**3. The production of reactive oxygen species**

#### **3.1. Endogenous production**

*The electron transfer in the respiratory chain* involves an incomplete reduction of molecular O2 at a rate of 1-2% with the formation of superoxide anion and of singlet oxygen.

If the anion is released in a low in protons environment, it will initiate peroxidation, the substrate being formed by polyunsaturated fatty acids from cell membranes.

If the anion will reach a proton rich environment, dismutation will take place; this following auditioning an electron from another anion and by proton reaction will form hydrogen peroxide. Dismutation can occur spontaneously, but in this case it takes place very slowly or catalyzed by SOD, which increases 1010 the reaction rate to the body's pH. There is an inversely proportional relationship between reaction rate and pH value. The efficiency of this enzyme is proven by its presence in all aerobic cells, and cells exposed to oxygen action, as hepatocytes and erythrocytes, contain large amounts of SOD [6].

$$\text{O}\_2 + \text{e}^- \rightarrow \text{O}\_2^- + 2\text{H} \rightarrow \text{H}\_2\text{O}\_2$$

Superoxide anion production during mitochondrial respiration has a self-regulation mechanism. Superoxide anion formed in part by autoxidation of NADH dehydrogenase, can then induce this enzyme's inactivation, so the presence of SOD in the membrane matrix to achieve dismutation is absolutely necessary. It results that the two enzymes SOD and NADPH dehydrogenase are a metabolic control and energy preservation couple in the presence of oxygen [7].

The release of hydrogen peroxide is proportional to the partial pressure of O2. In case of a cerebral or cardiac ischemia, extramitochondrial concentration decreases, disrupting oxidative phosphorylation and ATP levels. An inversely proportional relationship between mitochondrial H2O2 formation rate and lifetime exists. Thus, it was observed experimentally that old animals present an increase in lipid peroxide formation in the mitochondria as a result of increased production of superoxide anion compared with young animals.

*Antimicrobial defense*. Phagocytosis of bacterial germs is accompanied by a massive production of superoxide anions and other derivatives (OH- , HOCl, H2O2, 1O2) from the leukocyte metabolism. A NADPH-dependent oxidase, activated by protein kinase C and arachidonic acid released under the action of phospholipase A allow anion synthesis with an increased consumption of O2.

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

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,

*The electron transfer in the respiratory chain* involves an incomplete reduction of molecular O2

If the anion is released in a low in protons environment, it will initiate peroxidation, the

If the anion will reach a proton rich environment, dismutation will take place; this following auditioning an electron from another anion and by proton reaction will form hydrogen peroxide. Dismutation can occur spontaneously, but in this case it takes place very slowly or catalyzed by SOD, which increases 1010 the reaction rate to the body's pH. There is an inversely proportional relationship between reaction rate and pH value. The efficiency of this enzyme is proven by its presence in all aerobic cells, and cells exposed to oxygen action,


Superoxide anion production during mitochondrial respiration has a self-regulation mechanism. Superoxide anion formed in part by autoxidation of NADH dehydrogenase, can then induce this enzyme's inactivation, so the presence of SOD in the membrane matrix to achieve dismutation is absolutely necessary. It results that the two enzymes SOD and NADPH dehydrogenase are a metabolic control and energy preservation couple in the

The release of hydrogen peroxide is proportional to the partial pressure of O2. In case of a cerebral or cardiac ischemia, extramitochondrial concentration decreases, disrupting oxidative phosphorylation and ATP levels. An inversely proportional relationship between mitochondrial H2O2 formation rate and lifetime exists. Thus, it was observed experimentally that old animals present an increase in lipid peroxide formation in the mitochondria as a

*Antimicrobial defense*. Phagocytosis of bacterial germs is accompanied by a massive

leukocyte metabolism. A NADPH-dependent oxidase, activated by protein kinase C and arachidonic acid released under the action of phospholipase A allow anion synthesis with

, HOCl, H2O2, 1O2) from the

result of increased production of superoxide anion compared with young animals.

production of superoxide anions and other derivatives (OH-

at a rate of 1-2% with the formation of superoxide anion and of singlet oxygen.

substrate being formed by polyunsaturated fatty acids from cell membranes.

as hepatocytes and erythrocytes, contain large amounts of SOD [6].

hemoproteins, nucleic acids, carbohydrates or steroids [4].

**3. The production of reactive oxygen species** 

**3.1. Endogenous production** 

presence of oxygen [7].

an increased consumption of O2.

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 myeloperoxidase.

The enzyme present in phagosome, myeloperoxidase, will catalyze in the presence of H2O2 and chloride ions, forming toxic halogenated derivatives.

$$\text{H}\_2\text{O}\_2 + \text{Cl}^- \rightarrow \text{ClO} + \text{H}\_2\text{O}$$

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. These products of activated leukocytes have bactericidal properties.

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 involved in prostaglandin synthesis.

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

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


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 inducible NO synthase but not of the constitutive enzyme.

Nitrogen reactive radicals have a cytotoxic effect by inhibiting mitochondrial respiration, DNA synthesis, and mediate oxidation of protein and non-protein sulfhydryl groups.

#### 28 Lipid Metabolism

Although NO has a protective role at the vascular level by a relaxing effect (EDRF), under certain conditions it may exert a cytotoxic effect, causing pathological vasodilatation, tissue destructions, inhibits platelet aggregation, modulates lymphocyte and immune response function.

Oxidative Stress and Lipid Peroxidation – A Lipid Metabolism Dysfunction 29

and NO2 may react with H2O2 produced by alveolar

performed under the action of

Ingestion of alcohol causes ROS synthesis by different mechanisms: xanthine oxidase and aldehyde oxidase can oxidase the main metabolite of ethanol (acetaldehyde) resulting in

Ethanol also stimulates the production of superoxide anion and, by inducing NADPH-

The alcohol ingestion decreases the activity of protective enzymes (SOD, glutathione peroxidase). Also low serum concentrations of selenium and vitamin E have been found in

Toxic substances as nitrogen oxide and nitrogen dioxide in the environment are responsible for autoxidation of polyunsaturated fatty acids in lung alveoli. The reaction may be

cytochrome P450 or in the presence of Fe2 is another factor that induces autoxidation of

Anticancer drugs are able to synthesize free radicals, this process depending on the mode of

These drugs under the action of cytochrome P450-dependent enzymes produce the activation of O2 with the formation of ROS which will attack GSH and other thiols (hemoglobin), causing the formation of lipid peroxides and activation of Ca2+-dependent

These mechanisms can induce disturbances of the coagulation system (increased hemolysis), severe forms of cardiomyopathies, because of the low level of cardiac antioxidants (AO)

In physiological conditions a delicate balance exists between ROS production and the antioxidant capacity. A higher ROS production and/or a decreased antioxidant capacity is responsible for the harmful effects of free radicals or the oxidative stress (OS). Oxidative stress represents an important pathogenic mechanism involved in inflammation,

End products of free radicals action, aldehydes, inhibit the activity of membrane enzymes (glucose-6-phosphate, adenylate cyclase). These aldehydes react selectively with proteins or

The emergence of OS is one of the most important pathogenic mechanisms involved in

oxidase synthesis, NADPH cytochrome reductase and P450 cytochrome.

polyunsaturated fatty acids, increasing lipid hydroperoxides concentration.

superoxide anion.

alcoholics.

reversible or irreversible. NO.

action and their toxicity.

cancerogenesis or aging [24].

endonucleases.

[25].

macrophages and can generate the hydroxyl radical.

The reduction of carbon tetrachloride (CCl4) in CCl3.

**4. Reactive oxygen species and oxidative stress** 

enzymes containing SH groups and cause tissue destructions.

inflammation, carcinogenesis, radiation disease and aging.

*Synthesis of prostaglandins*. Phospholipase A2 catalyzes the degradation of membrane phospholipids with arachidonic acid formation. Stimuli such as phagocytosis, antibody production, and immune complex formation, the action of bacterial endotoxins or cytokines stimulate the activation of this enzyme. There are two enzymatic forms: type I PLA2, membrane bound, which is stimulated by Ca2+ at physiological pH, and the type II one, cytoplasmic, which is inhibited by Ca2+ and is active at acidic pH. Two enzymes, lipoxygenase and cyclooxygenase, bound to plasmic and microsomal membranes, convert arachidonic acid in derivatives such as: thromboxane, prostaglandins, leukotrienes [18].

Under the action of lipoxygenase, arachidonic acid is converted into a hydroperoxide: hydroperoxyeicosatetraenoic acid (HPETE) which will release the hydroxyl radical during its transformation into hydroxyeicosatetraenoic acid (HETE). Hill et al. have emphasized the role of glutathione peroxidase (GSH-Px) and of glutathione in this reaction: blocking the activity of this enzyme, they have noticed a significant decrease (of 66%) of HPETE conversion in HETE [14, 16].

Under the action of cyclooxygenase, arachidonic acid incorporates two oxygen molecules to form an endoperoxide, PGG; it loses the OH group to form PGH. This transformation, which is accompanied by the release of hydroxyl radical, exerts a negative retro-control to prostaglandin synthesis, inactivating the cyclooxygenase. Some of the products developed have a complex effect on the inflammatory process: thus, in the first phase, PGE2 acts on cells from the vascular wall with a procoagulant effect, and in the late phase it has an inflammatory effect by inhibiting leukocyte activation and oxidative metabolism of these cells during phagocytosis. The byproducts resulting from this process will be the ones to modulate the intensity of the next phase [15, 22].

The two endoperoxides formed, PGG2 and PGH2, have an inducible role on the production of PCI2 or TxA, being involved in the mechanism that ensures homeostasis of the vascular and platelet phase of hemostasis.

The other enzyme has a dual effect, and promotes the initiation of lipid peroxidation and the decomposition of resulting products of these reactions.

#### **3.2. Exogenous production**

The human body is subjected to aggression from various agents capable of producing free radicals. Thus, UVs induce the synthesis of ROS and free radicals generating molecules via photosensitizing agents.

Ingestion of alcohol causes ROS synthesis by different mechanisms: xanthine oxidase and aldehyde oxidase can oxidase the main metabolite of ethanol (acetaldehyde) resulting in superoxide anion.

28 Lipid Metabolism

function.

leukotrienes [18].

conversion in HETE [14, 16].

modulate the intensity of the next phase [15, 22].

decomposition of resulting products of these reactions.

and platelet phase of hemostasis.

**3.2. Exogenous production** 

photosensitizing agents.

Although NO has a protective role at the vascular level by a relaxing effect (EDRF), under certain conditions it may exert a cytotoxic effect, causing pathological vasodilatation, tissue destructions, inhibits platelet aggregation, modulates lymphocyte and immune response

*Synthesis of prostaglandins*. Phospholipase A2 catalyzes the degradation of membrane phospholipids with arachidonic acid formation. Stimuli such as phagocytosis, antibody production, and immune complex formation, the action of bacterial endotoxins or cytokines stimulate the activation of this enzyme. There are two enzymatic forms: type I PLA2, membrane bound, which is stimulated by Ca2+ at physiological pH, and the type II one, cytoplasmic, which is inhibited by Ca2+ and is active at acidic pH. Two enzymes, lipoxygenase and cyclooxygenase, bound to plasmic and microsomal membranes, convert arachidonic acid in derivatives such as: thromboxane, prostaglandins,

Under the action of lipoxygenase, arachidonic acid is converted into a hydroperoxide: hydroperoxyeicosatetraenoic acid (HPETE) which will release the hydroxyl radical during its transformation into hydroxyeicosatetraenoic acid (HETE). Hill et al. have emphasized the role of glutathione peroxidase (GSH-Px) and of glutathione in this reaction: blocking the activity of this enzyme, they have noticed a significant decrease (of 66%) of HPETE

Under the action of cyclooxygenase, arachidonic acid incorporates two oxygen molecules to form an endoperoxide, PGG; it loses the OH group to form PGH. This transformation, which is accompanied by the release of hydroxyl radical, exerts a negative retro-control to prostaglandin synthesis, inactivating the cyclooxygenase. Some of the products developed have a complex effect on the inflammatory process: thus, in the first phase, PGE2 acts on cells from the vascular wall with a procoagulant effect, and in the late phase it has an inflammatory effect by inhibiting leukocyte activation and oxidative metabolism of these cells during phagocytosis. The byproducts resulting from this process will be the ones to

The two endoperoxides formed, PGG2 and PGH2, have an inducible role on the production of PCI2 or TxA, being involved in the mechanism that ensures homeostasis of the vascular

The other enzyme has a dual effect, and promotes the initiation of lipid peroxidation and the

The human body is subjected to aggression from various agents capable of producing free radicals. Thus, UVs induce the synthesis of ROS and free radicals generating molecules via Ethanol also stimulates the production of superoxide anion and, by inducing NADPHoxidase synthesis, NADPH cytochrome reductase and P450 cytochrome.

The alcohol ingestion decreases the activity of protective enzymes (SOD, glutathione peroxidase). Also low serum concentrations of selenium and vitamin E have been found in alcoholics.

Toxic substances as nitrogen oxide and nitrogen dioxide in the environment are responsible for autoxidation of polyunsaturated fatty acids in lung alveoli. The reaction may be reversible or irreversible. NO. and NO2 may react with H2O2 produced by alveolar macrophages and can generate the hydroxyl radical.

The reduction of carbon tetrachloride (CCl4) in CCl3. performed under the action of cytochrome P450 or in the presence of Fe2 is another factor that induces autoxidation of polyunsaturated fatty acids, increasing lipid hydroperoxides concentration.

Anticancer drugs are able to synthesize free radicals, this process depending on the mode of action and their toxicity.

These drugs under the action of cytochrome P450-dependent enzymes produce the activation of O2 with the formation of ROS which will attack GSH and other thiols (hemoglobin), causing the formation of lipid peroxides and activation of Ca2+-dependent endonucleases.

These mechanisms can induce disturbances of the coagulation system (increased hemolysis), severe forms of cardiomyopathies, because of the low level of cardiac antioxidants (AO) [25].
