**2. Biochemistry of reactive oxygen species (ROS)**

Free radicals can be formed by three mechanisms:

 Homolytic cleavage of a covalent bond of a molecule, each fragment retaining one electron

$$\mathbb{X} \colon \mathbb{Y} \to \mathbb{X} + \mathbb{Y}^\cdot$$

Loss by a molecule of a single electron

$$\mathbf{A} \rightarrow \mathbf{A}^\cdot \mathbf{+e}^\cdot$$

Addition by a molecule of a single electron

$$\mathbf{A} \text{ e}^{\cdot} \to \mathbf{A}^{\cdot \cdot}$$

© 2013 Borza et al., licensee InTech. This is an open access chapter 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. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### 24 Lipid Metabolism

 Heterolytic cleavage – covalent bond electrons are held up by only one of the molecule's fragments. Basically, charged ions occur.

Oxidative Stress and Lipid Peroxidation – A Lipid Metabolism Dysfunction 25

*The reduction of O2 by two electrons* leads to the formation of hydrogen peroxide, H2O2.

H2O2 is often formed in biological systems via peroxide anion production.

way by which the bactericidal mechanism is achieved in phagocytosis.

presence of antioxidants, especially albumin.

 Different enzymatic catalyzed reactions Decomposition of endoperoxides

determining the complexity of the process [1].

Free radical recombination among them or,

The end of the reaction occurs by:

dismutase (SOD), catalase.

Singlet oxygen is formed in the following reactions:

Degradation of hydroperoxides in liver microsomes


2- 22 2 2O + 2H H O + O

H2O2 is not a free radical, but falls within the category of reactive oxygen species that include not only free radicals but also its non-radical derivatives involved in producing these ROS. Of all free radicals, H2O2 is the most stable and the easiest to quantify. Intracellular formation of hydrogen peroxide, depending on the content of catalase, is the

*The formation of singlet oxygen (1O2).* It represents an excited form of molecular oxygen, resulting from the absorption of an energy quantum. It is equated to a ROS due to its strong reactivity. Singlet oxygen has an electrophilic character, reacting with many organic compounds: polyunsaturated fatty acids, cholesterol, hydroperoxides or organic compounds containing S or N atoms, producing oxides. In plasma it is neutralized by the

Reaction of hydrogen peroxide or hydroxyl radical with the superoxide anion

**2.1. Free radicals resulting in lipid peroxidation propagation phase** 

stimulate the propagation of the reaction by forming a new radical.

Lipid peroxidation is a complex process consisting of three major phases: initiation, propagation and end of the reaction. The initiation phase is slow due to the need of accumulation of a sufficient quantity of ROS, followed by the activation process of oxygen which is the amplifier factor. The process' latency period is that which determines the continuation of reactions by altering the oxidative balance in favor of pro-oxidant factors. The evolution of these reactions is unpredictable due to the formation of own catalysts

Free radicals are very unstable, their lifetime being very short. Their reactivity results from their coupling at the end of the reaction, only for an unpaired electron to reappear, thus

Intervention of antioxidant systems with membrane or intracellular action: superoxide

$$\mathsf{X}\,\mathsf{Y} \to \mathsf{X}\colon \mathsf{Y}$$

Oxygen activation is the main factor that induces enhanced formation of ROS. Due to its presence in the atmosphere, but also in the body, free radicals reaction with oxygen is inevitable. A second characteristic of oxygen refers to its electronic structure. Thus, O2 has on the outer layer two unpaired electrons, each located on one orbital. Therefore, oxygen can be considered a free di-radical, but with a lower reactivity. Oxidation of this electron donor is achieved by spin inversion from the O2 reaction with transition metals or by univalent reduction in two phases of one electron [5]. These two mechanisms underlie oxidation reactions that occur in nature. Although this process represents only 5%, following the univalent reduction of O2, ROS occurs, with greater reactivity and toxicity, as is the hydroxyl radical OH.

In biological systems, the most important free radicals are oxygen derivate radicals formed by the following mechanisms:

*O2 reduction by the transfer of an electron* will result in the synthesis of the superoxide anion (O2.-). The formation of the superoxide anion is the first step of O2 activation and occurs in the body during normal metabolic processes. In some cells, its production is continuous, which implies the existence of intracellular antioxidants [3].

$$\text{O}\_2 + \text{e}^- \rightarrow \text{O}\_2$$

*Tissue alteration* by traumatic, chemical or infectious means causes cell lysis along with the release of iron from deposits or by the action of hydrolases on metalloproteinase.

*Reduced transition metal autooxidation* generates the superoxide anion. The reaction of transition metal ions with O2 can be considered a reversible redox reaction, important in promoting ROS formation.

$$\begin{array}{rcl} \text{Fe}^{2+} \text{O}\_{2} & \rightarrow & \text{Fe}^{3+} \text{O}\_{2-} \\\\ \text{Cu} + \text{O}\_{2} & \rightarrow & \text{Cu}^{2+} \text{O}\_{2-} \end{array}$$

Degradation of H2O2 in the presence of transition metal ions leads to the formation of the most reactive and toxic ROS: the hydroxyl radical (OH.) (Fenton and Haber-Weiss reaction). To this radical, the body does not present antioxidant defense systems such as for the superoxide anion or hydrogen peroxide (H2O2). Although metallothioneins (natural antioxidants) are proteins that bind to metal ions, including Fe2+, thus inhibiting the Haber-Weiss reaction, however they are found in too low concentrations in the body to be effective in the decomposition of the hydroxyl radical. But these reactions can be inhibited by specific scavengers for OH, such as mannitol and chelating agents: desferroxamine. However, chelators as EDTA stimulate this reaction.

*The reduction of O2 by two electrons* leads to the formation of hydrogen peroxide, H2O2.

$$\rm O\_2 + 2e^- + 2H \rightarrow H\_2O\_2$$

H2O2 is often formed in biological systems via peroxide anion production.

$$\text{2O}\_2 + \text{2H} \rightarrow \text{H}\_2\text{O}\_2 + \text{O}\_2$$

H2O2 is not a free radical, but falls within the category of reactive oxygen species that include not only free radicals but also its non-radical derivatives involved in producing these ROS. Of all free radicals, H2O2 is the most stable and the easiest to quantify. Intracellular formation of hydrogen peroxide, depending on the content of catalase, is the way by which the bactericidal mechanism is achieved in phagocytosis.

*The formation of singlet oxygen (1O2).* It represents an excited form of molecular oxygen, resulting from the absorption of an energy quantum. It is equated to a ROS due to its strong reactivity. Singlet oxygen has an electrophilic character, reacting with many organic compounds: polyunsaturated fatty acids, cholesterol, hydroperoxides or organic compounds containing S or N atoms, producing oxides. In plasma it is neutralized by the presence of antioxidants, especially albumin.

Singlet oxygen is formed in the following reactions:


24 Lipid Metabolism

is the hydroxyl radical OH.

by the following mechanisms:

promoting ROS formation.

chelators as EDTA stimulate this reaction.

Heterolytic cleavage – covalent bond electrons are held up by only one of the


In biological systems, the most important free radicals are oxygen derivate radicals formed

*O2 reduction by the transfer of an electron* will result in the synthesis of the superoxide anion (O2.-). The formation of the superoxide anion is the first step of O2 activation and occurs in the body during normal metabolic processes. In some cells, its production is continuous,


*Tissue alteration* by traumatic, chemical or infectious means causes cell lysis along with the

*Reduced transition metal autooxidation* generates the superoxide anion. The reaction of transition metal ions with O2 can be considered a reversible redox reaction, important in

> 2 3 <sup>2</sup> 2- Fe + O Fe + O

<sup>2</sup> Cu + O Cu + O <sup>2</sup> 2-

Degradation of H2O2 in the presence of transition metal ions leads to the formation of the most reactive and toxic ROS: the hydroxyl radical (OH.) (Fenton and Haber-Weiss reaction). To this radical, the body does not present antioxidant defense systems such as for the superoxide anion or hydrogen peroxide (H2O2). Although metallothioneins (natural antioxidants) are proteins that bind to metal ions, including Fe2+, thus inhibiting the Haber-Weiss reaction, however they are found in too low concentrations in the body to be effective in the decomposition of the hydroxyl radical. But these reactions can be inhibited by specific scavengers for OH, such as mannitol and chelating agents: desferroxamine. However,

release of iron from deposits or by the action of hydrolases on metalloproteinase.

molecule's fragments. Basically, charged ions occur.

which implies the existence of intracellular antioxidants [3].

Degradation of hydroperoxides in liver microsomes

#### **2.1. Free radicals resulting in lipid peroxidation propagation phase**

Lipid peroxidation is a complex process consisting of three major phases: initiation, propagation and end of the reaction. The initiation phase is slow due to the need of accumulation of a sufficient quantity of ROS, followed by the activation process of oxygen which is the amplifier factor. The process' latency period is that which determines the continuation of reactions by altering the oxidative balance in favor of pro-oxidant factors. The evolution of these reactions is unpredictable due to the formation of own catalysts determining the complexity of the process [1].

Free radicals are very unstable, their lifetime being very short. Their reactivity results from their coupling at the end of the reaction, only for an unpaired electron to reappear, thus stimulate the propagation of the reaction by forming a new radical.

The end of the reaction occurs by:


#### 26 Lipid Metabolism

Peroxides and their decomposition products (aldehydes, lipofuscin) are the most stable and represent the final link of O2 activation. They are produced directly by the hydroxyl or singlet oxygen radical. During these reactions, own catalysts are formed, represented by free radicals or degradation products that diversify and increase the oxidation reactions; the structures involved are diverse, and are represented by polyunsaturated fatty acids, hemoproteins, nucleic acids, carbohydrates or steroids [4].

Oxidative Stress and Lipid Peroxidation – A Lipid Metabolism Dysfunction 27

The sequence of reactions initiated in the membrane continues into the cytoplasm where a substantial amount of superoxide anion is formed which then is diffuses also extracellularly. Increased use of glucose occurs for energetic purposes and for restoring NADPH and

Hydrogen peroxide is toxic on the neutrophil, which is inhibited by the presence at this level of the three enzymes that degrade the excess of peroxide: GSH-peroxidase, catalase and

The enzyme present in phagosome, myeloperoxidase, will catalyze in the presence of H2O2


In turn, hypochlorous acid can react with aminic groups or with the ammonium ion (NH4) forming chloramines. In the presence of hydrogen peroxide, HOCl forms singlet oxygen.

Based on the properties of leukocytes to emit chemiluminescence during phagocytosis, this method has a clinic utility. Chemiluminescence emission is due to formation of free radicals, lipid peroxides and prostaglandin synthesis, a process associated with phagocytosis. This property is suppressed by anesthesia, cytostatic agents and anti-inflammatory preparations. Drugs with anti-inflammatory effect inhibit the activity of cyclooxygenase, the enzyme

A deficiency in the leukocyte production of free radicals (septic granulomatosis) or decrease of myeloperoxidase activity (following corticotherapy) is characterized by particularly

oxygen dependent mechanism involves activation of myeloperoxidase and other

 Nitrogen compounds dependent mechanism involving participation of NO, NO2, other nitrogen oxides and nitrites. In this mechanism both types of cytotoxic inorganic

 The third mechanism is independent of oxygen and nitrogen by changing phagolysosome pH that favors the action of antimicrobial substances present in the

The constitutive form of NO synthase is found in endothelial cells, neutrophils, neurons. The existence of the inducible form has been shown in macrophages, hepatocytes, endothelial cells, neutrophils and platelets. Glucocorticoids inhibit the expression of

Nitrogen reactive radicals have a cytotoxic effect by inhibiting mitochondrial respiration,

DNA synthesis, and mediate oxidation of protein and non-protein sulfhydryl groups.

During phagocytosis, three cytotoxic and antimicrobial effect mechanisms take place:

oxygen consumption necessary for the production of ROS [8].

and chloride ions, forming toxic halogenated derivatives.

involved in prostaglandin synthesis.

lysosomal or nuclear level.

sensitivity to infections.

peroxidases

These products of activated leukocytes have bactericidal properties.

oxidants interact: oxygen and nitrogen reactive radicals.

inducible NO synthase but not of the constitutive enzyme.

myeloperoxidase.
