mitochondrial damage affects the interior mitochondrial membrane, causing electron outflow from the transporting chain, inducing a further increase of

Mitochondria represent the most important place in generating ATPs, which serves as a fuel for sperm to move. This is why its proper function represents a fundamental key point in the mosaic of male infertility problems. Defects in the pathway for ATP production correlate with low sperm motility, known as

asthenozoospermia [114]. There is an inactivation of genes which encode constituting proteins of the electron transport chain, mainly those that are involved in ATP formation [115]. When the extent of such injury overwhelms DNA repair capacity mechanisms, the subsequent alterations in mitochondrial biology stimulate the activation of the genes responsible for stress–response, hereby inducing apoptosis [116].

Formation of radical amino acids is of the result of protein oxidation (PO), especially of the alpha-central carbon, causing disintegration of peptide skeletons [117]. Moreover, the SH-rich lateral chains of methionine and cysteine are inclined to be oxidised with propagation of methionine sulphoxide and disulphides, respectively [87]. Similarly, arginine, proline, threonine and lysine are oxidised, resulting in the formation of carbonylated proteins (aldehyde and ketones), markers of PO status [117]. These alterations impact the protein morphology and physiology, with

Antioxidants are defined as chemicals compounds with the ability to donate electrons and thereby neutralize an excessive production of ROS [118]. Humans possess a well-sophisticated antioxidant system to shelter the body's cells and tissues

As a physiological response to OS, seminal plasma is endowed with various scavengers acting enzymes indexed as total antioxidant capacity (TAC) measured

a wide impact on spermatogenesis and fertility potential.

**3. Antioxidants in male infertility treatment**

to be 10x higher comparing to blood plasma [120].

*2.4.3 DNA damage*

*Male Infertility, Oxidative Stress and Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.98204*

repair mechanisms [112].

*2.4.4 Mitochondrial dysfunction*

OS status [113].

*2.4.5 Protein oxidation*

against oxidation [119].

**269**

#### *2.3.5 Sperm-oocyte fusion*

ROS are also necessary in the finalization of the fertilization process. This final step is due to enhanced membrane fluidity, which is controlled and directed by ROS in inhibiting the protein tyrosine phosphatase activity, which prevents deactivation of PLA2, a necessary step for accomplishing sperm-oocyte fusion [93]. When the spermatozoon penetrates the zona pellucida and the corona radiata, the oocyte changes the composition of the vitelline layer [24]. This envelope is catalyzed by ovoperoxidase making o,o-dityrosine crosslinks to prevent polyspermy [94].

#### **2.4 Pathological repercussions of oxidative stress**

High levels of ROS have the potential to damage cellular components by mediating lipid peroxidation, apoptosis, DNA damage, mitochondrial dysfunction and protein oxidation.

#### *2.4.1 Lipid peroxidation (LPO)*

Sperm membranes are mostly constituted by poly-unsaturated fatty acid (PUFAs), which represents a disadvantage in terms of OS susceptibility [95].

Lipid peroxidation (LPO) is as a chemical reaction by which oxidants assault carbon double bond(s) in lipid compounds, especially PUFAs, by detaching hydrogen and adding oxygen to carbon, by generating LOO• and LOOH [96]. *In vitro* research highlighted a negative correlation between malondialdehyde (MDA - end product of LPO) concentration, and sperm morphology and motility [97–99]. LPO is a self-propagating process passing through three phases: (1) initiation; (2) propagation; (3) termination. Through all three phases free radicals enter in a radical-chain reaction [32].

The propagation of the oxidative wave can also result in DNA fragmentation and protein damage, affecting particularly sperm motility, morphology and fertilizing capacity.

#### *2.4.2 Apoptosis*

The programmed cell death, known as apoptosis, is a physiological phenomenon. In the male reproductive tract, apoptosis is responsible for supervising the excess production of male gametes, a process being regulated by extrinsic and intrinsic stimuli [80]. The intrinsic stimuli include apoptosis-including genes like p53, Bax and Fas, but also Bcl-2 and c-kit genes which act as apoptosis suppressors [100], while extrinsic stimuli consist of varicocele, infection, heat stress, environmental toxins, advanced male age lifestyle factors, ionizing and nonionizing radiations, defective protamination and idiopathic causes [101, 102] . During the process of spermatogenesis, spontaneous germ cell apoptosis in all developing stages of spermatozoa has been seen in the testis of normozoospermic and non-obstructive azoospermic men [20]. This guarantees that only functionally and genetically competent germ cells become mature spermatozoa [103]. Prolactin and insulin are considered as pro-survival hormones which bind to specific receptors on sperm membrane [104]. The inhibition of this cascade will result in increased ROS

generation by mitochondria, followed by the release of cytochrome C, which in turn activates the apoptotic caspases, triggering the apoptosis [74, 82, 105]. High levels of cytochrome C have been found in seminal plasma of infertile men [82, 106].

#### *2.4.3 DNA damage*

diacylglycerol (DAG), which are responsible for acrosomal exocytosis and activation of PKC. This further results in Ca2+ inflow and activation of PLA2 (phospholipase A2), which play a key role in the cleavage of secondary fatty and consequently

ROS are also necessary in the finalization of the fertilization process. This final step is due to enhanced membrane fluidity, which is controlled and directed by ROS in inhibiting the protein tyrosine phosphatase activity, which prevents deactivation of PLA2, a necessary step for accomplishing sperm-oocyte fusion [93]. When the spermatozoon penetrates the zona pellucida and the corona radiata, the oocyte changes the composition of the vitelline layer [24]. This envelope is catalyzed by ovoperoxidase making o,o-dityrosine crosslinks to prevent polyspermy [94].

High levels of ROS have the potential to damage cellular components by mediating lipid peroxidation, apoptosis, DNA damage, mitochondrial dysfunction and

Sperm membranes are mostly constituted by poly-unsaturated fatty acid (PUFAs), which represents a disadvantage in terms of OS susceptibility [95]. Lipid peroxidation (LPO) is as a chemical reaction by which oxidants assault carbon double bond(s) in lipid compounds, especially PUFAs, by detaching hydrogen and adding oxygen to carbon, by generating LOO• and LOOH [96]. *In vitro* research highlighted a negative correlation between malondialdehyde (MDA - end product of LPO) concentration, and sperm morphology and motility [97–99]. LPO is a self-propagating process passing through three phases: (1) initiation; (2) propagation; (3) termination. Through all three phases free radicals enter in a

The propagation of the oxidative wave can also result in DNA fragmentation and protein damage, affecting particularly sperm motility, morphology and fertilizing

The programmed cell death, known as apoptosis, is a physiological phenomenon. In the male reproductive tract, apoptosis is responsible for supervising the excess production of male gametes, a process being regulated by extrinsic and intrinsic stimuli [80]. The intrinsic stimuli include apoptosis-including genes like p53, Bax and Fas, but also Bcl-2 and c-kit genes which act as apoptosis suppressors [100], while extrinsic stimuli consist of varicocele, infection, heat stress, environmental toxins, advanced male age lifestyle factors, ionizing and nonionizing radiations, defective protamination and idiopathic causes [101, 102] . During the process of spermatogenesis, spontaneous germ cell apoptosis in all developing stages of spermatozoa has been seen in the testis of normozoospermic and non-obstructive azoospermic men [20]. This guarantees that only functionally and genetically competent germ cells become mature spermatozoa [103]. Prolactin and insulin are considered as pro-survival hormones which bind to specific receptors on sperm membrane [104]. The inhibition of this cascade will result in increased ROS

increasing the membrane fluidity, necessary sperm-oocyte fusion [92].

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

**2.4 Pathological repercussions of oxidative stress**

*2.3.5 Sperm-oocyte fusion*

protein oxidation.

*2.4.1 Lipid peroxidation (LPO)*

radical-chain reaction [32].

capacity.

**268**

*2.4.2 Apoptosis*

It is reported that infertile males with high seminal OS levels present high fragmentation of sperm DNA [107]. Numerous contributors can include lifestyle factors, radiation, advanced male age, varicocele, infection and idiopathic causes [108, 109]. Guanine base (G) is the most common DNA's organic base exposed to OS assault and converts into 8-hydroxy-deoxyguanosine (8-OHdG) by free radicals [110]. Mechanisms by which OS cause DNA damage involve warping single and double-stranded DNA crosslinks, direct oxidation of DNA bases and DNA mutations [111]. Comparing to nuclear DNA, mitochondrial DNA is more susceptible to DNA damage, due to the lack of histones and protamines, and nucleotide excision repair mechanisms [112].

In addition, mitochondrial damage affects the interior mitochondrial membrane, causing electron outflow from the transporting chain, inducing a further increase of OS status [113].

#### *2.4.4 Mitochondrial dysfunction*

Mitochondria represent the most important place in generating ATPs, which serves as a fuel for sperm to move. This is why its proper function represents a fundamental key point in the mosaic of male infertility problems. Defects in the pathway for ATP production correlate with low sperm motility, known as asthenozoospermia [114]. There is an inactivation of genes which encode constituting proteins of the electron transport chain, mainly those that are involved in ATP formation [115]. When the extent of such injury overwhelms DNA repair capacity mechanisms, the subsequent alterations in mitochondrial biology stimulate the activation of the genes responsible for stress–response, hereby inducing apoptosis [116].

#### *2.4.5 Protein oxidation*

Formation of radical amino acids is of the result of protein oxidation (PO), especially of the alpha-central carbon, causing disintegration of peptide skeletons [117]. Moreover, the SH-rich lateral chains of methionine and cysteine are inclined to be oxidised with propagation of methionine sulphoxide and disulphides, respectively [87]. Similarly, arginine, proline, threonine and lysine are oxidised, resulting in the formation of carbonylated proteins (aldehyde and ketones), markers of PO status [117]. These alterations impact the protein morphology and physiology, with a wide impact on spermatogenesis and fertility potential.

#### **3. Antioxidants in male infertility treatment**

Antioxidants are defined as chemicals compounds with the ability to donate electrons and thereby neutralize an excessive production of ROS [118]. Humans possess a well-sophisticated antioxidant system to shelter the body's cells and tissues against oxidation [119].

As a physiological response to OS, seminal plasma is endowed with various scavengers acting enzymes indexed as total antioxidant capacity (TAC) measured to be 10x higher comparing to blood plasma [120].

The anti-oxidant defense system implicates a co-action of different endo/exogenous players to scavenge the potential oxidative damage of ROS [121]. These consist of CAT, SOD, glutathione peroxidase (GPx), peroxiredoxins and glutathione-Stransferase [122], and water-soluble and fat-soluble vitamins [123]. The role and effect of endogenous and exogenous antioxidants are discussed below.

neutralizes peroxides [135]. It is mainly expressed in the mitochondrial sperm matrix, while nuclear isoform of GPx has been correlated with sperm DNA preservation from oxidative detrimental impact and chromatin condensation [136]. GPx reduces fat hydroperoxides into alcohols and free H2O2 to H2O, it is fundamental for protecting lipid integrity and maintaining sperm viability and membrane integrity [134].

Most common exogenous antioxidants refer to carnitines, α-tocopherol, ascorbic

L-carnitine (LC) and L-acetyl carnitine (LAC), a water-soluble antioxidant, are implicated in sperm metabolism, motility and viability [147]. It helps in preventing lipid peroxidation, sperm DNA protection and apoptosis [148]. The highest concentration of carnitine is found in the epididymis and spermatozoa [132]. Studies of the semen samples of infertile men, especially oligoasthenoteratozoospermic (OAT)

This is a water-soluble vitamin. Humans and other vertebrates lack the enzyme L-glucono-gamma lactone oxidase (LGGLO), which is essential for *in vivo* synthesis. Hence, its intake with diet or as a supplement is fundamental. Vitamin C concentration is 10-times higher in seminal plasma comparing to serum [149]. It nullifies the activity of •OH, O2•- and H2O2 radicals, thereby protecting against

Carotenoids can be found naturally in fruits and vegetables. Carotenoid cannot be synthesized by humans, by introduced by the diet. Lycopene, a fat-soluble aromatic

enoids seem to be more effective [151]. It can alter the levels of antioxidant enzymes by modification of the levels of ROS, making great contribution to the human antioxidant system [43, 119]. There are studies on fertile men that show high concentration of Lycopene, and reduced levels in seminal plasma of infertile men [152].

CoQ10 is an intermediate of the mitochondrial electron transport chain [153, 154]. Low seminal plasma/sperm concentrations of CoQ10 have been

Zn is one of the most abundant elements in human [156]. It acts as metalloprotein cofactor in the metabolism of nucleic acids transcription, signal transduction, protein synthesis and cell death regulation [157]. Moreover, Zn is fundamental

O2, but a combination of carot-

acid, carotenoids, zinc and selenium. Spermatozoa carry with them minimal endogenous antioxidant amounts, thus during the entire process of spermatogenesis, sperm rely on exogenous antioxidants [137]. Studies about their efficacy in

men, have shown lower carnitine levels compared to fertile men [133].

**3.2 Exogenous antioxidants**

*3.2.1 Carnitines*

clinical trials are presented in **Table 4**.

*Male Infertility, Oxidative Stress and Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.98204*

*3.2.2 Vitamin C (L-ascorbic acid)*

carotenoid, is reported to be strong neutralizer of <sup>1</sup>

associated with reduced sperm motility [155].

oxidative damage [150].

*3.2.4 Coenzyme Q-10 (CoQ10)*

*3.2.3 Carotenoids*

*3.2.5 Zinc (Zn)*

**271**

#### **3.1 Endogenous antioxidants**

The major endogenous antioxidant enzymes are: (1) CAT, (2) SOD and (3) GPx. Studies about their efficacy in clinical trials are presented in **Table 3**.

#### *3.1.1 Catalase*

Activity of catalase (tetrameric protein) is consisted in dissolving hydrogen peroxide into water and oxygen, through the oxidation of hydrogen ion donors, such as methanol (CH3OH), ethanol (CH3CH2OH), with the consumption of 1 mol of H2O2 [128]. In addition, CAT has an important role in terms of physiological effects during sperm capacitation, inducing NO activity and the removal of ROS [129].

#### *3.1.2 Superoxide dismutase (SOD)*

SOD is known as metallo-enzyme, as it has the catalytic metal in the active site [130]. The SOD enzyme consists of three different classes existing in both extraand intracellular compartments. SOD-1 or CuZnSOD is the first intracellular enzyme, with Cu and Zn in the active center; it is usually localized in the cytosol [131]. SOD-2 or MnSOD is the second intracellular isoform, localizing in mitochondria and showing Mn in the active center [132]. The extracellular form of SOD (EC-SOD or SOD-3) is a glycosylated homotetramer mainly secreted into the extracellular area. It is upregulated by cytokines, downregulated by TNF-α, and anchored to the extracellular matrix [133]. CuZnSOD is highly active (75%) in comparison with SOD-3 (25%) [119, 130].

#### *3.1.3 Glutathione peroxidase (GPX)*

GPx is a cytosolic antioxidant seleno-enzyme mainly expressed in the epididymis and testis [134]. GPx catalyzes the reduction of detrimental hydroperoxides with thiol cofactors [119]. A "catalytic triad" is formed by the selenocysteine in the active site with tryptophan and glutamine: this activates the selenium portion and


#### **Table 3.**

*The role of endogenous antioxidants enzymes.*

neutralizes peroxides [135]. It is mainly expressed in the mitochondrial sperm matrix, while nuclear isoform of GPx has been correlated with sperm DNA preservation from oxidative detrimental impact and chromatin condensation [136]. GPx reduces fat hydroperoxides into alcohols and free H2O2 to H2O, it is fundamental for protecting lipid integrity and maintaining sperm viability and membrane integrity [134].

#### **3.2 Exogenous antioxidants**

Most common exogenous antioxidants refer to carnitines, α-tocopherol, ascorbic acid, carotenoids, zinc and selenium. Spermatozoa carry with them minimal endogenous antioxidant amounts, thus during the entire process of spermatogenesis, sperm rely on exogenous antioxidants [137]. Studies about their efficacy in clinical trials are presented in **Table 4**.

#### *3.2.1 Carnitines*

The anti-oxidant defense system implicates a co-action of different endo/exogenous players to scavenge the potential oxidative damage of ROS [121]. These consist of CAT, SOD, glutathione peroxidase (GPx), peroxiredoxins and glutathione-Stransferase [122], and water-soluble and fat-soluble vitamins [123]. The role and

The major endogenous antioxidant enzymes are: (1) CAT, (2) SOD and (3) GPx.

Activity of catalase (tetrameric protein) is consisted in dissolving hydrogen peroxide into water and oxygen, through the oxidation of hydrogen ion donors, such as methanol (CH3OH), ethanol (CH3CH2OH), with the consumption of 1 mol of H2O2 [128]. In addition, CAT has an important role in terms of physiological effects during

SOD is known as metallo-enzyme, as it has the catalytic metal in the active site [130]. The SOD enzyme consists of three different classes existing in both extraand intracellular compartments. SOD-1 or CuZnSOD is the first intracellular enzyme, with Cu and Zn in the active center; it is usually localized in the cytosol [131]. SOD-2 or MnSOD is the second intracellular isoform, localizing in mitochondria and showing Mn in the active center [132]. The extracellular form of SOD (EC-SOD or SOD-3) is a glycosylated homotetramer mainly secreted into the extracellular area. It is upregulated by cytokines, downregulated by TNF-α, and anchored to the extracellular matrix [133]. CuZnSOD is highly active (75%) in comparison with

GPx is a cytosolic antioxidant seleno-enzyme mainly expressed in the epididymis and testis [134]. GPx catalyzes the reduction of detrimental hydroperoxides with thiol cofactors [119]. A "catalytic triad" is formed by the selenocysteine in the active

[124, 125]

[126]

[127]

site with tryptophan and glutamine: this activates the selenium portion and

• Positive correlation between levels of CAT and fertilization rates.

SOD • Its levels are positively associated with sperm concentration (p<0.001) and

• Negative relationship was found with DNA fragmentation (p=0.014).

GPx • 10x greater GPx activity in the fertile group comparing with the GPx activity

control samples that received placebo.

• Studies are limited in this field.

• Statistically significant (p<0.001).

motility (p=0.008).

in infertile men.

*The role of endogenous antioxidants enzymes.*

**Table 3.**

**270**

**Enzyme Study findings Ref.** CAT • ↑ CAT activity in the group that received antioxidant therapy, comparing to

effect of endogenous and exogenous antioxidants are discussed below.

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

Studies about their efficacy in clinical trials are presented in **Table 3**.

sperm capacitation, inducing NO activity and the removal of ROS [129].

**3.1 Endogenous antioxidants**

*3.1.2 Superoxide dismutase (SOD)*

SOD-3 (25%) [119, 130].

*3.1.3 Glutathione peroxidase (GPX)*

*3.1.1 Catalase*

L-carnitine (LC) and L-acetyl carnitine (LAC), a water-soluble antioxidant, are implicated in sperm metabolism, motility and viability [147]. It helps in preventing lipid peroxidation, sperm DNA protection and apoptosis [148]. The highest concentration of carnitine is found in the epididymis and spermatozoa [132]. Studies of the semen samples of infertile men, especially oligoasthenoteratozoospermic (OAT) men, have shown lower carnitine levels compared to fertile men [133].

#### *3.2.2 Vitamin C (L-ascorbic acid)*

This is a water-soluble vitamin. Humans and other vertebrates lack the enzyme L-glucono-gamma lactone oxidase (LGGLO), which is essential for *in vivo* synthesis. Hence, its intake with diet or as a supplement is fundamental. Vitamin C concentration is 10-times higher in seminal plasma comparing to serum [149]. It nullifies the activity of •OH, O2•- and H2O2 radicals, thereby protecting against oxidative damage [150].

#### *3.2.3 Carotenoids*

Carotenoids can be found naturally in fruits and vegetables. Carotenoid cannot be synthesized by humans, by introduced by the diet. Lycopene, a fat-soluble aromatic carotenoid, is reported to be strong neutralizer of <sup>1</sup> O2, but a combination of carotenoids seem to be more effective [151]. It can alter the levels of antioxidant enzymes by modification of the levels of ROS, making great contribution to the human antioxidant system [43, 119]. There are studies on fertile men that show high concentration of Lycopene, and reduced levels in seminal plasma of infertile men [152].

#### *3.2.4 Coenzyme Q-10 (CoQ10)*

CoQ10 is an intermediate of the mitochondrial electron transport chain [153, 154]. Low seminal plasma/sperm concentrations of CoQ10 have been associated with reduced sperm motility [155].

#### *3.2.5 Zinc (Zn)*

Zn is one of the most abundant elements in human [156]. It acts as metalloprotein cofactor in the metabolism of nucleic acids transcription, signal transduction, protein synthesis and cell death regulation [157]. Moreover, Zn is fundamental


depends on the integrity of the mitochondrial sheath) [162]. Effects and the roles of

Various vitamin e isoforms have been found, but their role and importance remains enigmatic, and of the eight naturally occurring forms, only α-tocopherol is maintained in the plasma [163]. Therefore, vitamin E is crucial in maintaining all the necessary functions of healthy sperm and protecting it from detrimental effects of OS. Studies show lower levels of vitamin E in infertile men compared to fertile men [135], allowing somehow to increase concentration of the peroxidation byproduct MDA in the seminal fluid [164]. It is mainly used in combination with other vitamins and minerals. In vitro and in vivo studies which show improvements exclusively in the sperm motility and other semen parameters, successful pregnan-

cies and mitigation of oxidative stress markers, presented in **Table 5**.

spermatogonia (72 3 days), or for three to six months [175, 176].

Vitamin E intake and its dosage should exclusively be determined by a healthcare professional because of adverse events due to vitamin toxicity. The recommended daily dose of vitamin E is 15 mg (30 IU) for adults [173], a dose of 200–800 mg/day may cause gastrointestinal distress, while a daily dose greater than 1000 mg (1500 IU) is associated with increased risk of hemorrhage (antiplatelet effects), thrombophlebitis,, elevated creatinine, gonadal dysfunction and death

Infertile patients which want to increase concentrations vitamin E, its sources can be found in nuts, seeds, vegetable oils, leafy vegetables and fortified cereals. It needs proper and critical analysis for establishing the correct dosage and duration of antioxidants administration. In case of raised OS status, remedy must be administered at least for 12 weeks, according to the proper minimal period for

Referring to the studies analyzed above, vitamin E consumption has its obvious beneficial effects. But, the question here is whether vitamin E is more effective solely or in a combination? If used solely, is the efficacy more accentuated in *in vivo*

vitamin E are presented in **Figure 3**.

*Male Infertility, Oxidative Stress and Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.98204*

[163, 174].

**Figure 3.**

**273**

*Effects of vitamin E in male reproduction physiology.*

#### **Table 4.**

*The role and effect of exogenous antioxidants enzymes.*

for optimal sustain of spermatogenesis and adequate function of the male reproductive organs [158]. It also plays a key role in preventing LPO and preserves sperm structure, by reducing generation of H2O2 and •OH, through separating active redox transition metals, such as Fe and Cu [144].

#### *3.2.6 Selenium (Se)*

Se is an important trace mineral, implicated in many biological processes. Se is the constituent of enzymes such as GPx and seleno-proteins, it shows a major impact in redox defense system, spermatogenesis and increased fertility capacity in both males and females [159]. It protects sperm DNA against OS damage, although the mechanism is still unclear [160].

#### *3.2.7 Role and effect of vitamin E in male reproduction*

Vitamin E is the major lipophilic antioxidant [156] and it has been recognized as an essential nutrient for reproduction since its discovery in 1922 [161]. It neutralizes •OH and O2•- by lessening lipid per-oxidation commenced by ROS, thus protecting cell membranes from oxidation [160]. Vitamin E ameliorates other scavenging oxidants manners and helps maintaining sperm morphology and motility (which

#### *Male Infertility, Oxidative Stress and Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.98204*

depends on the integrity of the mitochondrial sheath) [162]. Effects and the roles of vitamin E are presented in **Figure 3**.

Various vitamin e isoforms have been found, but their role and importance remains enigmatic, and of the eight naturally occurring forms, only α-tocopherol is maintained in the plasma [163]. Therefore, vitamin E is crucial in maintaining all the necessary functions of healthy sperm and protecting it from detrimental effects of OS. Studies show lower levels of vitamin E in infertile men compared to fertile men [135], allowing somehow to increase concentration of the peroxidation byproduct MDA in the seminal fluid [164]. It is mainly used in combination with other vitamins and minerals. In vitro and in vivo studies which show improvements exclusively in the sperm motility and other semen parameters, successful pregnancies and mitigation of oxidative stress markers, presented in **Table 5**.

Vitamin E intake and its dosage should exclusively be determined by a healthcare professional because of adverse events due to vitamin toxicity. The recommended daily dose of vitamin E is 15 mg (30 IU) for adults [173], a dose of 200–800 mg/day may cause gastrointestinal distress, while a daily dose greater than 1000 mg (1500 IU) is associated with increased risk of hemorrhage (antiplatelet effects), thrombophlebitis,, elevated creatinine, gonadal dysfunction and death [163, 174].

Infertile patients which want to increase concentrations vitamin E, its sources can be found in nuts, seeds, vegetable oils, leafy vegetables and fortified cereals.

It needs proper and critical analysis for establishing the correct dosage and duration of antioxidants administration. In case of raised OS status, remedy must be administered at least for 12 weeks, according to the proper minimal period for spermatogonia (72 3 days), or for three to six months [175, 176].

Referring to the studies analyzed above, vitamin E consumption has its obvious beneficial effects. But, the question here is whether vitamin E is more effective solely or in a combination? If used solely, is the efficacy more accentuated in *in vivo*

**Figure 3.** *Effects of vitamin E in male reproduction physiology.*

for optimal sustain of spermatogenesis and adequate function of the male reproductive organs [158]. It also plays a key role in preventing LPO and preserves sperm structure, by reducing generation of H2O2 and •OH, through separating active

**Antioxidants Study findings Ref.**

remarkable increase in sperm motility and morphology.

Vit. C • Studies suggest positive association between levels of ascorbic acid in seminal plasma and sperm morphology and viability. • Very effective in controlling sperm agglutination.

Carotenoids • In a randomized clinical trial, Nouri et al. included 44 patients with

CoQ10 • Alahmar et al., study treated 65 oligoasthenozoospermic men and 40 fertile control groupwith 200 mg/day CoQ10 for 3 months. • Authors observed a significant improvement in total sperm motility, sperm concentration, TAC, and GPx levels as well as reduced SDF.

Zn • Randomized cross-sectional study and case study, combined antioxidant

• No significant change of Protein Carbonyl (PC) (p=0.554).

• Intake (two times daily, not more than 30 weeks) is associated with a

• Kobori et al. treated 169 males for 6 months with vitamin C, E and CoQ10, and reported a noteworthy improvement of sperm

• Tretament with 25 mg lycopene resulted in increased sperm count,

• Significantly correlated with sperm density (r = 0.341, p < 0.0001), motility (r = 0.253, p < 0.0001) and viability (r = 0.286, p < 0.0001) • Decrease levels of MDA, enhancing sperm motility and concentration

• Included 12 males, treated twice daily with 50 microgram in 3 months

• Significantly increase in sperm count (39.24 27.4–58.1 21.6; p<0.01), motility (22.14 12.9–50.7 17.6; p<0.01) and morphology [138, 139]

[140, 141]

[142]

[143]

[144, 145]

[146]

LC & LAC • Analyzed in certain systematic reviews and meta-analysis.

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

concentration and sperm motility.

concentration, total motility and TAC.

oligozoospermia.

formula.

(p < 0.001).

period.

*The role and effect of exogenous antioxidants enzymes.*

Se • Longitudinal study by Mossa et al.

(68 5.7–82.1 6.4; p<0.01).

Se is an important trace mineral, implicated in many biological processes. Se is

Vitamin E is the major lipophilic antioxidant [156] and it has been recognized as an essential nutrient for reproduction since its discovery in 1922 [161]. It neutralizes •OH and O2•- by lessening lipid per-oxidation commenced by ROS, thus protecting cell membranes from oxidation [160]. Vitamin E ameliorates other scavenging oxidants manners and helps maintaining sperm morphology and motility (which

the constituent of enzymes such as GPx and seleno-proteins, it shows a major impact in redox defense system, spermatogenesis and increased fertility capacity in both males and females [159]. It protects sperm DNA against OS damage, although

redox transition metals, such as Fe and Cu [144].

*3.2.7 Role and effect of vitamin E in male reproduction*

the mechanism is still unclear [160].

*3.2.6 Selenium (Se)*

**Table 4.**

**272**


or *in vitro* studies? Data presented above from different studies demonstrate the complexity and the unpredictability of vitamin E or antioxidant supplementation, even though there are studies that suggest improvements in sperm parameters, decrease of oxidative stress status, improvements in zona pellucida binding test and

citrate 25 mg s.1x1/6 months

Vit. E 400 mg s.1x1/2 months Vit. C 1000 mg s.1x1/2 months

**Dose/duration Results Ref.**

total sperm motility (p<0.001).

(p≤0.05);

parameters.

Increased sperm total motility

[172]

No significant effect on other

Vitamin E doesn't work only as an antioxidant, but it is also involved in the modulation of cellular responses by modulating enzymes or by regulating the

ROS are very important in certain physiological processes; however they can be very dangerous for male fertility potential if the levels overcome a physiological

Therefore, normal fine redox equilibrium between ROS and antioxidants is extremely important. The understanding of this fine balance will facilitate steps towards proper diagnosis and treatment in ideal dosages of antioxidant treatment. The most widely utilized antioxidants either as single therapy or combined are:

According to current literature we can conclude that vitamin E used alone is more effective when used for *in vitro* procedures, and very effective used in a dual, triple or more combinations in terms of sperm parameters and oxidative stress

Further augmentative clinical trials are needful to ascertain the right and effective antioxidant combination, for reliable and appropriate guiding of this sensitive

vitamin C, E, NAC, carnitines, CoQ10, zinc, selenium, and lycopene.

higher pregnancy rates.

**Study design Number of study**

men

**subjects/abnormality**

*Male Infertility, Oxidative Stress and Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.98204*

60 asthenozoospermic

*The role and effect of vitamin E solely and in combination.*

**4. Conclusion**

Randomized controlled trial

**Table 5.**

threshold.

status.

**275**

medical issue.

activity of specific transcription factors [173, 177].


**Table 5.**

**Study design Number of study**

Vitamin E *in vivo* studies

Double-blind, placebocontrolled, randomized study

Randomized placebocontrolled double-blind trial

Randomized controlled study

Double-blind randomized placebo crossover controlled

trial

Evaluation study

Experimental study

Randomized controlled trial

Comparative prospective randomized trial

**274**

Vitamin E *in vitro* studies

**subjects/abnormality**

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

101 couples (50 in the vitamin E group and 51 in the placebo group)

87 asthenospermic men (52 treated with vitamin E; 35 placebo treatment)

45 infertile men after varicocelectomy, n=22 receiving vitamin E and n=23 control group without supplementation.

30 healthy men with high levels of ROS in semen.

43 subjects, normal (n=23) and abnormal (n=20).

50 asthenoteratozoospermic men

Vitamin E in combination with one or more vitamins

90 idiopathic

men

oligoastheno-zoospermic

54 voluntary infertile men Vit. E 100 mg

**Dose/duration Results Ref.**

group;

birth);

control group.

Improvement of the performance of the spermatozoa in the zona pellucida binding test (p=0.004);

No significant effect was demonstrated in the conventional semen parameters and levels of ROS;

↑ post-thaw motility (p=0.041);

fragmentation.

(p<0.001).

parameters;

No improvements in sperm vitality and the degree of DNA

Significantly higher total sperm motility (p<0.001), progressive motility (p<0.001) and viability (p<0.001) compared with control group after 2, 4 and 6 hours of incubation; MDA levels were decreased significantly after 6 hours

Significant improve in sperm motility (p<0.05), without significant effects on other

Significant decrease in the MDA concentration.

Significant increase in sperm concentration (p=0.001); Improvement in the mean

placebo group;

↑motility in the vitamin E

Morphology was better in the

[165]

[162]

[166]

[167]

[168]

[169]

[170]

[171]

Statistically significant higher live-birth rate per transfer in the vitamin e group.

↑motility in the vitamin E group, comparing to placebo group (p<0.001);

↑Pregnancy (81% with a live

↓ MDA levels (sperm LPO).

No significant differences were found in terms of sperm count, sperm motility and pregnancy rates comparing to

400 mg/daily p.o

100 mg s.3x1 p.o./ 6 months

300 mg s.2x1 p.o./ 12 months

300 mg s.2x1 p.o./ 3 months

100 or 200 μmol Vitamin E to cryopreservation medium

2 mM (milli-molar) vitamin E.

s.2x2/3 months Selenium 35 μg s.3x2/3 months

Vit. E 400 mg s.1x1/6 months Clomiphene

*The role and effect of vitamin E solely and in combination.*

or *in vitro* studies? Data presented above from different studies demonstrate the complexity and the unpredictability of vitamin E or antioxidant supplementation, even though there are studies that suggest improvements in sperm parameters, decrease of oxidative stress status, improvements in zona pellucida binding test and higher pregnancy rates.

Vitamin E doesn't work only as an antioxidant, but it is also involved in the modulation of cellular responses by modulating enzymes or by regulating the activity of specific transcription factors [173, 177].

### **4. Conclusion**

ROS are very important in certain physiological processes; however they can be very dangerous for male fertility potential if the levels overcome a physiological threshold.

Therefore, normal fine redox equilibrium between ROS and antioxidants is extremely important. The understanding of this fine balance will facilitate steps towards proper diagnosis and treatment in ideal dosages of antioxidant treatment.

The most widely utilized antioxidants either as single therapy or combined are: vitamin C, E, NAC, carnitines, CoQ10, zinc, selenium, and lycopene.

According to current literature we can conclude that vitamin E used alone is more effective when used for *in vitro* procedures, and very effective used in a dual, triple or more combinations in terms of sperm parameters and oxidative stress status.

Further augmentative clinical trials are needful to ascertain the right and effective antioxidant combination, for reliable and appropriate guiding of this sensitive medical issue.

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*Male Infertility, Oxidative Stress and Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.98204*

> review of pharmacologic management. Expert Opin Pharmacother. 2012; 13:

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[12] Samal S, Dhadwe K, Gupta U. Epidemiological Study of Male Infertility. Indian Medical Gazette, 2012;5:174–80. DOI: 10.18203/ 2320-1770.ijrcog20161710

[13] Chandra A, Mosher WD. The demography of infertility and the use of medical care for infertility. Infertility and Reproductive Medicine Clinics of North America 1993; 52(2): 283–296. DOI: 10.1016/j.fertnstert.2015.10.007

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648–660 PMCID: 1684057.

2511–2531. DOI: 10.1517/ 14656566.2012.740011.

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[3] McDonald Evens E. A global perspective on infertility: An under recognized public health issue. Carolina Papers International Health.2004; 18: 1– 42 http://cgi.unc.edu/research/pdf/Eve

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[5] Inhorn MC, Patrizio P. Infertility around the globe: new thinking on gender, reproductive technologies and global movements in the 21st century. Hum Reprod Update. 2015; 21: 411–426.

DOI: 10.1093/humupd/dmv016

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**277**

[6] Sharlip ID, Jarow JP, Belker AM, Lipshultz LI, Sigman M, Thomas AJ, Schlegel PN, Howards SS, Nehra A, Damewood MD, Overstreet JW,

Sadovsky R. Best practice policies for male infertility. Fertil Steril. 2002;77(5):873–82. DOI: 10.1016/s0015-0282(02)03105-9.

10.1186/1742-4755-10-3

fertnstert.2009.09.009.

htm

ns.pdf

### **Author details**

Vegim Zhaku1 \*, Ashok Agarwal<sup>2</sup> , Sheqibe Beadini<sup>3</sup> , Ralf Henkel2,4,5, Renata Finelli2 , Nexhbedin Beadini<sup>6</sup> and Sava Micic<sup>7</sup>

1 Department of Physiology, Faculty of Medical Sciences, University of Tetovo, Tetovo, North Macedonia

2 American Center for Reproductive Medicine, Andrology Center, Cleveland, Ohio, USA

3 Department of Physiology and Biochemistry, Faculty of Medical Sciences, University of Tetovo, Tetovo, North Macedonia

4 Department of Metabolism, Digestion and Reproduction, Imperial College London, London, United Kingdom

5 Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa

6 Department of Cell and Molecular Biology and Human Genetics, Faculty of Medical Sciences, University of Tetovo, Tetovo, North Macedonia

7 Andrology Department, Uromedica Polyclinic, Belgrade, Serbia

\*Address all correspondence to: vegim.zhaku@unite.edu.mk

© 2021 The Author(s). Licensee IntechOpen. 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.

*Male Infertility, Oxidative Stress and Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.98204*

### **References**

**Author details**

\*, Ashok Agarwal<sup>2</sup>

University of Tetovo, Tetovo, North Macedonia

Nexhbedin Beadini<sup>6</sup> and Sava Micic<sup>7</sup>

London, London, United Kingdom

provided the original work is properly cited.

Tetovo, North Macedonia

, Sheqibe Beadini<sup>3</sup>

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

1 Department of Physiology, Faculty of Medical Sciences, University of Tetovo,

3 Department of Physiology and Biochemistry, Faculty of Medical Sciences,

4 Department of Metabolism, Digestion and Reproduction, Imperial College

5 Department of Medical Bioscience, University of the Western Cape, Bellville,

6 Department of Cell and Molecular Biology and Human Genetics, Faculty of

© 2021 The Author(s). Licensee IntechOpen. 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,

Medical Sciences, University of Tetovo, Tetovo, North Macedonia

7 Andrology Department, Uromedica Polyclinic, Belgrade, Serbia

\*Address all correspondence to: vegim.zhaku@unite.edu.mk

2 American Center for Reproductive Medicine, Andrology Center, Cleveland, Ohio,

, Ralf Henkel2,4,5, Renata Finelli2

,

Vegim Zhaku1

USA

South Africa

**276**

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[2] Convention on the Rights of Persons with Disabilities. 2007. Accessed on 30th December, 2020. URL: https://www.un. org/esa/socdev/enable/rights/convtexte. htm

[3] McDonald Evens E. A global perspective on infertility: An under recognized public health issue. Carolina Papers International Health.2004; 18: 1– 42 http://cgi.unc.edu/research/pdf/Eve ns.pdf

[4] Mumtaz Z, Shahid U, Levay A. Understanding the impact of gendered roles on the experiences of infertility amongst men and women in Punjab. Reprod Health. 2013;10: 3. DOI: 10.1186/1742-4755-10-3

[5] Inhorn MC, Patrizio P. Infertility around the globe: new thinking on gender, reproductive technologies and global movements in the 21st century. Hum Reprod Update. 2015; 21: 411–426. DOI: 10.1093/humupd/dmv016

[6] Sharlip ID, Jarow JP, Belker AM, Lipshultz LI, Sigman M, Thomas AJ, Schlegel PN, Howards SS, Nehra A, Damewood MD, Overstreet JW, Sadovsky R. Best practice policies for male infertility. Fertil Steril. 2002;77(5):873–82. DOI: 10.1016/s0015-0282(02)03105-9.

[7] Hamada AJ, Montgomery B, Agarwal A. Male infertility: a critical review of pharmacologic management. Expert Opin Pharmacother. 2012; 13: 2511–2531. DOI: 10.1517/ 14656566.2012.740011.

[8] Zhaku V, Beadini Sh, Beadini N, Xhaferi V, Golaboska J. The role of semen analysis in the expression of male infertility in southwestern part of North Macedonia (Experiences from 7 municipalities). UNIVERSI– International Journal of Education Science Technology Innovation Health and Enviroment. 2019;5(2):96–104.

[9] Taylor A. ABC of subfertility: extent of the problem. BMJ. 2003;327(7412): 434–436. DOI:10.1136/bmj.327.7412.434

[10] Gnoth C, Godehardt D, Godehardt E, Frank-Herrmann P, Freundl G. Time to pregnancy: results of the German prospective study and impact on the management of infertility. Hum Reprod. 2003;18(9): 1959–66. DOI: 10.1093/humrep/deg366.

[11] Zhao Y., Kolp L., Yates M., Zacur H. Clinical Evaluation of Female Factor Infertility. In: Carrell D., Peterson C. (eds) Reproductive Endocrinology and Infertility. Springer, New York, 2010. DOI:10.1007/978-1-4419-1436-1\_10

[12] Samal S, Dhadwe K, Gupta U. Epidemiological Study of Male Infertility. Indian Medical Gazette, 2012;5:174–80. DOI: 10.18203/ 2320-1770.ijrcog20161710

[13] Chandra A, Mosher WD. The demography of infertility and the use of medical care for infertility. Infertility and Reproductive Medicine Clinics of North America 1993; 52(2): 283–296. DOI: 10.1016/j.fertnstert.2015.10.007

[14] Lian Z, Zack M. M, Erickson J.D. Paternal age and the occurrence of birth defects. Am J Hum Genet. 1986; 39: 648–660 PMCID: 1684057.

[15] Sengupta P, Borges E Jr, Dutta S, Krajewska-Kulak E. Decline in sperm count in European men during the past 50 years. Hum Exp Toxicol. 2018; 37(3): 247–255. DOI: 10.1177/ 0960327117703690.

[16] Aggarwal R, Puri M, Dada R, Saurabh G. Correlation between leukocytospermia and oxidative stress in male partners of infertile couples with leukocytospermia. Int J Reprod Contracept Obstet Gynecol 2015;4:168–72. DOI: 10.5455/2320-1770.ijrcog 20150230.

[17] Forrester SJ, Kikuchi DS, Hernandes MS, Xu Q, Griendling KK. Reactive Oxygen Species in Metabolic and Inflammatory Signaling. Circ Res. 2018 Mar 16;122(6):877–902. DOI: 10.1161/CIRCRESAHA.117.311401.

[18] Papa S, Skulachev VP. Reactive oxygen species, mitochondria, apoptosis and aging. Mol Cell Biochem. 1997; 174 (1–2):305–19. PMID: 9309704.

[19] Sharma RK, Pasqualotto AE, Nelson DR, Thomas AJ Jr, Agarwal A. Relationship between seminal white blood cell counts and oxidative stress in men treated at an infertility clinic. J Androl. 2001; 22(4):575–583.PMID: 11451354.

[20] Agarwal A, Saleh RA, and Bedaiwy MA: Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril. 2003; 79(4): 829–843. DOI: 10.1016/s0015-0282(02) 04948-8.

[21] Sikka SC: Relative impact of oxidative stress on male reproductive function. Curr Med Chem. 2001; 8(7): 851–62. DOI: 10.2174/ 0929867013373039.

[22] Agarwal A, Prabakaran S, Allamaneni S. What an andrologist/ urologist should know about free radicals and why. Urology.2006;67:2–8. DOI:10.1016/j.urology.2005.07.012

[23] Doshi S. B, Khullar K., Sharma R. K., Agarwal A. Role of reactive nitrogen species in male infertility. Reproductive Biology and Endocrinology. 2012; 10, 109. DOI:10. 1186/1477–7827–10-109

[31] Mesa-Garcia M. D, Plaza-Diaz,J, Gomez-Llorente C. Molecular Basis of Oxidative Stress and Inflammation. In: Del Moral A.M, Garcia C.M.A. Obesity:

*Male Infertility, Oxidative Stress and Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.98204*

> plasma? A-meta analysis. Oncotarget. 2018;9(36):24494–24513. Published

[39] Nakamura BN, Lawson G, Chan JY, Banuelos J, Cortés MM, Hoang YD, Ortiz L, Rau BA, Luderer U. Knockout of the transcription factor NRF2 disrupts spermatogenesis in an agedependent manner. Free Radic Biol Med. 2010; 49(9):1368–1379. DOI:10. 1016/j.freeradbiomed.2010.07.019

[40] Rolf C, Cooper TG, Yeung CH, Nieschlag E. Antioxidant treatment of patients with asthenozoospermia or moderate oligoasthenozoospermia with high-dose vitamin C and vitamin E: a randomized, placebo-controlled, double-blind study. Hum Reprod.1999; 14: 1028–1033. DOI: 10.1093/humrep/

[41] Silver EW, Eskenazi B, Evenson DP, Block G, Young S, Wyrobek AJ. Effect

nonsmoking men. J Androl. 2005; 26: 550–556. DOI: 10.2164/jandrol.04165

[42] Menezo YJ, Hazout A, Panteix G, Robert F, Rollet J, Cohen-Bacrie P, Chapuis F, Clement P, Benkhalifa M. Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect. Reprod Biomed Online. 2007; 14: 418–421. DOI: 10.1016/s1472-6483(10)

[43] Castagne V, Lefevre K, Natero R, Clarke PG, Bedker DA. An optimal redox status for the survival of axotomized ganglion cells in the developing retina. Neuroscience. 1999; 93: 313–320. DOI: 10.1016/s0306-4522

[44] Madkour LH. Nanoparticles induce oxidative and endoplasmic reticulum

stresses. Springer, Cham,

Switzerland.2020; 329–401. DOI: 10.1007/978-3-030-37297-2

of antioxidant intake on sperm chromatin stability in healthy

2018 May 11. DOI:10.18632/

oncotarget.25075

14.4.1028

60887-5

(99)00138-4

antioxidants. Elsevier. 2018; 41–62. DOI: 10.1016/b978-0-12-812504-5.00003-9.

[32] Szabo C, Ischiropoulos H, Radi R.

pathophysiology and development of Aprioku JS therapeutics. Nat Rev Drug Discov. 2007;6(8):662–80. DOI:

Apoptosis, Autophagy, and Ferroptosis. Oxid Med Cell Longev. 2019 Oct 13; 2019:5080843. DOI: 10.1155/2019/

[34] Desai N, Sabanegh Jr, Kim E t, Agarwal A. Free radical theory of aging:

Urology. 2010; 75:14–19. DOI:10.1016/j.

[35] Ayala A, Muñoz MF, Argüelles S. Lipid peroxidation. In: Production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2 nonenal. Oxidative Medicine and Cellular Longevity. 2014; 1–31. DOI:

[36] Kothari S, Thompson A, Agarwal A,

du Plessis SS. Free radicals: their beneficial and detrimental effect on sperm function. Indian J EXP Biol. 2010;

48(5):425–35. PMID: 20795359.

DOI:10.5653/cerm.2018.45.2.57.

**279**

[38] Huang C, Cao X, Pang D, et al. Is male infertility associated with increased oxidative stress in seminal

[37] Alahmar AT. The effects of oral antioxidants on the semen of men with idiopathic oligoasthenoteratozoospermia. Clin Exp Reprod Med. 2018; 45(2):57–66.

Impli-cations in male infertility.

urology.2009.05.025

10.1155/2014/360438.

Oxidative stress and dietary

Peroxynitrite: biochemistry,

[33] Su LJ, Zhang JH, Gomez H, Murugan R, Hong X, Xu D, Jiang F, Peng ZY. Reactive Oxygen Species-Induced Lipid Peroxidation in

10.1038/nrd2222

5080843.

[24] Pryor WA, Houk KN, Foote CS, Fukuto JM, Ignarro LJ, Squadrito GL, Davies KJ. Free radical biology and medicine: it's a gas, man! Am J Physiol Regul Integr Comp Physiol. 2006;291 (3):R491–511. DOI: 10.1152/ ajpregu.00614.2005.

[25] Aruoma OI, Halliwell B, Gajewski E, Dizdaroglu M. Copper-ion-dependent damage to the bases in DNA in the presence of hydrogen peroxide. Biochem J. 1991;273 ( Pt 3):601–604. DOI:10.1042/bj2730601

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[27] Tvrdá E, Massanyi P, Lukáč N. Physiological and Pathological Roles of Free Rad-icals in Male Reproduction In: Rosaria Meccariello, Rosanna Chianese (Eds.), Sperma-tozoa - Facts and Perspectives. 2018. DOI:10.5772/ intechopen.70793

[28] Lampiao F, Opperman CJ, Agarwal A, du Plessis SS. Oxidative stress. In: S.J. Parekattil and A. Agarwal (eds.), Male Infertility: Contemporary Clinical Approaches, Andrology, ART & Antioxidants, 1st edn. Springer, New York, 2012; 225–235. DOI: 10.1007/ 978-1-4614-3335-4\_22.

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[31] Mesa-Garcia M. D, Plaza-Diaz,J, Gomez-Llorente C. Molecular Basis of Oxidative Stress and Inflammation. In: Del Moral A.M, Garcia C.M.A. Obesity: Oxidative stress and dietary antioxidants. Elsevier. 2018; 41–62. DOI: 10.1016/b978-0-12-812504-5.00003-9.

[15] Sengupta P, Borges E Jr, Dutta S, Krajewska-Kulak E. Decline in sperm count in European men during the past 50 years. Hum Exp Toxicol. 2018; 37(3):

*Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects*

[23] Doshi S. B, Khullar K., Sharma R. K., Agarwal A. Role of reactive nitrogen species in male infertility. Reproductive Biology and Endocrinology. 2012; 10, 109. DOI:10. 1186/1477–7827–10-109

[24] Pryor WA, Houk KN, Foote CS, Fukuto JM, Ignarro LJ, Squadrito GL, Davies KJ. Free radical biology and medicine: it's a gas, man! Am J Physiol Regul Integr Comp Physiol. 2006;291

[25] Aruoma OI, Halliwell B, Gajewski E, Dizdaroglu M. Copper-ion-dependent damage to the bases in DNA in the presence of hydrogen peroxide. Biochem J. 1991;273 ( Pt 3):601–604.

(3):R491–511. DOI: 10.1152/ ajpregu.00614.2005.

DOI:10.1042/bj2730601

DOI: 10.1071/rd9950659.

intechopen.70793

978-1-4614-3335-4\_22.

158–172. PMCID: 3911811.

[29] Schrader M, Fahimi HD. Mammalian peroxisomes and reactive oxygen species. Histochem Cell Biol. 2004;122(4):383–93. DOI: 10.1007/s00418-004-0673-1.

[30] Aprioku J.S. Pharamacology of free radicals and the impact of reactive oxygen species on the testis. Journal of reproduction & infertility. 2013; 14(4),

[26] Aitken RJ. Free radicals, lipid peroxidation and sperm function. Reprod Fertil Dev. 1995;7(4):659–68.

[27] Tvrdá E, Massanyi P, Lukáč N. Physiological and Pathological Roles of Free Rad-icals in Male Reproduction In: Rosaria Meccariello, Rosanna Chianese (Eds.), Sperma-tozoa - Facts and Perspectives. 2018. DOI:10.5772/

[28] Lampiao F, Opperman CJ, Agarwal A, du Plessis SS. Oxidative stress. In: S.J. Parekattil and A. Agarwal (eds.), Male Infertility: Contemporary Clinical Approaches, Andrology, ART & Antioxidants, 1st edn. Springer, New York, 2012; 225–235. DOI: 10.1007/

247–255. DOI: 10.1177/ 0960327117703690.

[16] Aggarwal R, Puri M, Dada R, Saurabh G. Correlation between

[17] Forrester SJ, Kikuchi DS,

leukocytospermia and oxidative stress in male partners of infertile couples with leukocytospermia. Int J Reprod

Contracept Obstet Gynecol 2015;4:168–72. DOI: 10.5455/2320-1770.ijrcog 20150230.

Hernandes MS, Xu Q, Griendling KK. Reactive Oxygen Species in Metabolic and Inflammatory Signaling. Circ Res. 2018 Mar 16;122(6):877–902. DOI: 10.1161/CIRCRESAHA.117.311401.

[18] Papa S, Skulachev VP. Reactive oxygen species, mitochondria, apoptosis and aging. Mol Cell Biochem. 1997; 174

(1–2):305–19. PMID: 9309704.

[19] Sharma RK, Pasqualotto AE, Nelson DR, Thomas AJ Jr, Agarwal A. Relationship between seminal white blood cell counts and oxidative stress in men treated at an infertility clinic. J Androl. 2001; 22(4):575–583.PMID:

[20] Agarwal A, Saleh RA, and Bedaiwy MA: Role of reactive oxygen species in

reproduction. Fertil Steril. 2003; 79(4): 829–843. DOI: 10.1016/s0015-0282(02)

the pathophysiology of human

[21] Sikka SC: Relative impact of oxidative stress on male reproductive function. Curr Med Chem. 2001; 8(7):

[22] Agarwal A, Prabakaran S, Allamaneni S. What an andrologist/ urologist should know about free radicals and why. Urology.2006;67:2–8. DOI:10.1016/j.urology.2005.07.012

851–62. DOI: 10.2174/ 0929867013373039.

11451354.

04948-8.

**278**

[32] Szabo C, Ischiropoulos H, Radi R. Peroxynitrite: biochemistry, pathophysiology and development of Aprioku JS therapeutics. Nat Rev Drug Discov. 2007;6(8):662–80. DOI: 10.1038/nrd2222

[33] Su LJ, Zhang JH, Gomez H, Murugan R, Hong X, Xu D, Jiang F, Peng ZY. Reactive Oxygen Species-Induced Lipid Peroxidation in Apoptosis, Autophagy, and Ferroptosis. Oxid Med Cell Longev. 2019 Oct 13; 2019:5080843. DOI: 10.1155/2019/ 5080843.

[34] Desai N, Sabanegh Jr, Kim E t, Agarwal A. Free radical theory of aging: Impli-cations in male infertility. Urology. 2010; 75:14–19. DOI:10.1016/j. urology.2009.05.025

[35] Ayala A, Muñoz MF, Argüelles S. Lipid peroxidation. In: Production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2 nonenal. Oxidative Medicine and Cellular Longevity. 2014; 1–31. DOI: 10.1155/2014/360438.

[36] Kothari S, Thompson A, Agarwal A, du Plessis SS. Free radicals: their beneficial and detrimental effect on sperm function. Indian J EXP Biol. 2010; 48(5):425–35. PMID: 20795359.

[37] Alahmar AT. The effects of oral antioxidants on the semen of men with idiopathic oligoasthenoteratozoospermia. Clin Exp Reprod Med. 2018; 45(2):57–66. DOI:10.5653/cerm.2018.45.2.57.

[38] Huang C, Cao X, Pang D, et al. Is male infertility associated with increased oxidative stress in seminal

plasma? A-meta analysis. Oncotarget. 2018;9(36):24494–24513. Published 2018 May 11. DOI:10.18632/ oncotarget.25075

[39] Nakamura BN, Lawson G, Chan JY, Banuelos J, Cortés MM, Hoang YD, Ortiz L, Rau BA, Luderer U. Knockout of the transcription factor NRF2 disrupts spermatogenesis in an agedependent manner. Free Radic Biol Med. 2010; 49(9):1368–1379. DOI:10. 1016/j.freeradbiomed.2010.07.019

[40] Rolf C, Cooper TG, Yeung CH, Nieschlag E. Antioxidant treatment of patients with asthenozoospermia or moderate oligoasthenozoospermia with high-dose vitamin C and vitamin E: a randomized, placebo-controlled, double-blind study. Hum Reprod.1999; 14: 1028–1033. DOI: 10.1093/humrep/ 14.4.1028

[41] Silver EW, Eskenazi B, Evenson DP, Block G, Young S, Wyrobek AJ. Effect of antioxidant intake on sperm chromatin stability in healthy nonsmoking men. J Androl. 2005; 26: 550–556. DOI: 10.2164/jandrol.04165

[42] Menezo YJ, Hazout A, Panteix G, Robert F, Rollet J, Cohen-Bacrie P, Chapuis F, Clement P, Benkhalifa M. Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect. Reprod Biomed Online. 2007; 14: 418–421. DOI: 10.1016/s1472-6483(10) 60887-5

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s0015-0282(16)57861-3.

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**288**

Elshenoufy A, Elghamrawi H, Fayad A, Abdelrahman S. Combination of vitamin E and clomiphene citrate in

motility and viability in

doi: 10.1111/and.13891.

2020.09.006.

### *Edited by Pınar Erkekoglu and Júlia Scherer Santos*

Vitamin E is a group of fat-soluble compounds found in a wide variety of foods. Daily requirements of vitamin E can be met with a balanced diet. High-dose supplementation may be hazardous rather than beneficial. Vitamin E serves as an antioxidant, participates in anti-inflammatory processes, inhibits platelet aggregation, and enhances immunity. Vitamin E supplementation can be beneficial against coronary artery disease, eye disorders, cognitive decline, cancer, and skin aging. This book will mainly focus on the diverse functions of vitamin E, importance of vitamin E status to provide a healthy lifespan, and the interaction between vitamin E and several pathological conditions. Readers will receive a general overview of the importance of vitamin E in health and different pathological conditions.

Published in London, UK © 2021 IntechOpen © Elenasfotos / iStock

Vitamin E in Health and Disease - Interactions, Diseases and Health Aspects

IntechOpen Book Series

Biochemistry, Volume 22

Vitamin E in Health

and Disease

Interactions, Diseases and Health Aspects

*Edited by Pınar Erkekoglu and Júlia Scherer Santos*