**Lipid Peroxidation: Chemical Mechanism, Biological Implications and Analytical Determination**

Marisa Repetto, Jimena Semprine and Alberto Boveris

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/45943

## **1. Introduction**

Currently, lipid peroxidation is considered as the main molecular mechanisms involved in the oxidative damage to cell structures and in the toxicity process that lead to cell death. First, lipid peroxidation was studied for food scientists as a mechanism for the damage to alimentary oils and fats, nevertheless other researchers considered that lipid peroxidation was the consequence of toxic metabolites (e.g. CCl4) that produced highly reactive species, disruption of the intracellular membranes and cellular damage (Dianzani & Barrera, 2008).

Lipid peroxidation is a complex process known to occur in both plants and animals. It involves the formation and propagation of lipid radicals, the uptake of oxygen, a rearrangement of the double bonds in unsaturated lipids and the eventual destruction of membrane lipids, with the production of a variety of breakdown products, including alcohols, ketones, alkanes, aldehydes and ethers (Dianzani & Barrera, 2008).

In pathological situations the reactive oxygen and nitrogen species are generated at higher than normal rates, and as a consequence, lipid peroxidation occurs with -tocopherol deficiency. In addition to containing high concentrations of polyunsaturated fatty acids and transition metals, biological membranes of cells and organelles are constantly being subjected to various types of damage (Chance et al., 1979; Halliwell & Gutteridge, 1984). The mechanism of biological damage and the toxicity of these reactive species on biological systems are currently explained by the sequential stages of reversible oxidative stress and irreversible oxidative damage. Oxidative stress is understood as an imbalance situation with increased oxidants or decreased antioxidants (Sies, 1991a; Boveris et al., 2008). The concept implies the recognition of the physiological production of oxidants (oxidizing free-radicals and related species) and the existence of operative antioxidant defenses. The imbalance

© 2012 Repetto 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. © 2012 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.

concept recognizes the physiological effectiveness of the antioxidant defenses in maintaining both oxidative stress and cellular damage at a minimum level in physiological conditions (Boveris et al., 2008).

Lipid peroxidation is a chain reaction initiated by the hydrogen abstraction or addition of an oxygen radical, resulting in the oxidative damage of polyunsaturated fatty acids (PUFA). Since polyunsaturated fatty acids are more sensitive than saturated ones, it is obvious that the activated methylene (RH) bridge represents a critical target site. The presence of a double bond adjacent to a methylene group makes the methylene C-H bond weaker and therefore the hydrogen in more susceptible to abstraction. This leaves an unpaired electron on the carbon, forming a carbon-centered radical, which is stabilized by a molecular rearrangement of the double bonds to form a conjugated diene which then combines with oxygen to form a peroxyl radical. The peroxyl radical is itself capable of abstracting a hydrogen atom from another polyunsaturated fatty acid and so of starting a chain reaction (Halliwell & Gutteridge, 1984) (Fig. 1).

**Figure 1.** Initiation step of lipid peroxidation process.

Molecular oxygen rapidly adds to the carbon-centered radicals (R. ) formed in this process, yielding lipid peroxyl radicals (ROO. ). Decomposition of lipid peroxides is catalyzed by transition metal complexes yielding alcoxyl (RO. ) or hydroxyl (HO. ) radicals. These participate in chain reaction initiation that in turn abstract hydrogen and perpetuate the chain reaction of lipid peroxidation. The formation of peroxyl radicals leads to the production of organic hydroperoxides, which, in turn, can subtract hydrogen from another PUFA. This reaction is termed propagation, implying that one initiating hit can result in the conversion of numerous PUFA to lipid hydroperoxides. In sequence of their appearance, alkyl, peroxyl and alkoxyl radicals are involved. The resulting fatty acid radical is stabilized by rearrangement into a conjugated diene that retains the more stable products including hydroperoxides, alcohols, aldehydes and alkanes. Lipid hydroperoxide (ROOH) is the first, comparatively stable, product of the lipid peroxidation reaction (Halliwell & Gutteridge, 1984) (Fig. 2).

**Figure 2.** Initial phase of the propagation step of lipid peroxidation process indicating the oxygen uptake.

Reduced iron complexes (Fe2+) react with lipid peroxides (ROOH) to give alkoxy radicals, whereas oxidized iron complexes (Fe3+) react more slowly to produce peroxyl radicals. Both radicals can take part in the propagation of the chain reaction. The end products of these complex metal ion-catalyzed breakdowns of lipid hydroperoxides include the cytotoxic aldehydes and hydrocarbon gases such as ethane.

The free radical chain reaction propagates until two free radicals conjugate each other to terminate the chain. The reaction can also terminate in the presence of a chain-breaking antioxidant such as vitamin E (α-tocopherol) (Halliwell & Gutteridge, 1984).

In conditions in which lipid peroxidation is continuously initiated it gives non-radical products destroying two radicals at a time. In the presence of transition metal ions, ROOH can give rise to the generation of radicals capable of re-initiating lipid peroxidation by redox-cycling of these metal ions (Halliwell & Gutteridge, 1984).

Lipid peroxidation causes a decrease in membrane fluidity and in the barrier functions of the membranes. The many products of lipid peroxidation such as hydroperoxides or their aldehyde derivatives inhibit protein synthesis, blood macrophage actions and alter chemotactic signals and enzyme activity (Fridovich & Porter, 1981).

## **2. Biological implications of lipid peroxidation**

4 Lipid Peroxidation

conditions (Boveris et al., 2008).

(Halliwell & Gutteridge, 1984) (Fig. 1).

**Figure 1.** Initiation step of lipid peroxidation process.

transition metal complexes yielding alcoxyl (RO.

yielding lipid peroxyl radicals (ROO.

uptake.

Molecular oxygen rapidly adds to the carbon-centered radicals (R.

concept recognizes the physiological effectiveness of the antioxidant defenses in maintaining both oxidative stress and cellular damage at a minimum level in physiological

Lipid peroxidation is a chain reaction initiated by the hydrogen abstraction or addition of an oxygen radical, resulting in the oxidative damage of polyunsaturated fatty acids (PUFA). Since polyunsaturated fatty acids are more sensitive than saturated ones, it is obvious that the activated methylene (RH) bridge represents a critical target site. The presence of a double bond adjacent to a methylene group makes the methylene C-H bond weaker and therefore the hydrogen in more susceptible to abstraction. This leaves an unpaired electron on the carbon, forming a carbon-centered radical, which is stabilized by a molecular rearrangement of the double bonds to form a conjugated diene which then combines with oxygen to form a peroxyl radical. The peroxyl radical is itself capable of abstracting a hydrogen atom from another polyunsaturated fatty acid and so of starting a chain reaction

participate in chain reaction initiation that in turn abstract hydrogen and perpetuate the chain reaction of lipid peroxidation. The formation of peroxyl radicals leads to the production of organic hydroperoxides, which, in turn, can subtract hydrogen from another PUFA. This reaction is termed propagation, implying that one initiating hit can result in the conversion of numerous PUFA to lipid hydroperoxides. In sequence of their appearance, alkyl, peroxyl and alkoxyl radicals are involved. The resulting fatty acid radical is stabilized by rearrangement into a conjugated diene that retains the more stable products including hydroperoxides, alcohols, aldehydes and alkanes. Lipid hydroperoxide (ROOH) is the first, comparatively

stable, product of the lipid peroxidation reaction (Halliwell & Gutteridge, 1984) (Fig. 2).

**Figure 2.** Initial phase of the propagation step of lipid peroxidation process indicating the oxygen

) formed in this process,

) radicals. These

). Decomposition of lipid peroxides is catalyzed by

) or hydroxyl (HO.

The biological production of reactive oxygen species primarily superoxide anion (O2.-) and hydrogen peroxide (H2O2) is capable of damaging molecules of biochemical classes including nucleic acids and aminoacids. Exposure of reactive oxygen to proteins produces denaturation, loss of function, cross-linking, aggregation and fragmentation of connective tissues as collagen (Chance et al., 1979). However, the most damaging effect is the induction of lipid peroxidation. The cell membrane which is composed of poly-unsaturated fatty acids is a primary target for reactive oxygen attack leading to cell membrane damage.

The lipid peroxidation of polyunsaturated fatty acids may be enzymatic and non-enzymatic. Enzymatic lipid peroxidation is catalyzed by the lipoxygenases family, a family of lipid peroxidation enzymes that oxygenates free and esterified PUFA generating as a consequence, peroxy radicals. Non enzymatic lipid peroxidation and formation of lipidperoxides are initiated by the presence of molecular oxygen and is facilitated by Fe2+ ions (Repetto et al., 2010a).

Oxidative breakdown of biological phospholipids occurs in most cellular membranes including mitochondria, microsomes, peroxisomes and plasma membrane. The toxicity of lipid peroxidation products in mammals generally involves neurotoxicity, hepatotoxicity and nephrotoxicity (Boveris et al., 2008). The principal mechanism involves detoxification process in liver. Toxicity from lipid peroxidation affect the liver lipid metabolism where cytochrome P-450s is an efficient catalyst in the oxidative transformation of lipid derived aldehydes to carboxylic acids adding a new facet to the biological activity of lipid oxidation metabolites. Cytochrome P-450-mediated metabolism operates in parallel with other metabolic transformations of aldehydes; hence, the P450s could serve as reserve or

compensatory mechanisms when other high capacity pathways of aldehyde elimination are compromised due to disease or toxicity. Finally, 4-hydroxynonenal (HNE), unsaturated aldehydes, such as acrolein, trans-2-hexenal, and crotonaldehyde, are also food constituents or environmental pollutants, P-450s may be significant in favoring lipid peroxidation that has significant downstream effects and possibly play a major role in cell signaling pathways. Oxidized lipids appear to have a signaling function in pathological situations, are proinflammatory agonists and contribute to neuronal death under conditions in which membrane lipid peroxidation occurs. For example, mitochondrial lipid cardiolipin makes up to 18% of the total phospholipids and 90% of the fatty acyl chains are unsaturated. Oxidation of cardiolipin may be one of the critical factors initiating apoptosis by liberating cytochrome c from the mitochondrial inner membrane and facilitating permeabilization of the outer membrane. The release of cytochrome c activates a proteolytic cascade that culminates in apoptotic cell death (Navarro & Boveris, 2009).

Previous results indicate that lipid peroxidation has a role in the pathogenesis of several pathologies as neurodegenerative (Dominguez et al., 2008; Famulari et al., 1996; Fiszman et al., 2003), inflammatory (Farooqui & Farooqui, 2011), infectious (Repetto et al., 1996), gastric (Repetto et al., 2003) and nutritional diseases (Repetto et al., 2010b).

Oxidative damage in liver is associated with hepatic lipid metabolism, and may be affecting the absorption and transport mechanisms of -tocopherol in this organ. In the liver, the morphological damage is previous to the lipid peroxidation and the consumption of endogenous antioxidants. In kidney and heart, indeed, lipid peroxidation and oxidative damage preceded necrosis (Repetto et al., 2010b).

Lipid peroxidation is a chain reaction process characterized by repetitive hydrogen abstraction by HO. and RO. , and addition of O2 to alkyl radicals (R. ) resulting in the generation of ROO. and in the oxidative destruction of polyunsaturated fatty acids, in which the methylene group (=RH-) is the main target (Halliwell & Gutteridge, 1984).

The association between increased phospholipid oxidation, free-radical mediated reactions and pathological states was early recognized (Cadenas, 1989; Verstraeten et al., 1997; Liu et al., 2003). The contribution by Sies of the concept of oxidative stress followed (Sies, 1991a,1991b) with the implication that increased free-radical mediated reactions, basically by HO. and RO. , would produce phospholipid, protein, lipid, DNA, RNA or carbohydrate oxidation, whatever is close (Halliwell & Gutteridge, 1984). The increased oxidation of the cell biochemical constituents is associated with ultra structural changes in mitochondrial morphology with mitochondrial swelling and increased matrix volume (Boveris et al., 2008). In human liver, the morphological changes can affect the organ structure and function as it is the case for the bile canaliculi that are damaged in liver transplanted patients; a fact that is interpreted as consequence of the oxidative damage that is associated to ischemiareperfusion (Cutrin et al., 1996). Interestingly, there are reports in rat liver experimental models, of increased peroxidation secondary to increased mitochondrial production of O2 and H2O2 (Fridovich, 1978; Navarro &Boveris, 2007; Navarro et al., 2009).

#### **3. Chemical mechanisms for lipid peroxidation process**

6 Lipid Peroxidation

compensatory mechanisms when other high capacity pathways of aldehyde elimination are compromised due to disease or toxicity. Finally, 4-hydroxynonenal (HNE), unsaturated aldehydes, such as acrolein, trans-2-hexenal, and crotonaldehyde, are also food constituents or environmental pollutants, P-450s may be significant in favoring lipid peroxidation that has significant downstream effects and possibly play a major role in cell signaling pathways. Oxidized lipids appear to have a signaling function in pathological situations, are proinflammatory agonists and contribute to neuronal death under conditions in which membrane lipid peroxidation occurs. For example, mitochondrial lipid cardiolipin makes up to 18% of the total phospholipids and 90% of the fatty acyl chains are unsaturated. Oxidation of cardiolipin may be one of the critical factors initiating apoptosis by liberating cytochrome c from the mitochondrial inner membrane and facilitating permeabilization of the outer membrane. The release of cytochrome c activates a proteolytic cascade that

Previous results indicate that lipid peroxidation has a role in the pathogenesis of several pathologies as neurodegenerative (Dominguez et al., 2008; Famulari et al., 1996; Fiszman et al., 2003), inflammatory (Farooqui & Farooqui, 2011), infectious (Repetto et al., 1996), gastric

Oxidative damage in liver is associated with hepatic lipid metabolism, and may be affecting the absorption and transport mechanisms of -tocopherol in this organ. In the liver, the morphological damage is previous to the lipid peroxidation and the consumption of endogenous antioxidants. In kidney and heart, indeed, lipid peroxidation and oxidative

Lipid peroxidation is a chain reaction process characterized by repetitive hydrogen

The association between increased phospholipid oxidation, free-radical mediated reactions and pathological states was early recognized (Cadenas, 1989; Verstraeten et al., 1997; Liu et al., 2003). The contribution by Sies of the concept of oxidative stress followed (Sies, 1991a,1991b) with the implication that increased free-radical mediated reactions, basically

oxidation, whatever is close (Halliwell & Gutteridge, 1984). The increased oxidation of the cell biochemical constituents is associated with ultra structural changes in mitochondrial morphology with mitochondrial swelling and increased matrix volume (Boveris et al., 2008). In human liver, the morphological changes can affect the organ structure and function as it is the case for the bile canaliculi that are damaged in liver transplanted patients; a fact that is interpreted as consequence of the oxidative damage that is associated to ischemiareperfusion (Cutrin et al., 1996). Interestingly, there are reports in rat liver experimental models, of increased peroxidation secondary to increased mitochondrial production of O2-

the methylene group (=RH-) is the main target (Halliwell & Gutteridge, 1984).

and H2O2 (Fridovich, 1978; Navarro &Boveris, 2007; Navarro et al., 2009).

, and addition of O2 to alkyl radicals (R.

and in the oxidative destruction of polyunsaturated fatty acids, in which

, would produce phospholipid, protein, lipid, DNA, RNA or carbohydrate

) resulting in the

culminates in apoptotic cell death (Navarro & Boveris, 2009).

damage preceded necrosis (Repetto et al., 2010b).

and RO.

abstraction by HO.

generation of ROO.

and RO.

by HO.

(Repetto et al., 2003) and nutritional diseases (Repetto et al., 2010b).

The spectrum of oxygen reactive species that are considered responsible for biological oxygen toxicity include the intermediates of the partial reduction of oxygen, superoxide radical (O2.-), hydrogen peroxide (H2O2), and other reactive species as hydroxyl radicals (HO**.** ), peroxyl radical (ROO**.** ), nitric oxide (NO), peroxinitrite (ONOO-) and singlet oxygen (1O2).

The biological effects of excess levels of the spectrum of these species are quite similar, and that is the reason they are collectively called reactive oxygen species (ROS). The main freeradical mediated chain reactions in biological systems are summarized in Fig. 3. The Beckman-Radi-Freeman pathway and the Cadenas-Poderoso shunt have been added to the original consecutive reactions of the Fenton/Haber-Weiss pathway and lipid peroxidation process to incorporate NO and ONOO. to the biochemical free-radical mediated chain reaction (Moncada et al., 1991; Boveris et al., 2008) (Fig. 3).

In the last years the denominations "reactive oxygen species" (ROS) and "reactive nitrogen species" (RNS) had became very popular. The ROS denomination involves the three chemical species of the Fenton/Haber-Weiss pathway (O2- , H2O2 and HO. ), the products of the partial reduction of oxygen. Similarly, the RNS denomination is loosely referring to the three chemical species of the Beckman-Radi-Freeman pathway (NO, ONOO. , and NO2) (Moncada et al., 1991). The reference as a whole to either group, ROS and RNS, is usually made to explain or to refer to their biological activity, what reflects the fact that each group, ROS and RNS, are auto-propagated in biological systems from their promoters, O2 and NO. Nevertheless, the advantage and facility in referring to the biological effects implies the ignorance of the biochemistry of the process.

**Figure 3.** The free-radical mediated chain reaction in biochemistry. O2.-, superoxide radical; H2O2, hydrogen peroxide, HO·, hydroxyl radical; NO, nitric oxide; ONOO. , peroxinitrite; ·NO2, nitrogen dioxide; UQH2, ubiquinol; UQH·, ubisemiquinone; R·, alkyl radical; ROO·, peroxyl radical; 1O2, singlet oxygen.

The individual steps of the free-radical mediated chain reaction of biological systems (Fig. 3) are in majority non-enzymatic second order reactions with fast reaction rates, about 107 M-1 s-1. The exceptions are the enzymatic dismutation of O2- (1010 M-1 s-1, catalyzed by the antioxidant enzyme superoxide dismutase, SOD), the first order reaction of decomposition of ONOO- , and the relatively lower rate (105 M-1 s-1) of the homolysis of H2O2 catalyzed by Fe2+ (Boveris et al., 2008).

Concerning the molecular mechanisms that produces lipid peroxidation in biological systems previous, it is accepted that lipid peroxidation may be a consequence of a) intermediates of the partial reduction of oxygen (homolysis of H2O2 and HO. generation), b) direct autoxidation of lipids, c) intermediates of the nitric oxide metabolism, and d) modifications of lipid membrane surface structure (Fridovich & Porter, 1981; Boveris et al., 2008; Navarro & Boveris, 2009; Repetto et al., 2010a;).

The lipid peroxidation process is induced for the pro-oxidant effect of transition metals. A vast evidence supports the occurrence of reactions of metal ions with H2O2, and hydroperoxides in the cytosol and in biological membranes. The latter ones are the main target of oxidative damage. In other words, by one mechanism, transition metals produce lipid peroxidation by stimulation of the oxidative capacity of H2O2 by promoting freeradical mediated processes (Fridovich, 1978; Moncada et al., 1991; Verstraeten et al., 1997; Repetto et al., 2010a; Repetto & Boveris, 2012), and by another mechanism, they bind to negatively charged phospholipids which alters the physical properties of the bilayer and favors the initiation and propagation reactions of lipid peroxidation (Repetto et al., 2010a; Repetto & Boveris, 2012).

Lipid peroxidation is a chain reaction initiated by hydrogen abstraction or by addition of an oxygen radical, resulting in the oxidative damage of polyunsaturated fatty acids (PUFA). Since polyunsaturated fatty acids are more sensitive than saturated ones, it is obvious that the activated methylene (RH) bridge represents a critical target site. This initiation is usually performed by a radical of sufficient reactivity (Eq.1):

$$\text{R} \text{H} + \text{R} \cdot \rightarrow \quad \text{R} \cdot + \text{RH} \tag{1}$$

Molecular oxygen rapidly adds to the carbon-centred radical (R. ) formed in this process, yielding the lipid peroxyl radical (ROO) (Eq. 2):

$$\text{R} \cdot \text{Oz} \quad \text{\rightarrow\\_ROO} \tag{2}$$

The formation of peroxyl radicals leads to the production of organic hydroperoxides, which, in turn, can abstract hydrogen from another PUFA, analogous to reaction 1:

$$\text{RiH} + \text{ROO} \cdot \rightarrow \text{R} \cdot + \text{ROOH} \tag{3}$$

This reaction is termed propagation, implying that one initiating hit results in the conversion of numerous PUFA to lipid hydroperoxides.

In the sequence of their appearance, alkyl, peroxyl, and alkoxyl radicals are generated in the free radical chain reaction.

The alkyl radical is stabilized by rearrangement into a conjugated diene that is a relatively stable product*.*

Lipid hydroperoxide (ROOH) is the first stable product of the lipid peroxidation reaction. Under conditions where lipid peroxidation is continuously initiated, radical anhilation or termination occurs with the destroying of two radicals at once:

$$\text{ROO} \cdot \quad + \quad \text{ROO} \cdot \quad \rightarrow \quad \text{ROH} + \text{RO} + \text{@z} \tag{4}$$

In the presence of transition metal ions, ROOH gives raise to the generation of radicals capable of (re-)initiating the lipid peroxidation by redox-cycling of the metal ions (Repetto et al., 2010a; Repetto & Boveris, 2012):

$$\text{ROOH} + \text{Me}^{\text{n}+} \quad \rightarrow \quad \text{RO} \cdot \text{ } + \text{Me}^{\text{(n-1)}+} \tag{5}$$

$$\text{ROOH} + \text{Me}^{\text{(n-l)+}} \rightarrow \begin{array}{c} \text{ROO} \cdot \text{ } + \text{Me}^{\text{n+}} \end{array} \tag{6}$$

#### **3.1. Autoxidation of lipids: Non-enzymatic lipid peroxidation**

8 Lipid Peroxidation

of ONOO-

Fe2+ (Boveris et al., 2008).

Repetto & Boveris, 2012).

The individual steps of the free-radical mediated chain reaction of biological systems (Fig. 3) are in majority non-enzymatic second order reactions with fast reaction rates, about 107 M-1

antioxidant enzyme superoxide dismutase, SOD), the first order reaction of decomposition

Concerning the molecular mechanisms that produces lipid peroxidation in biological systems previous, it is accepted that lipid peroxidation may be a consequence of a) intermediates of the partial reduction of oxygen (homolysis of H2O2 and HO. generation), b) direct autoxidation of lipids, c) intermediates of the nitric oxide metabolism, and d) modifications of lipid membrane surface structure (Fridovich & Porter, 1981; Boveris et al.,

The lipid peroxidation process is induced for the pro-oxidant effect of transition metals. A vast evidence supports the occurrence of reactions of metal ions with H2O2, and hydroperoxides in the cytosol and in biological membranes. The latter ones are the main target of oxidative damage. In other words, by one mechanism, transition metals produce lipid peroxidation by stimulation of the oxidative capacity of H2O2 by promoting freeradical mediated processes (Fridovich, 1978; Moncada et al., 1991; Verstraeten et al., 1997; Repetto et al., 2010a; Repetto & Boveris, 2012), and by another mechanism, they bind to negatively charged phospholipids which alters the physical properties of the bilayer and favors the initiation and propagation reactions of lipid peroxidation (Repetto et al., 2010a;

Lipid peroxidation is a chain reaction initiated by hydrogen abstraction or by addition of an oxygen radical, resulting in the oxidative damage of polyunsaturated fatty acids (PUFA). Since polyunsaturated fatty acids are more sensitive than saturated ones, it is obvious that the activated methylene (RH) bridge represents a critical target site. This initiation is usually

R. + O2 ROO (2)

The formation of peroxyl radicals leads to the production of organic hydroperoxides, which,

R1.

This reaction is termed propagation, implying that one initiating hit results in the

In the sequence of their appearance, alkyl, peroxyl, and alkoxyl radicals are generated in the

R1 . + RH (1)

+ ROOH (3)

) formed in this process,

, and the relatively lower rate (105 M-1 s-1) of the homolysis of H2O2 catalyzed by

(1010 M-1 s-1, catalyzed by the

s-1. The exceptions are the enzymatic dismutation of O2-

2008; Navarro & Boveris, 2009; Repetto et al., 2010a;).

performed by a radical of sufficient reactivity (Eq.1):

Molecular oxygen rapidly adds to the carbon-centred radical (R.

in turn, can abstract hydrogen from another PUFA, analogous to reaction 1:

R1H + R .

R1H + ROO .

free radical chain reaction.

conversion of numerous PUFA to lipid hydroperoxides.

yielding the lipid peroxyl radical (ROO) (Eq. 2):

Non-enzymatic lipid peroxidation is a free radical driven chain reaction in which one free radical induces the oxidation of lipids, mainly phospholipids containing polyunsaturated fatty acids. Autoxidation of lipids in biological systems is a direct process that occurs by homolysis of endogenous hydroperoxides by scission of ROOH and production of RO. and ROO. .

The polyunsaturated fatty acids such as linoleic and arachidonic acids, which are present as phosphoglyceride esters in lipid membranes, are particularly susceptible to autoxidation. Moreover, autoxidation in biological systems has been associated with such important pathological events as damage to cellular membranes in the process of aging and the action of certain toxic substance. The autoxidation of most organic substrates in homogeneous solution is a spontaneous free-radical chain process at oxygen partial pressures above 100 torr (Repetto et al., 2010a).

Lipid hydroperoxides, in presence or absence of catalytic metal ions, produce a large variety of products including short and long chain aldehydes and phospholipids and cholesterol ester aldehydes, which provide an equivalent hydrogen abstraction from an unsaturated fatty acid and formation of free radical. The secondary products can be used to assess the degree of lipid peroxidation in a system (Sies, 1991a) (Eq. 7 to 9).

Eq. 7 requires some comments. As written is thermodinamically non spontaneous since it involves the breaking of a C-H bond (435 kJ/mol). However, polyunsaturated fatty acids in solutions are readily autooxidized, likely catalized by transition metal ions. The R· radicals reaction with O2 yielding ROO. .

$$\text{RH} \rightarrow \text{ R} \cdot + \text{H} \cdot \tag{7}$$

$$\text{R} \cdot + \text{ Or} \rightarrow \begin{array}{c} \text{ROO} \end{array} \tag{8}$$

$$\text{ROO} + \text{RH} \rightarrow \text{ROOH} + \text{R} \cdot \tag{9}$$

Transition metal ions Fe2+ and Cu+ stimulate lipid peroxidation by the reductive cleavage of endogenous lipid hydroperoxides (ROOH) of membrane phospholipids to the corresponding alkoxyl (RO. ) and peroxyl (ROO. ) radicals in a process that is known as ROOH-dependent lipid peroxidation (Eqs. 10 and 11):

$$\text{Fe}^{2+} + \text{ROOH} \rightarrow \text{RO}^{\cdot} + \text{OH}^{\cdot} \cdot + \text{Fe}^{3+} \tag{10}$$

$$\text{Fe}^{3+} + \text{ROOH} \rightarrow \text{ROr} + \text{H}^{+} + \text{Fe}^{2+} \tag{11}$$

The mechanisms of these two reactions appear to involve the formation of Fe(II)-Fe(III) or Fe(II)-O2-Fe(III) complexes with maximal rates of HO· radical formation at a ratio Fe(II)/Fe(III) of 1 (Repetto et al., 2010a; Repetto & Boveris, 2012).

Cu2+ and Cu+ are known for their capacity to decompose organic hydroperoxides (ROOH) to form RO. and ROO· (Eqs. 12 and 13) (Sies, 1991a; Repetto et al., 2010a; Repetto & Boveris, 2012).

$$\mathrm{Cu^{+} + ROOH \rightarrow \cdot \cdot RO + OH^{\cdot} + Cu^{2+}} \tag{12}$$

$$\text{Cu}^{2+} + \text{ROOH} \rightarrow \text{ROr} + \text{H}^{+} + \text{Cu}^{+} \tag{13}$$

#### **3.2. Lipid peroxidation generated for intermediates of the partial reduction of oxygen**

The physiological generation of the products of the partial reduction of oxygen, O2 and H2O2, constitute the biological basis of the process of lipid peroxidation in mammalian aerobic cells. From a molecular point of view hydroxyl radical (HO·) generation, formed from H2O2 and Fe2+ by the Fenton reaction, has been considered for a long time as the likely rate-limiting step for physiological lipid peroxidation (Verstraeten et al., 1997; Repetto Boveris, 2012). The Fenton reaction and Fenton-like reactions (Eq. 14) are frequently used to explain the toxic effects of redox-active metals (Eq. 5), where M(n)+ is usually a transition metal ion:

$$\text{Fe}^{2+} + \text{H}\text{O}\text{I} \rightarrow [\text{Fe(II)H}\text{O}] \rightarrow \text{Fe}^{3+} + \text{HO}^{\cdot} + \text{HO} \tag{14}$$

Trace (nM) levels of cellular and circulating active transition metal ions seem enough for the catalysis of a slow Fenton reaction *in vivo*, at the physiological levels of H2O2 (0.1-1.0 M) (Chance et al., 1979).

Reactive oxygen species mainly include O2.- and H2O2, which are physiologically generated as by-products of mitochondrial electron transfer. The formation of O2.- is originated from the auto-oxidation of the ubisemiquinone of complexes I and III and the production of H2O2 occurs by intramitochondrial Mn-SOD catalysis (Navarro Boveris, 2004; Navarro et al., 2007, 2010). When the electron transfer process is blocked at complexes I and III, electrons pass directly to O2 producing O2.-. The reactive oxygen and nitrogen species, although kept in low steady-state concentrations by antioxidant systems, are able to react and damage biomolecules (Fig. 3). Mitochondria are considered the main intracellular source of oxidizing reactive oxygen species (Navarro Boveris, 2004; Navarro et al., 2005, 2009, 2010).

At low level of H2O2, Fe2+ induces lipid peroxide decomposition, generating peroxyl and alkoxyl radicals and favoring lipid peroxidation. These results indicate that the onset of the Fe3+ stimulatory effect on Fe2+-dependent lipid peroxidation is due to reactive oxygen species production via Fe2+ oxidation with endogenous ROOH (Repetto Boveris, 2012).

The Cu+ ion is considered an effective catalyst for the Fenton reaction (Eq. 15) [3].

10 Lipid Peroxidation

form RO.

**oxygen** 

(Chance et al., 1979).

2012).

ROO.

and Cu+

ROOH-dependent lipid peroxidation (Eqs. 10 and 11):

Fe2+ + ROOH RO.

Fe3+ + ROOH RO2.

Cu+ + ROOH RO.

Cu2+ + ROOH RO2.

Fe(II)/Fe(III) of 1 (Repetto et al., 2010a; Repetto & Boveris, 2012).

endogenous lipid hydroperoxides (ROOH) of membrane phospholipids to the

The mechanisms of these two reactions appear to involve the formation of Fe(II)-Fe(III) or Fe(II)-O2-Fe(III) complexes with maximal rates of HO· radical formation at a ratio

Cu2+ and Cu+ are known for their capacity to decompose organic hydroperoxides (ROOH) to

**3.2. Lipid peroxidation generated for intermediates of the partial reduction of** 

constitute the biological basis of the process of lipid peroxidation in mammalian aerobic cells. From a molecular point of view hydroxyl radical (HO·) generation, formed from H2O2 and Fe2+ by the Fenton reaction, has been considered for a long time as the likely rate-limiting step for physiological lipid peroxidation (Verstraeten et al., 1997; Repetto Boveris, 2012). The Fenton reaction and Fenton-like reactions (Eq. 14) are frequently used to explain the toxic effects of

Trace (nM) levels of cellular and circulating active transition metal ions seem enough for the catalysis of a slow Fenton reaction *in vivo*, at the physiological levels of H2O2 (0.1-1.0 M)

Reactive oxygen species mainly include O2.- and H2O2, which are physiologically generated as by-products of mitochondrial electron transfer. The formation of O2.- is originated from the auto-oxidation of the ubisemiquinone of complexes I and III and the production of H2O2 occurs by intramitochondrial Mn-SOD catalysis (Navarro Boveris, 2004; Navarro et al., 2007, 2010). When the electron transfer process is blocked at complexes I and III, electrons pass directly to O2 producing O2.-. The reactive oxygen and nitrogen species, although kept

The physiological generation of the products of the partial reduction of oxygen, O2-

redox-active metals (Eq. 5), where M(n)+ is usually a transition metal ion:

Fe2+ + H2O2 [Fe(II)H2O2] Fe3+ + HO-

and ROO· (Eqs. 12 and 13) (Sies, 1991a; Repetto et al., 2010a; Repetto & Boveris,

+ OH-

+ OH-

) and peroxyl (ROO.

Transition metal ions Fe2+

corresponding alkoxyl (RO.

+ RH ROOH + R. (9)

) radicals in a process that is known as

+ H+ + Fe2+ (11)

+ Fe3+ (10)

+ Cu2+ (12)

+ HO (14)

and H2O2,

+ H+ + Cu+ (13)

stimulate lipid peroxidation by the reductive cleavage of

$$\text{Cu}^+ + \text{HxO} \rightarrow [\text{Cu(l)-H}\text{O}] \rightarrow \text{Cu}^{2+} + \text{HO}^- + \text{HO}^-\tag{15}$$

The process of lipid peroxidation has been recognized as a free radical-mediated and physiologically occurring with the supporting evidence of in situ organ chemiluminescence (Boveris et al., 1980). The main initiation reaction is understood to be mediated by HO. or by a ferryl intermediate, both with the equivalent potential for hydrogen abstraction from an unsaturated fatty acid, with formation of an alkyl radical (R. ) (Repetto Boveris, 2012) (Eq. 16):

$$\text{H}\text{HO}\cdot + \text{RH} \rightarrow \text{HxO} + \text{R}\cdot \tag{16}$$

One effect of the reaction of hydroxyl radicals, their formation catalyzed by iron ions, with lipids is to make those lipids insoluble or fibrotic that can be considered causative of membrane disruption and oxidative damage associated in different pathologies.

#### **3.3. Lipid peroxidation generated from intermediates of the nitric oxide metabolism**

An area of interest that has currently increased over the past decades is the study of nitric oxide (NO) since the demonstration, in 1987, of its formation by the enzyme NO synthase in vascular endothelial cells. This NO radical accounts for the properties of the called endothelial derived relaxing factor, is the endogenous stimulator of the soluble guanylate cyclase and is a potent vasodilator *in vitro* (Moncada et al., 1991). Unsaturated fatty acids are susceptible to nitration reactions. The nitric oxide (NO)-derived species are diffusible across membranes, their concentration in the hydrophobic core of membranes and lipoproteins lead to react fast with fatty acids and lipid peroxyl radicals (ROO. ) during the lipid oxidadation process generating oxidized and nitrated products of free lipids (arachidonic acid, arachidonate oleate, linoleate) and esterified (cholesteryl linoleate). Lipid nitration process includes *in vivo* different molecular mechanisms: a) NO autooxidation to nitrite, which has oxidant and nitrating properties, b) electrophilic addition of NO relates species to unsaturated fatty acids, c) radical reactions between ROO. and NO, d) peroxynitrite (ONOO. ) derived free radicals mediate oxidation, nitrosation and nitration reactions. These species are considered currently as mediators of adaptative inflammatory responses.

NO is an endogenous mediator of many physiological functions through stimulation of the guanylate cyclase enzyme including the regulation of vascular relaxing, post-traslational

protein changes, gene expression and inflammatory cell function (Moncada et al., 1991). Free and esterified fatty acids as arachidonic and linoleic acids are important components of lipoproteins and membranes that may be oxidized for different compounds. The NO and NO-derived radicals react with fatty acids generating oxidized and nitrated species as nitroalkenes and consequently, nitroalcohols. At low oxygen concentrations the most important biological NO derivatives is ONOO. . The nitroalkylation process occurs *in vitro* and in vivo, is involved in redox processes and cell signaling through the reversible covalent bound and post-traslational modifications responsible for structure, function and subcellular distribution of proteins (Valdez et al., 2011) and regulating the pro-inflammatory effect of oxidant exposure (Nair et al., 2007).

A novel mechanism for hydroxyl radical production, which is not dependent on the presence of transition metals, has recently been proposed. This involves the production of peroxynitrite (Beckman et al., 1990, 1994; Rachmilewitz et al., 1993) which has proinflammatory effects *in vitro* (Moncada et al., 1991), from the reaction of NO with O2.- (Eqs. 17 to 20):

$$\text{NO} + \text{O} \cdot \text{H}^{\cdot} \quad \rightarrow \qquad \text{ONOO} \cdot \tag{17}$$

$$\text{ONOO}^{\cdot} + \text{H}^{\cdot} \rightarrow \text{ONOH} \tag{18}$$

ONOOH HO. + NO2 (19)

$$2\,\mathrm{H}^{\ast}\,+\,\mathrm{Or}\,^{\cdot}\,+\,\mathrm{Or}\,^{\cdot}\to\,\mathrm{H}\mathrm{O}\,\mathrm{Zr}\,\,\mathrm{\cdot}\,\mathrm{Or}\,\,\tag{20}$$

In pathological situations, macrophages and neutrophils, recruited to a site of injury, are activated to produce NO as part of the inflammatory response. Furthermore, SOD activity rapidly scavenges O2.- and also prolongs the vaso-relaxant effects of NO (Murphy Sies, 1991; Hogg et al., 1992; Rachmilewitz et al., 1993).

#### **3.4. Modifications of lipid membrane structure**

The presence of cholesterol in cell surface membranes influences their susceptibility to peroxidation, probably both by intercepting some of the radicals present and by affecting the internal structure of the membrane by interaction of its large hydrophobic ring structure with fatty acid-side-chains. As lipid peroxidation precedes in any membrane, several of the products produced have a detergent-like activity, specially released fatty acids or phospholipids with one of their fatty-acid side-chains removed. This will contribute to increased membrane disruption and further peroxidation.

The onset of lipid peroxidation within biological membranes is associated with changes in their physicochemical properties and with alteration of biological function of lipids and proteins. Polyunsaturated fatty acids and their metabolites play physiological roles: energy provision, membrane structure, fluidity, flexibility and selective permeability of cellular membranes, and cell signaling and regulation of gene expression (Catala, 2006). The hydroxyl radical generated as a consequence of the Fenton reaction, oxidizes the cellular components of biological membranes (Fig. 4).

**Figure 4.** Lipid, DNA and protein oxidative damage from reactive hydroxyl radical.

(Eqs. 17 to 20):

important biological NO derivatives is ONOO.

effect of oxidant exposure (Nair et al., 2007).

ONOO-

1991; Hogg et al., 1992; Rachmilewitz et al., 1993).

**3.4. Modifications of lipid membrane structure** 

increased membrane disruption and further peroxidation.

components of biological membranes (Fig. 4).

protein changes, gene expression and inflammatory cell function (Moncada et al., 1991). Free and esterified fatty acids as arachidonic and linoleic acids are important components of lipoproteins and membranes that may be oxidized for different compounds. The NO and NO-derived radicals react with fatty acids generating oxidized and nitrated species as nitroalkenes and consequently, nitroalcohols. At low oxygen concentrations the most

and in vivo, is involved in redox processes and cell signaling through the reversible covalent bound and post-traslational modifications responsible for structure, function and subcellular distribution of proteins (Valdez et al., 2011) and regulating the pro-inflammatory

A novel mechanism for hydroxyl radical production, which is not dependent on the presence of transition metals, has recently been proposed. This involves the production of peroxynitrite (Beckman et al., 1990, 1994; Rachmilewitz et al., 1993) which has proinflammatory effects *in vitro* (Moncada et al., 1991), from the reaction of NO with O2.-

In pathological situations, macrophages and neutrophils, recruited to a site of injury, are activated to produce NO as part of the inflammatory response. Furthermore, SOD activity rapidly scavenges O2.- and also prolongs the vaso-relaxant effects of NO (Murphy Sies,

The presence of cholesterol in cell surface membranes influences their susceptibility to peroxidation, probably both by intercepting some of the radicals present and by affecting the internal structure of the membrane by interaction of its large hydrophobic ring structure with fatty acid-side-chains. As lipid peroxidation precedes in any membrane, several of the products produced have a detergent-like activity, specially released fatty acids or phospholipids with one of their fatty-acid side-chains removed. This will contribute to

The onset of lipid peroxidation within biological membranes is associated with changes in their physicochemical properties and with alteration of biological function of lipids and proteins. Polyunsaturated fatty acids and their metabolites play physiological roles: energy provision, membrane structure, fluidity, flexibility and selective permeability of cellular membranes, and cell signaling and regulation of gene expression (Catala, 2006). The hydroxyl radical generated as a consequence of the Fenton reaction, oxidizes the cellular

. The nitroalkylation process occurs *in vitro*

NO + O2.- ONOO- (17)

ONOOH HO. + NO2 (19)

2 H+ + O2.- + O2.- H2O2 + O2.- (20)

+ H+ ONOOH (18)

The binding of positively charged species to a membrane (to the negatively-charged headgroups of phospholipids) can alter the susceptibility of the membrane to oxidative damage. This can be seen as either an enhancement or an inhibition of the rate of lipid peroxidation. Several metal ions such as Ca2+, Co2+, Cd2+, Al3+, Hg2+ and Pb2+ alter the rate of peroxidation in liposomes, erythrocytes and microsomal membranes, often stimulating the peroxidation induced by iron ions.

In the lipid peroxidation of the brain phosphatidylcholine-phosphatidylserine (PC-PS) liposomes (Repetto et al., 2010a) hydrogen abstraction occurred at the allilic carbons 9 and 10 of the oleic acid chain. Secondary initiation reactions are provided by hydrogen abstraction by RO· and ROO· (Eqs. 21 to 23) at the mentioned tertiary carbons:

$$\text{RO} + \text{RH} \rightarrow \text{ROH} + \text{R} \tag{21}$$

$$\text{ROr} + \text{RH} \rightarrow \text{ROOH} + \text{R} \tag{22}$$

$$\text{R} \cdot \text{+ Or} \to \text{ROr} \tag{23}$$

The R· and ROO· radicals (Eqs.21-23) are central to the free radical-mediated process of lipid peroxidation. The addition reaction of R· with O2 to yield ROO· (Eq. 23) yield a product that is able to abstract hydrogen atoms and to regenerate R· for a new cycle of the free-radical chain-reaction [38]. The whole process, by repetition of reaction 23, consumes O2 and produces malondialdehyde (O=HC-CH2-CH=O), 4-hydroxynonenal and other dialdehydes as secondary and end products of lipid peroxidation. The process produces TBARS at an approximate ratio of 0.12 TBARS/O2 and normally utilized as measurement of the rate and extent of lipid peroxidation (Junqueira et al., 2004).

There are two consequences of lipid peroxidation: structural damage to membranes and generation of secondary products. Membrane damage derives from the production of broken fatty acyl chains, lipid-lipid or lipid-protein cross-links, and endocyclization reactions to produce isoprostanes and neuroprostanes (Catala, 2006). This effect is severe for biological systems, produce damage of membrane function, enzymatic inactivation and toxic effects on cellular division and function.

### **4. Role of transition metal on lipid peroxidation process**

Studies in the past two decades have shown that redox active metals undergo redox cycling reactions and possess the ability to produce reactive radicals such as superoxide anion radical and nitric oxide in biological systems. Disruption of metal ion homeostasis leads to oxidative stress, a state with increased formation of reactive oxygen species that overwhelms antioxidant protection and subsequently induces DNA damage, lipid peroxidation, protein modification and other effects, all symptomatic for numerous diseases, involving cancer, cardiovascular disease, diabetes, atherosclerosis, neurological disorders, and chronic inflammation.

The mechanism of lipid peroxidation in biological systems caused by free radicals has been the focus of scientific interest for many years (Chance et al., 1976; Fridovich Porter, 1981; Fraga et al., 1988; Gonzalez-Flecha et al., 1991a, 1991b; Famulari et al., 1996; Fiszman et al., 2003; Junqueira et al., 2004; Catala, 2006; Boveris et al., 2008; Dianzani Barrera, 2008; Dominguez et al., 2008; Repetto, 2008; Repetto et al., 2010b). Currently, it is known that the OH. radical, is formed mainly by the Haber-Weiss reaction, and it is responsible for the biological damage (Repetto et al., 2010a; Repetto et al., 2010b) (Eq.24):

$$\text{Or} + \text{H}\text{O} \rightarrow \text{Or} + \text{HO} \cdot + \text{HO} \tag{24}$$

However, this reaction would not proceed significantly *in vivo* because the rate constant for the reaction is lower than that of the dismutation reaction. Nevertheless, a modification of the Haber-Weiss reaction, the Fenton reaction and Fenton-like reactions, utilizes the redox cycling ability of iron to increase the rate of reaction, is more feasible *in vivo* (Chance et al., 1979; Boveris et al., 1980; Gonzalez Flecha et al., 1991b), and is frequently used to explain the toxic effects of redox-active metals where M(n)+ is usually a transition metal ion.

As a transition metal that can exist in several valences and that can bind up to six ligands, iron is an important component of industrial catalysts in the chemical industry especially for redox reactions (Repetto et al., 2010a; Repetto Boveris, 2012).

There are several reports on the role of transition metals in lipid peroxidation process associated with cellular toxicities, because once they enter our physiological systems, these metals play a role in oxidative adverse effects. Some transition metals including iron, chromium, lead, and cadmium generate lipid peroxidation *in vitro* e *in vivo*: fatty acids, cod liver oil, biological membranes, tissues and organs, suggesting that metals contribute to the oxidative effects of lipid peroxidation observed in various diseases (Repetto et al., 2010a; Repetto Boveris, 2012).

The Fenton reaction occurs *in vivo* at a very low rate, and hence cannot account for any substantial production of OH. radicals in biology. On the other hand, when catalysed by transition metal ions, OH. radicals can be formed through reactions 25 and 26:

$$\rm M^{(n)+} + \rm O\natural \rm \rightarrow \rm M^{(n\cdot 1)+} + \rm O\imath \tag{25}$$

$$\rm M^{(n\text{1)+}} + \rm H\rm O\rightarrow\rm M^{(n)+} + \rm HO\cdot +\rm HO\cdot\tag{26}$$

The concentration of intracellular redox active transition metals is either low or negligible: free Fe2+ is 0.2-0.5 M and the pool of free Cu2+ is about a single ion per cell. However, trace (nM) levels of cellular and circulating active transition metal ions seem enough for the catalysis of a slow Fenton reaction *in vivo* at the physiological levels of hydrogen peroxide (H2O2, 0.1-1.0 M) (Repetto et al., 2010a; Repetto Boveris, 2012).

14 Lipid Peroxidation

OH.

and chronic inflammation.

O2-

Repetto Boveris, 2012).

transition metal ions, OH.

substantial production of OH.

M(n) + + O2-

**4. Role of transition metal on lipid peroxidation process** 

biological damage (Repetto et al., 2010a; Repetto et al., 2010b) (Eq.24):

toxic effects of redox-active metals where M(n)+ is usually a transition metal ion.

redox reactions (Repetto et al., 2010a; Repetto Boveris, 2012).

Studies in the past two decades have shown that redox active metals undergo redox cycling reactions and possess the ability to produce reactive radicals such as superoxide anion radical and nitric oxide in biological systems. Disruption of metal ion homeostasis leads to oxidative stress, a state with increased formation of reactive oxygen species that overwhelms antioxidant protection and subsequently induces DNA damage, lipid peroxidation, protein modification and other effects, all symptomatic for numerous diseases, involving cancer, cardiovascular disease, diabetes, atherosclerosis, neurological disorders,

The mechanism of lipid peroxidation in biological systems caused by free radicals has been the focus of scientific interest for many years (Chance et al., 1976; Fridovich Porter, 1981; Fraga et al., 1988; Gonzalez-Flecha et al., 1991a, 1991b; Famulari et al., 1996; Fiszman et al., 2003; Junqueira et al., 2004; Catala, 2006; Boveris et al., 2008; Dianzani Barrera, 2008; Dominguez et al., 2008; Repetto, 2008; Repetto et al., 2010b). Currently, it is known that the

radical, is formed mainly by the Haber-Weiss reaction, and it is responsible for the

+ H2O2 O2 + HO· + HO-

However, this reaction would not proceed significantly *in vivo* because the rate constant for the reaction is lower than that of the dismutation reaction. Nevertheless, a modification of the Haber-Weiss reaction, the Fenton reaction and Fenton-like reactions, utilizes the redox cycling ability of iron to increase the rate of reaction, is more feasible *in vivo* (Chance et al., 1979; Boveris et al., 1980; Gonzalez Flecha et al., 1991b), and is frequently used to explain the

As a transition metal that can exist in several valences and that can bind up to six ligands, iron is an important component of industrial catalysts in the chemical industry especially for

There are several reports on the role of transition metals in lipid peroxidation process associated with cellular toxicities, because once they enter our physiological systems, these metals play a role in oxidative adverse effects. Some transition metals including iron, chromium, lead, and cadmium generate lipid peroxidation *in vitro* e *in vivo*: fatty acids, cod liver oil, biological membranes, tissues and organs, suggesting that metals contribute to the oxidative effects of lipid peroxidation observed in various diseases (Repetto et al., 2010a;

The Fenton reaction occurs *in vivo* at a very low rate, and hence cannot account for any

M(n-1) + + H2O2 M(n)+ + HO· + HO- (26)

radicals can be formed through reactions 25 and 26:

radicals in biology. On the other hand, when catalysed by

M(n-1)+ + O2 (25)

(24)

It is well known that iron serves as a catalyst for the formation of the highly reactive hydroxyl radical via Fenton reaction. In addition to ferrous ion, many metal ions including Cu (I), Cr (II), and Co (II) were found to have the oxidative features of the Fenton reagent. Therefore, the mixtures of these metal compounds with H2O2 were named ''Fenton like reagents". In actual in vivo systems, once organic peroxides (ROOH) are formed by the action of ROS, heat, and/or photo-irradiation, ROOH can be substituted for HO. , where ROOH reacts with metal ions to form alkoxyl radicals. Subsequently, a chain reaction of lipid peroxidation occurs.

The mechanisms for metal transition ions promoted lipid peroxidation are H2O2 decomposition and direct homolysis of endogenous hydroperoxides. The Fe2+-H2O2 mediated lipid peroxidation takes place by a pseudo-second order process, and the Cu2+ mediated process by a pseudo-first order reaction. Co2+ and Ni2+ alone, do not induce lipid peroxidation. Nevertheless, when they are combined with Fe2+, Fe2+-H2O2-mediated lipid peroxidation is stimulated in the presence of Ni2+ and is inhibited in the presence of Co2+ (Fig. 5) (Repetto et al., 2010a).

**Figure 5.** Phospholipid oxidation at different concentrations of transition metals.

There are many factors influencing lipid peroxidation products formation from lipids catalyzed by various metals. For example, the quantitative measurement of the reaction of Fe (II) and H2O2 has shown that a stoichiometric amount of hydroxyl radical is spin-trapped when ion concentration was less than 1 μM, suggesting that the strength of the Fenton system depended on the metal concentration. Since Fenton reported that a mixture of hydrogen peroxide and ferrous salts was an effective oxidant of a large variety of organic substrates in 1894 this reagent (the Fenton's reagent) has been used to investigate many subjects related to *in vitro* oxidation of organic substrates including lipids (Repetto et al., 2010a; Repetto Boveris, 2012).

In the *in vitro* model of phosphatidylcholine/phosphatidyserine (60:40) liposomes and hydrogen peroxide (H2O2), Fe and Cu promote lipid peroxidation, interpreted as the consequence of the homolytic scission of H2O2 and of endogenous hydroperoxides (ROOH) and of the generation of hydroxyl (HO•) and alcoxyl (RO•) radicals (Cadenas, 1989) depending strictly on the participation of Fe and Cu as redox-reactive metals . However, Co2+ and Ni2+ alone, do not induce lipid peroxidation. Nevertheless, when they are combined with Fe2+, Fe2+-H2O2-mediated lipid peroxidation is stimulated in the presence of Ni2+ and inhibited in the presence of Co2+ (Repetto et al., 2010a; Repetto Boveris, 2012).

Cr(III) occurs in nature and is an essential trace element utilized in the regulation of blood glucose levels. Cr(III) reacts with superoxide, subsequently Cr(II) yields hydroxyl radical via Fenton-like reaction with H2O2 to initiate lipid peroxidation.

Cadmium intoxication was shown to increase lipid peroxidation in rat liver, kidney and heart. However, the mechanisms of cadmium toxicity are not fully understood. Cadmium indirectly affects the generation of various radicals including superoxide and hydroxyl radical. The generation of hydrogen peroxide by cadmium ion may become a source of radicals in the Fenton system (Jomova Valko, 2011).

## **5. Toxic effects of secondary products of lipid peroxidation**

Many aldehydes are produced during the peroxidative decomposition of unsaturated fatty acids. Compared with free radicals, aldehydes are highly stable and diffuse out from the cell and attack targets far from the site of their production. About 32 aldehydes were identified as products of lipid peroxidation: a) saturated aldehydes (propanal, butanal, hexanal, octanal, being the decanal the most important); b) 2,3-trans-unsaturated-aldehydes (hexenal, octenal, nonenal, decenal and undecenal); c) a series of 4-hydroxylated,2,3-trans-unsaturated aldehydes: 4-hydroxyundecenal, being 4-hydroxinonenal (HNE) the most important quantitatively. Malonyldialdehyde (MDA) was considered for a long time as the most important lipid peroxidation metabolite. However, MDA is practically no toxic.

Recent studies have demonstrated that the most effective product of lipid peroxidation causing cellular damage is HNE. HNE produces different effects: acts as an intracellular signal able to modulate gene expression, cell proliferation, differentiation and apoptosis. The hydroxyl-group close to a carbonyl group present in HNE chemical structure is related to its high reactivity with different targets (thiol and amine groups). HNE is easily diffusible specie, but its biological effect depends on the molecule target and behavior as a signal to produce the damage.

Oxidative stress is a well known mechanism of cellular injury that occurs with increased lipoperoxidation of cell phospholipids and that has been implicated in various cell dysfunctions (Sies, 1991a,b; Catala, 2006). Aldehydes exhibit high reactivity with biomolecules, such as proteins, DNA and phospholipids generating intra and intermolecular adducts.

The physiological concentrations of these products are low; however, higher concentrations correspond to pathological situations. Therefore, DNA damage caused by lipid peroxidation end products could provide promising markers for risk prediction and targets for preventive measures. DNA-reactive aldehydes can damage DNA either by reacting directly with DNA bases or by generating more reactive bifunctional intermediates, which form exocyclic DNA adducts. Of these, HNE and MDA, acrolein, and crotonaldehyde have been shown to modify DNA bases, yielding promutagenic lesions and to contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced lipid peroxidation and HNE and MDA implicated carcinogenesis.

The end-products of lipid peroxidation (HNE and MDA) cause protein damage by addition reactions with lysine amino groups, cysteine sulfhydryl groups, and histidine imidazole groups (Esterbauer et al., 1991; Esterbauer, 1996). Modifications of protein by aldehyde products of lipid peroxidation contribute to neurodegenerative disorders, activation of kinases (Uchida et al., 1999; Uchida, 2003) and inhibition of the nuclear transcription factor (Camandola et al., 2000).

## **6. Lipid peroxidation of subcellular fragments**

#### **6.1. Microsomes**

16 Lipid Peroxidation

produce the damage.

adducts.

In the *in vitro* model of phosphatidylcholine/phosphatidyserine (60:40) liposomes and hydrogen peroxide (H2O2), Fe and Cu promote lipid peroxidation, interpreted as the consequence of the homolytic scission of H2O2 and of endogenous hydroperoxides (ROOH) and of the generation of hydroxyl (HO•) and alcoxyl (RO•) radicals (Cadenas, 1989) depending strictly on the participation of Fe and Cu as redox-reactive metals . However, Co2+ and Ni2+ alone, do not induce lipid peroxidation. Nevertheless, when they are combined with Fe2+, Fe2+-H2O2-mediated lipid peroxidation is stimulated in the presence of Ni2+ and inhibited in the presence of Co2+ (Repetto et al., 2010a; Repetto Boveris, 2012).

Cr(III) occurs in nature and is an essential trace element utilized in the regulation of blood glucose levels. Cr(III) reacts with superoxide, subsequently Cr(II) yields hydroxyl radical

Cadmium intoxication was shown to increase lipid peroxidation in rat liver, kidney and heart. However, the mechanisms of cadmium toxicity are not fully understood. Cadmium indirectly affects the generation of various radicals including superoxide and hydroxyl radical. The generation of hydrogen peroxide by cadmium ion may become a source of

Many aldehydes are produced during the peroxidative decomposition of unsaturated fatty acids. Compared with free radicals, aldehydes are highly stable and diffuse out from the cell and attack targets far from the site of their production. About 32 aldehydes were identified as products of lipid peroxidation: a) saturated aldehydes (propanal, butanal, hexanal, octanal, being the decanal the most important); b) 2,3-trans-unsaturated-aldehydes (hexenal, octenal, nonenal, decenal and undecenal); c) a series of 4-hydroxylated,2,3-trans-unsaturated aldehydes: 4-hydroxyundecenal, being 4-hydroxinonenal (HNE) the most important quantitatively. Malonyldialdehyde (MDA) was considered for a long time as the most

Recent studies have demonstrated that the most effective product of lipid peroxidation causing cellular damage is HNE. HNE produces different effects: acts as an intracellular signal able to modulate gene expression, cell proliferation, differentiation and apoptosis. The hydroxyl-group close to a carbonyl group present in HNE chemical structure is related to its high reactivity with different targets (thiol and amine groups). HNE is easily diffusible specie, but its biological effect depends on the molecule target and behavior as a signal to

Oxidative stress is a well known mechanism of cellular injury that occurs with increased lipoperoxidation of cell phospholipids and that has been implicated in various cell dysfunctions (Sies, 1991a,b; Catala, 2006). Aldehydes exhibit high reactivity with biomolecules, such as proteins, DNA and phospholipids generating intra and intermolecular

The physiological concentrations of these products are low; however, higher concentrations correspond to pathological situations. Therefore, DNA damage caused by lipid peroxidation

via Fenton-like reaction with H2O2 to initiate lipid peroxidation.

**5. Toxic effects of secondary products of lipid peroxidation** 

important lipid peroxidation metabolite. However, MDA is practically no toxic.

radicals in the Fenton system (Jomova Valko, 2011).

Microsomes isolated from liver have been shown to catalyze an NADPH-dependent peroxidation of endogenous unsaturated fatty acids in the presence of ferric ions and metal chelators, such as ADP or pyrophosphates. Microsomal membranes are particularly susceptible to lipid peroxidation owing to the presence of high concentrations of polyunsaturated fatty acids (Poyer McCay, 1971). The mechanism involved in the initiation of peroxidation in the NADPH-dependent microsomal system do not appear to involve neither superoxide nor hydrogen peroxide, since neither superoxide dismutase nor catalase cause inhibition of peroxidation. Nevertheless, reduced iron plays an important role in both the initiation and propagation of NADPH-dependent microsomal lipid peroxidation (Shires, 1975).

Microsomal membrane lipids, particularly the polyunsaturated fatty acids, undergo degradation during NADPH-dependent lipid peroxidation. The degradation of membrane lipids during lipid peroxidation has been observed to result in the production of singlet oxygen, which is detected as chemiluminescence (Boveris et al., 1980).

Nonenzymatic peroxidation of microsomal membranes also occurs and is probably mediated in part by endogenous hemoproteins and transition metals. High concentrations of transition metals (50 M) promote auto-oxidation of phospholipids (Repetto et al., 2010a).

#### **6.2. Mitochondria**

It is currently accepted that mitochondrial complex I is particularly sensitive to inactivation by oxygen free radicals and reactive nitrogen species. This special characteristic is frequently referred as complex I syndrome, with the symptoms of reduced mitochondrial respiration with malate-glutamate and ADP and of reduced complex I activity. This complex I syndrome has been observed in aging (Navarro et al., 2005; Navarro Boveris, 2004, 2008),

in ischemia-reperfusion (Gonzalez-Flecha et al., 1993), in Parkinson's disease, and in other neurodegenerative diseases (Schapira et al., 1990a, 1990b; Sayre et al., 1999; Carreras et al., 2004; Schapira, 2008; Navarro et al., 2009), and in this study, with the addition of the increased rates of production of O2.- and H2O2 by complex I mediated reactions, reactions with the free radicals intermediates of the lipid peroxidation process (mainly ROO·), and amine-aldehyde adduction reactions. It is now understood that the three processes above mentioned alter the native non-covalent polypeptide interactions of complex I and promote synergistically protein damage and inactivation by shifting the noncovalent bonding to covalent cross linking (Navarro et al., 2005). Complex I oxidative protein damage has also been considered the result of protein modification by reaction with malonaldehyde and 4- HO-nonenal (Sayre et al., 1999). It was hypothesized that protein damage in the subunits of complexes I and IV follows to free radical-mediated cross-linking and inactivation. The subunits that are normally held together by noncovalent forces are shifted to covalent crosslinking after reaction with the hydroperoxyl radicals (ROO·) and the stable aldehydes produced during the lipid peroxidation process.

The hypothesis that cumulative free radical-mediated protein damage is the chemical basis of respiratory complexes I and IV inactivation (Berlett Stadtman, 1997) offers the experimental approach of the chronic use of vitamin E, as an antioxidant for the lipid phase of the inner mitochondrial membrane and for the prevention of the mitochondrial /damage associated with aging. The adduction reactions of malonaldehyde and 4-HO-nonenal with protein evolve to stable advanced lipid peroxidation products (Sayre et al., 1999) and protein carbonyls (Nair et al., 2007; Navarro et al., 2008). The molecular mechanism involved in the inactivation of complex I is likely accounted for by ROO. and ONOO- . Upon aging, frontal cortex and hippocampal mitochondria show a decreased rate of respiration, especially marked with NAD-dependent substrates, and decreased enzymatic activities of complexes I and IV associated with an increase in the content of oxidation products (TBARS and protein carbonyls) (Navarro et al., 2008) (Fig. 6).

**Figure 6.** Lipid peroxidation and protein peroxidation by secondary products of lipid peroxidation in mitochondria.

## **7. Lipid peroxidation and human pathologies**

18 Lipid Peroxidation

mitochondria.

produced during the lipid peroxidation process.

and protein carbonyls) (Navarro et al., 2008) (Fig. 6).

in ischemia-reperfusion (Gonzalez-Flecha et al., 1993), in Parkinson's disease, and in other neurodegenerative diseases (Schapira et al., 1990a, 1990b; Sayre et al., 1999; Carreras et al., 2004; Schapira, 2008; Navarro et al., 2009), and in this study, with the addition of the increased rates of production of O2.- and H2O2 by complex I mediated reactions, reactions with the free radicals intermediates of the lipid peroxidation process (mainly ROO·), and amine-aldehyde adduction reactions. It is now understood that the three processes above mentioned alter the native non-covalent polypeptide interactions of complex I and promote synergistically protein damage and inactivation by shifting the noncovalent bonding to covalent cross linking (Navarro et al., 2005). Complex I oxidative protein damage has also been considered the result of protein modification by reaction with malonaldehyde and 4- HO-nonenal (Sayre et al., 1999). It was hypothesized that protein damage in the subunits of complexes I and IV follows to free radical-mediated cross-linking and inactivation. The subunits that are normally held together by noncovalent forces are shifted to covalent crosslinking after reaction with the hydroperoxyl radicals (ROO·) and the stable aldehydes

The hypothesis that cumulative free radical-mediated protein damage is the chemical basis of respiratory complexes I and IV inactivation (Berlett Stadtman, 1997) offers the experimental approach of the chronic use of vitamin E, as an antioxidant for the lipid phase of the inner mitochondrial membrane and for the prevention of the mitochondrial /damage associated with aging. The adduction reactions of malonaldehyde and 4-HO-nonenal with protein evolve to stable advanced lipid peroxidation products (Sayre et al., 1999) and protein carbonyls (Nair et al., 2007; Navarro et al., 2008). The molecular mechanism

aging, frontal cortex and hippocampal mitochondria show a decreased rate of respiration, especially marked with NAD-dependent substrates, and decreased enzymatic activities of complexes I and IV associated with an increase in the content of oxidation products (TBARS

**Figure 6.** Lipid peroxidation and protein peroxidation by secondary products of lipid peroxidation in

and ONOO-

. Upon

involved in the inactivation of complex I is likely accounted for by ROO.

The organism must confront and control the balance of both pro-oxidants and antioxidants continuously. The balance between these is tightly regulated and extremely important for maintaining vital cellular and biochemical functions. This balance often referred to as the redox potential, is specific for each organelle and biological site, and any interference of the balance in any direction might be deleterious for the cell and organism. Changing the balance towards an increase in the pro-oxidant over the capacity of the antioxidant is defined as oxidative stress and might lead to oxidative damage. Changing the balance towards an increase in the reducing power, or the antioxidant, might also cause damage and can be defined as reductive stress.

Oxidative stress and damage have been implicated in numerous disease processes, including inflammation, degenerative diseases, and tumor formation and involved in physiological phenomena, such as aging and embryonic development. The dual nature of these species with their beneficial and deleterious characteristics implies the complexities of their effects at a biological site.

Lipid peroxidation has been pointed out as a key chemical event in the oxidative stress associated with several inborn and acquired pathologies. Disruption of organelle and cell membranes together with calcium homeostasis alterations are the main supramolecular events linked to lipid peroxidation. However, it is not clear if lipid peroxidation process is a cause, triggering step of the clinical manifestations of the disease, or a consequence of toxic effects of lipid peroxidation products.

In pathological situations the reactive oxygen species are generated and as a consequence lipid peroxidation occurs with -tocopherol deficiency. In addition to containing high concentrations of polyunsaturated fatty acids and transitional metals, red blood cells are constantly being subjected to various types of oxidative stress. Red blood cells however are protected by a variety of antioxidant systems which are capable of preventing most of the adverse effects under normal conditions. Among the antioxidant systems in the red cells, tocopherol possesses an important and unique role. -tocopherol may protect the red cells from oxidative damage via a free radical scavenging mechanism and as a structural component of the cell membrane (Chitra Shyamaladevi, 2011).

Levels of Met-Hb. are regarded as an index of intracellular damage to the red cell and it is increased when -tocopherol is consumed and the rate of lipid peroxidation is increased. Scavenging of free radicals by -tocopherol is the first and the most critical step in defending against oxidative damage to the red cells. When -tocopherol is adequate, GSH and ascorbic acid may complement the antioxidant functions of -tocopherol by providing reducing equivalents necessary for its recycling/regeneration.

On the other hand, when -tocopherol is absent, GSH and ascorbic acid release transitional metals from the bound forms and/or maintain metal ions in a catalytic state. Free radical generation catalysed by transition metal ions in turn initiates oxidative damage to cell

membranes. Membrane damage can lead to release of heme compounds from erythrocytes. The heme compounds released may further promote oxidative damage especially when reducing compounds are present (Boveris et al., 2008).

## **8. Lipid peroxidation and aging**

Aging is a process directly related to systemic oxidative stress. Two components of the oxidative stress situation have been recognized in human aging: a decrease in availability of nutritional molecular antioxidants and an accumulation of products derived from the oxidation of biological structures. Oxidation of biomolecules is related to susceptibility to diseases, such as cancer and heart disease, as well as associated with the process of aging (Navarro et al., 2005; Navarro Boveris, 2007, 2008).

The products derived from lipid peroxidation, measured in plasma by Junqueira et al., (2004) as fluorescent products, were higher in elderly than younger human subjects and even higher in disabled octogenarians and nonagenarians. This increase in lipid peroxidation products was directly correlated with age, and was associated with decreases in vitamin E and C.

## **9. Analytical determination of lipid peroxidation**

Since the acceptation of the oxidative stress concept, scientists and physicians have been searching for a simple assay or a small group of determination that would result useful for the assessment of oxidative stress and lipid peroxidation in clinical situations. The determinations of marker metabolites are usually performed in blood, red blood cells or plasma. The markers for systemic oxidative stress are normally present in healthy humans and the assays for systemic oxidative stress are comparative, which makes necessary to have reference values from normal individuals.

At present, the plasma levels of oxidation products derived from free-radical mediated reactions and of antioxidants are used as indicators of systemic oxidative stress in humans and experimental animals. The more utilized determination of an oxidation product is MDA, which is determined with low specificity but with great efficiency by the simple and useful assay of TBARS with measurements made by spectrophotometry or spectrofluorometry. The normal plasma levels of TBARS are 2-3 M (Junqueira et al., 2004).

Oxidative damage is characterized by increases in the levels of the oxidation products of macromolecules, such as thiobarbituric acid reactive substances (TBARS), and protein carbonyls. Many of these products can be found in biological fluids, as well as additionderivatives of these reactive end-products. As a result of lipid peroxidation a great variety of aldehydes can be produced, including hexanal, malondialdehyde (MDA) and 4 hydroxynonenal (Catala, 2006).

Oxidation of an endogenous antioxidant reflects an oxidative stress that is evaluated by measuring the decrease in the total level of the antioxidant or the increase in the oxidative

in vitamin E and C.

membranes. Membrane damage can lead to release of heme compounds from erythrocytes. The heme compounds released may further promote oxidative damage especially when

Aging is a process directly related to systemic oxidative stress. Two components of the oxidative stress situation have been recognized in human aging: a decrease in availability of nutritional molecular antioxidants and an accumulation of products derived from the oxidation of biological structures. Oxidation of biomolecules is related to susceptibility to diseases, such as cancer and heart disease, as well as associated with the process of aging

The products derived from lipid peroxidation, measured in plasma by Junqueira et al., (2004) as fluorescent products, were higher in elderly than younger human subjects and even higher in disabled octogenarians and nonagenarians. This increase in lipid peroxidation products was directly correlated with age, and was associated with decreases

Since the acceptation of the oxidative stress concept, scientists and physicians have been searching for a simple assay or a small group of determination that would result useful for the assessment of oxidative stress and lipid peroxidation in clinical situations. The determinations of marker metabolites are usually performed in blood, red blood cells or plasma. The markers for systemic oxidative stress are normally present in healthy humans and the assays for systemic oxidative stress are comparative, which makes necessary to have

At present, the plasma levels of oxidation products derived from free-radical mediated reactions and of antioxidants are used as indicators of systemic oxidative stress in humans and experimental animals. The more utilized determination of an oxidation product is MDA, which is determined with low specificity but with great efficiency by the simple and useful assay of TBARS with measurements made by spectrophotometry or spectrofluorometry. The normal plasma levels of TBARS are 2-3 M (Junqueira et al., 2004).

Oxidative damage is characterized by increases in the levels of the oxidation products of macromolecules, such as thiobarbituric acid reactive substances (TBARS), and protein carbonyls. Many of these products can be found in biological fluids, as well as additionderivatives of these reactive end-products. As a result of lipid peroxidation a great variety of aldehydes can be produced, including hexanal, malondialdehyde (MDA) and 4-

Oxidation of an endogenous antioxidant reflects an oxidative stress that is evaluated by measuring the decrease in the total level of the antioxidant or the increase in the oxidative

reducing compounds are present (Boveris et al., 2008).

(Navarro et al., 2005; Navarro Boveris, 2007, 2008).

**9. Analytical determination of lipid peroxidation** 

reference values from normal individuals.

hydroxynonenal (Catala, 2006).

**8. Lipid peroxidation and aging** 

form. The only way not to be influenced by nutritional status is to measure the ratio between oxidized and reduced antioxidants present in blood. The published literature provides compelling evidence that a) MDA represents a side product of enzymatic PUFAoxygenation and a secondary end product of no enzymatic (autoxidative) fatty peroxide formation and decomposition and b) sensitive analytical methods exist for the unambiguous isolation and direct quantification of MDA. Conceptually, these two facts indicate that MDA is an excellent index of lipid peroxidation. However, this conclusion is limited in practice by several important consideration: a) MDA yield as a result of lipid peroxidation varies with the nature of the PUFA peroxidised (specially its degree of instauration) and the peroxidation stimulus, b) only certain lipid oxidation products decompose yield MDA, c) MDA is only one of several end product of fatty peroxide formation and decomposition, d) the peroxidation environment influences both the formation of lipid-derived precursors and their decomposition to MDA, e) MDA itself is a reactive substance which can be oxidative and metabolically degraded, f) oxidative injury to no lipid biomolecules has the potential to generate MDA. With biological materials, it appears prudent to consider the TBARS test more than an empirical indicator of the potential occurrence of peroxidative lipid damage and not as a measure of lipid peroxidation (Repetto, 2008). The thiobarbituric acid test (TBARS) has been employed to a uniquely great degree over the last five decades to detect and quantify lipid peroxidation in a variety of chemical as well as biological material. Two underlying assumptions are implicit from the widespread use of the TBARS test to assess lipid peroxidation: a) an operative and quantitative relationship exists between lipid peroxidation and MDA, b) product formation during the TBARS test is diagnostic of the presence and amount of fatty peroxides.

Lipid peroxidation proceeds by a free-radical mediated chain reaction that includes initiation, propagation and termination reactions. The chain reaction is initiated by the abstraction of a hydrogen atom from a methylene group of an unsaturated fatty acid. Propagation is cycled through rounds of lipid peroxyl radical abstraction of the bismethylene hydrogen atoms of a polyunsaturated fatty acyl chain to generate new radicals, after O2 addition, resulting in the conversion of alkyl radical in hydroperoxyl radical. Termination involves the reaction of two hydroperoxyl radicals to form non-radical products. This reaction is particularly interesting since it is accompanied, although at low yield, by emission of light or chemiluminiscence. Some lipid peroxidation products are light-emitting species and their luminescence is used as an internal marker of oxidative stress (Chance et al., 1979; Boveris et al., 1980, Gonzalez-Flecha et al., 1991b; Sies, 1991a; Repetto, 2008). The measurement of light emission derived from 1O2 and excited triplet carbonyl compounds, which are the most important chemiluminiscent species in the lipid peroxidation of biological systems, is directly related to the rate of lipid peroxidation and allows an indirect assay of the content of lipophilic antioxidants in the sample (Gonzalez-Flecha et al., 1991a). Lipophilic antioxidants react with lipid peroxyl radicals and lower antioxidant content is associated with higher chemiluminescence (Repetto, 2008).

The low-level chemiluminescence which accompanies the peroxidation of polyunsaturated fatty acids has been used as a tool in kinetic and mechanistic studies of biological samples to estimate the extent of the reactions and even to indicate tissue damage promoted by oxidants. Triplet carbonyls and singlet oxygen formed in the annihilation of intermediate peroxyl radicals (ROO. ) have been identified as the chemiluminescence emitters.

Chemiluminescence is a very interesting way to evaluate an oxidative stress and lipid peroxidation in biological samples and living systems. The emission of light has been observed during stress in different experimental models. Chemiluminescence is very sensitive and thus can be applied to measure free radical production in human tissues.

Chemiluminescent systems may be classified in two classes based on the origin of the emitting molecule. In the first class, the emitter is a product of the chemical reaction (direct chemiluminescence). In the second class, there is energy transfer between an electronically excited product molecule and a second substance which then becomes the emitter (sensitized chemiluminescence) (Boveris et al., 1980; Gonzalez-Flecha et al., 1991b; Repetto, 2008).

The chemical mechanism responsible for spontaneous organ light emission is provided by the Russell's reaction in which two secondary or tertiary peroxyl radicals (ROO•) yield 1O2 and excited carbonyl groups (=CO\*) as products. In turn, two 1O2, through dimol emission, lead to photoemission at 640 and 670 nm, whereas =CO\* yields photons at the 460-470 nm band (Boveris et al., 1980). The main sources of the chemiluminescence detected in the direct and sensitized chemiluminescence is the dimol emission of ¹O2 (reaction 27) and the photon emission from excited carbonyl groups (reaction 28) (Boveris et al., 1980).

$$\begin{array}{cccc} \text{2} \upharpoonright & \rightarrow & \text{2} \upharpoonright + \text{họ́ (634-703 nm)} \end{array} \tag{27}$$

$$\text{RO}^\* \qquad \rightarrow \quad \text{RO} + \text{hv} \text{ (380-460 nm)} \tag{28}$$

These reactions are accompanied by chemiluminescence whose intensity may serve as an indirect measure of peroxide free radical and -tocopherol concentration in the sample.

Lipid peroxidation has been recognized as free radical-mediated and physiologically occurring (Navarro Boveris, 2004, Navarro et al., 2010; Repetto Boveris, 2012) with the supporting evidence of *in situ* organ chemiluminescence (Repetto, 2008). Spontaneous chemiluminescence of *in situ* organs directly reports the intracellular formation of singlet oxygen (1O2) (Boveris et al., 1980) and represents an issue of direct chemiluminescence. The generation of 1O2 implies the collision of two peroxyl radicals (ROO·) with formation of excited species, 1O2 itself and excited carbonyls, followed by photoemission. Light emission from *in situ* organs is a physiological phenomenon that provides a determination of the steady state concentration of singlet oxygen and indirectly of the rate of oxidative free radical reactions (Boveris et al., 1980). *In situ* liver chemiluminescence has been recognized as a reliable indicator of oxidative stress and damage in rat liver upon hydroperoxide infusion (Gonzalez-Flecha et al., 1991b), ischemia-reperfusion (Gonzalez-Flecha et al., 1993), and chronic and acute alcohol intoxication (Videla et al., 1983). The increases in photoemission observed were parallel to increased contents of indicators of lipid peroxidation (malonaldehyde and 4-HO-nonenal) but with a higher experimental/control ratio in organ chemiluminescence (Boveris et al., 1980).

22 Lipid Peroxidation

2008).

peroxyl radicals (ROO.

The low-level chemiluminescence which accompanies the peroxidation of polyunsaturated fatty acids has been used as a tool in kinetic and mechanistic studies of biological samples to estimate the extent of the reactions and even to indicate tissue damage promoted by oxidants. Triplet carbonyls and singlet oxygen formed in the annihilation of intermediate

Chemiluminescence is a very interesting way to evaluate an oxidative stress and lipid peroxidation in biological samples and living systems. The emission of light has been observed during stress in different experimental models. Chemiluminescence is very sensitive and thus can be applied to measure free radical production in human tissues.

Chemiluminescent systems may be classified in two classes based on the origin of the emitting molecule. In the first class, the emitter is a product of the chemical reaction (direct chemiluminescence). In the second class, there is energy transfer between an electronically excited product molecule and a second substance which then becomes the emitter (sensitized chemiluminescence) (Boveris et al., 1980; Gonzalez-Flecha et al., 1991b; Repetto,

The chemical mechanism responsible for spontaneous organ light emission is provided by the Russell's reaction in which two secondary or tertiary peroxyl radicals (ROO•) yield 1O2 and excited carbonyl groups (=CO\*) as products. In turn, two 1O2, through dimol emission, lead to photoemission at 640 and 670 nm, whereas =CO\* yields photons at the 460-470 nm band (Boveris et al., 1980). The main sources of the chemiluminescence detected in the direct and sensitized chemiluminescence is the dimol emission of ¹O2 (reaction 27) and the photon

These reactions are accompanied by chemiluminescence whose intensity may serve as an indirect measure of peroxide free radical and -tocopherol concentration in the sample.

Lipid peroxidation has been recognized as free radical-mediated and physiologically occurring (Navarro Boveris, 2004, Navarro et al., 2010; Repetto Boveris, 2012) with the supporting evidence of *in situ* organ chemiluminescence (Repetto, 2008). Spontaneous chemiluminescence of *in situ* organs directly reports the intracellular formation of singlet oxygen (1O2) (Boveris et al., 1980) and represents an issue of direct chemiluminescence. The generation of 1O2 implies the collision of two peroxyl radicals (ROO·) with formation of excited species, 1O2 itself and excited carbonyls, followed by photoemission. Light emission from *in situ* organs is a physiological phenomenon that provides a determination of the steady state concentration of singlet oxygen and indirectly of the rate of oxidative free radical reactions (Boveris et al., 1980). *In situ* liver chemiluminescence has been recognized as a reliable indicator of oxidative stress and damage in rat liver upon hydroperoxide infusion (Gonzalez-Flecha et al., 1991b), ischemia-reperfusion (Gonzalez-Flecha et al., 1993),

2 ¹O2 2 O2 + h (634-703 nm) (27)

RO\* RO + h (380-460 nm) (28)

emission from excited carbonyl groups (reaction 28) (Boveris et al., 1980).

) have been identified as the chemiluminescence emitters.

Tert-butyl hydroperoxide initiated chemiluminescence is an example of sensitized chemiluminescence, and it has been used to enhance the chemiluminescence accompanying lipid peroxidation and the -tocopherol content of tissues. This method has been successfully utilized to detect the existence of oxidative damage associated to experimental or pathological situations in tissue homogenates, subcellular fractions, and in human heart, liver and muscle biopsies (Gonzalez-Flecha et al., 1991b).

Tissue homogenates or blood samples are subjected to *in vitro* oxidative damage by supplementation with tert-butyl hydroperoxide. It reacts with hemoproteins and Fe2+ producing peroxyl and alcoxyl free radicals, which enter to the propagation phase of the lipid peroxidation radical chain reaction. The termination steps of the chain reaction generate compounds in an excited state: singlet oxygen and carbonyl groups. This assay is useful to evaluate the integral level of the non-enzymatic antioxidant defenses of a tissue (Gonzalez-Flecha et al., 1991a, 1993).

The increase of tert-butyl hydroperoxide-initiated chemiluminescence is indicative that tocopherol is the antioxidant consumed in erythrocytes and suggest that reactive oxygen species and lipid peroxidation catalyzed by reduced transition metals may be responsible for the onset of oxidative damage and the occurrence of systemic oxidative stress in patients suffering oxidative damage associated to neurological pathologies as Parkinson (Famulari et al., 1996, Dominguez et al., 2008), Alzheimer disease (Famulari et al., 1996; Repetto et al., 1999; Dominguez et al., 2008; Serra et al., 2009), and vascular dementia (Famulari et al., 1996, Dominguez et al., 2008; Serra et al., 2009); immunological diseases as HIV infection and AIDS (Repetto et al., 1996), hyperthyroidism and hypothyroidism (Abalovich et al., 2003). These methods were used to evaluate lipid peroxidation and oxidative damage in experimental models of oxidative stress in rats (Repetto et al., 2003, 2010; Ossani et al., 2007; Repetto Ossani, 2008; Repetto Boveris, 2010).

A common question of the researchers in the field is which the method of choice is. The answer is: none of them, and all of them. Each assay measures something different. Diene conjugation tells one about the early stages of peroxidation, as a direct measurement of lipid peroxides. In the absence of metal ions to decompose lipid peroxides there will be little formation of hydrocarbon gases, carbonyl compounds, or their fluorescent complexes, which does not necessarily mean therefore that nothing is happening. Even if peroxides do not decompose, the TBARS test can still detect them because of decomposition of peroxides. Changes in the mechanism of peroxide decomposition might alter the amount generated without any change in the overall rate of lipid peroxidation. Whatever method is chosen, one should think clearly what is being measured and how it relates to the overall lipid peroxidation process. Whatever possible, two or more different assay methods should be used.

## **10. Conclusion**

Lipid peroxidation is a physiological process that takes place in all aerobic cells. Unsaturated fatty acids which are structural part of cell membranes are subjected to lipid peroxidation by a non enzymatic and free-radical mediated reaction chain. The molecular mechanisms of the lipid peroxidation process are known and it can be estimated that about 1 % of the total oxygen uptake of cells, organs and bodies in taken up by the reactions of lipid peroxidation. The initiation reactions are provided by the transition-metal catalyzed hemolytic scission of H2O2 and ROOH. In turn, H2O2 is mainly generated from the mitochondrial dismutation of superoxide radical (O2.-). The products and by-products of lipid peroxidation are cytotoxic and lead in successive steps to oxidative stress, oxidative damage and apoptosis. In a long series of physiological and pathophysiological processes, including aging and neurodegenerative diseases, the rates of mitochondrial O2.- and H2O2 are increased with a parallel increase in the rate of the lipid peroxidation process. It is expected that supplementation with adequate antioxidants, as for instance, α-tocopherol, will keep sensitive cells and organs in healthy conditions and increase lifespan.

## **Author details**

Marisa Repetto, Jimena Semprine and Alberto Boveris *University of Buenos Aires, School of Pharmacy and Biochemistry, General and Inorganic Chemistry, Institute of Biochemistry and Molecular Medicine (IBIMOL-UBA-CONICET), Argentina* 

## **Acknowledgement**

We thank to Dr. Jorge Serra for helping in the revision of this version.

## **11. References**


Boveris, A.; Cadenas, E.; Reiter, R.; Filipkowski, M.; Nakase, Y. & Chance, B. (1980) Organ chemiluminescence: noninvasive assay for oxidative radical reactions *Proceeding of the National Academy of Sciences of the United States.* Vol. 177, pp. 347-351, ISSN: 0027-8424

24 Lipid Peroxidation

**10. Conclusion** 

**Author details** 

*General and Inorganic Chemistry,* 

**Acknowledgement** 

**11. References** 

Lipid peroxidation is a physiological process that takes place in all aerobic cells. Unsaturated fatty acids which are structural part of cell membranes are subjected to lipid peroxidation by a non enzymatic and free-radical mediated reaction chain. The molecular mechanisms of the lipid peroxidation process are known and it can be estimated that about 1 % of the total oxygen uptake of cells, organs and bodies in taken up by the reactions of lipid peroxidation. The initiation reactions are provided by the transition-metal catalyzed hemolytic scission of H2O2 and ROOH. In turn, H2O2 is mainly generated from the mitochondrial dismutation of superoxide radical (O2.-). The products and by-products of lipid peroxidation are cytotoxic and lead in successive steps to oxidative stress, oxidative damage and apoptosis. In a long series of physiological and pathophysiological processes, including aging and neurodegenerative diseases, the rates of mitochondrial O2.- and H2O2 are increased with a parallel increase in the rate of the lipid peroxidation process. It is expected that supplementation with adequate antioxidants, as for instance, α-tocopherol,

will keep sensitive cells and organs in healthy conditions and increase lifespan.

*Institute of Biochemistry and Molecular Medicine (IBIMOL-UBA-CONICET), Argentina* 

Abalovich, M.; Llesuy, S.; Gutierrez, S. Repetto, M. (2003) Peripheral markers of oxidative stress in Graves´ disease. The effects of methimazole and 131 Iodine treatments. *Clinical* 

Beckman, J.; Beckman, T.; Chen, J.; Marshall, P. Freeman, B. (1990) Apparent hydroxyl radical production from peroxynitrite. Implications for endothelial injury from nitric oxide oxide and superoxide. *Proceeding of the National Academy of Sciences of the United* 

Beckman, J.; Chen, J.; Ischiropulos, H. Crow, J. (1994) Oxidative chemistry of

Berlett, B.S. Stadtman, E.R. (1997) Protein oxidation in aging, disease, and oxidative stress.

peroxynitrite. *Methods in Enzymology.* Vol. 233, pp. 229-240, ISSN: 0076-6879

*The Journal of Biological Chemistry.* Vol. 272, pp. 20313–20316, ISSN: 0021-9258

We thank to Dr. Jorge Serra for helping in the revision of this version.

*Endocrinology*. Vol. 59, pp. 321-327, ISSN: 1365-2265

*States.* Vol. 87, pp. 1620-1624, ISSN: 0027-8424

Marisa Repetto, Jimena Semprine and Alberto Boveris *University of Buenos Aires, School of Pharmacy and Biochemistry,* 


Jomova, K. & Valko, M. (2011) Advances in metal-induced oxidative stress and human disease. *Toxicology.* Vol. 283, pp. 65-87, ISSN: 0300-483X.

26 Lipid Peroxidation

pp. 69-78, ISSN: 0022-510X

597, ISSN 0003-9942

ISSN: 0891-5849

0021-9258

1022, ISSN: 0049-8254

Famulari, A.; Marschoff, E.; Llesuy, S.; Kohan, S.; Serra, J.; Domínguez, R.; Repetto, M.G.; Reides, C. & Lustig, E.S. de (1996). Antioxidant enzymatic blood profiles associated with risk factors in Alzheimer's and vascular diseases. A predictive assay to differentiate demented subjects and controls. *Journal of the Neurological Sciences*, Vol. 141,

Farooqui, T. & Farooqui, A. (2011) Lipid-mediated oxidative stress and inflammation in the pathogenesis of Parkinson´s disease. *Parkinson´s disease*. DOI: 10.4061/2011/247467 Fiszman, M.; D´Eigidio, M.; Ricart, K.; Repetto, M.G.; Llesuy, S.; Borodinsky, L.; Trigo, R.; Riedstra, S.; Costa, P.; Saizar, R.; Villa, A. & Sica, R. (2003). Evidences of oxidative stress in Familial Amyloidotic Polyneuropathy Type 1. *Archives of Neurology*, Vol. 60, pp. 593-

Fraga, C.; Leibovitz, B. & Tappel, A. (1988). Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes. *Free Radicals in Biology and Medicine*, Vol. 4, pp. 155-161,

Fridovich, I. (1978) Superoxide radicals, superoxide dismutases and the aerobic lifestyle.

Fridovich, S. & Porter, N. (1981) Oxidation of arachidonic acid in micelles by superoxide and hydrogen peroxide. *The Journal of Biological Chemistry.* Vol. 256, pp. 260-265, ISSN:

Gatto, E.; Carreras, M.C.; Pargament, G.; Reides, C.; Repetto, M.G.; Llesuy, S.; Fernández Pardal, M. & Poderoso, J. (1996). Neutrophil function nitric oxide and blood oxidative stress in Parkinson's disease. *Movement Disorders*, Vol. 11, pp. 261-267, ISSN: 0885-3185 Gatto, E.; Carreras, C.; Pargament, G.; Riobó, N.; Reides, C.; Repetto, M.; Fernández Pardal, N.; Llesuy, S. & Poderoso, J. (1997). Neutrophyl function nitric oxide and blood oxidative stress in Parkinson´s Disease. *Focus Parkinson´s Disease,* Vol. 9, pp. 12-14 Gonzalez Flecha, B., Repetto, M.; Evelson, P. & Boveris, A. (1991a) Inhibition of microsomal lipid peroxidation by -tocopherol and -tocopherol acetate. *Xenobiotica*. 21: 1013–

González Flecha, B.; Llesuy, S. & Boveris, A. (1991b). Hydroperoxide-initiated chemiluminescence: assay for oxidative stress in biopsies of heart, liver and muscle.

Gonzalez-Flecha, B.; Cutrin, J.C. & Boveris, A. (1993) Time course and mechanism of oxidative stress and tissue damage in rat liver subjected to in vivo ischemia-reperfusion.

Halliwell, B. & Gutteridge, J.M.C. (1984). Oxygen toxicity, oxygen radicals, transition metals

Hogg, N.; Darley-Usmar, V.; Wilson, M. & Moncada, S. (1992) Production of hydroxyl radicals from the simultaneous generation of superoxide and nitric oxide. *Biochemical* 

Free Radicals in Biology and Medicine, Vol. 10, pp. 93-100, ISSN: 0891-5849

*Journal of Clinical Investigation.* Vol. 91, pp. 456–464, ISSN:�0021-9738

and disease. *Biochemical Journal*, Vol. 218, pp. 1-14, ISSN: 0264-6021

*Journal.* Vol. 281, pp. 419-424, ISSN: 0264-6021

*Photochemistry and Photobiology*. Vol. 28, pp. 733-741, ISSN: 1010-6030


nitric oxide synthase functional activity. *Journal of Bioenergetics and Biomembranes*, Vol. 42, pp. 405-412, ISSN: 0145-479X


Sayre, L.M.; Perry, G. & Smith, M.A. (1999) In situ methods for detection and localization of markers of oxidative stress: application in neurodegenerative disorders. *Methods in Enzymology.* Vol. 309, pp. 133–152, ISSN: 0076-6879

28 Lipid Peroxidation

42, pp. 405-412, ISSN: 0145-479X

pp. 107-117, ISSN: 0009-8981

163-194, ISBN: 978-81-7895-334-2

*Chemistry.* Vol. 246, pp. 263-269, ISSN: 0021-9258

ISSN:1093-9946

0022-3573

81-7895-311-3

ISSN: 0340-5761

nitric oxide synthase functional activity. *Journal of Bioenergetics and Biomembranes*, Vol.

Ossani, G.; Dalghi, M. & Repetto, M. (2007) Oxidative damage and lipid peroxidation in the kidney of choline-defficient rats. *Frontiers in Bioscience.* Vol. 12, pp. 1174-1183,

Poyer, J. & McCay, P. (1971) Reduced triphosphopyridine nucleotide oxidase-catalyzed alterations of membrane phospholipids. Dependence on Fe3+. *The Journal of Biological* 

Rachmilewitz, D.; Stamler, J.; Karmeli, F.; Mollins, M.; Singel, D.; Loscalo, J.; Xavier, R. & Podolsky, D. (1993) Peroxynitrite induced rat colitis-a new model of colonic

Repetto, M.; Reides, C.; Gomez Carretero, M.; Costa, M.; Griemberg, G., & Llesuy S. (1996) Oxidative Stress in Erythrocytes of HIV infected patients. *Clinica Chimica Acta.* Vol. 255,

Repetto, M.G.; Reides, C.; Evelson, P.; Kohan, S.; Lustig, E.S. de & Llesuy, S. (1999). Peripheral markers of oxidative stress in probable Alzheimer patients. *European Journal* 

Repetto, M.; María, A.; Giordano, O.; Guzmán,J.; Guerreiro, E. & Llesuy, S. (2003) Protective effect of Artemisia douglasiana Besser extracts on ethanol induced oxidative stress in gastric mucosal injury. *Journal of Pharmacy and Pharmacology.* Vol. 55, pp. 551-557, ISSN:

Repetto, M.G. (2008). Clinical use of chemiluminescence assays for the determination of systemic oxidative stress. In: Popov, I.; Lewin, G. (ed.), *Handbook of chemiluminescent methods in oxidative stress assessment*. Transworld Research Network: Kerala, India; pp.

Repetto, M.G. & Ossani, G. (2008) Sequential histopathological and oxidative damage in different organs in choline deficient rats. In: Álvarez, S.; Evelson P. (ed.), *Free Radical Pathophysiology*. Transworld Research Network: Kerala, India; pp. 433-450, ISBN: 978-

Repetto, M.G.; Ferrarotti, N.F. & Boveris, A. (2010a) The involvement of transition metal ions on iron- dependent lipid peroxidation. *Archives of Toxicology.* Vol. 84, pp. 255-262,

Repetto, M.; Ossani, G.; Monserrat, A. & Boveris, A. (2010b) Oxidative damage: The biochemical mechanism of cellular injury and necrosis in choline deficiency.

Repetto, M. & Boveris, A. (2010) Bioactivity of sesquiterpenes: novel compounds that protect from alcohol-induced gastric mucosal lesions and oxidative damage. *Mini* 

Repetto, M.G. & Boveris A. (2012). Transition metals: bioinorganic and redox reactions in biological systems. In: *Transition metals: uses and characteristics*. Nova Science Publishers

*Experimental and Molecular Pathology.* Vol. 88, pp. 143-149. ISSN: 0014-4800.

*Reviews in Medicinal Chemistry.* Vol. 10, pp. 615-623. ISSN: 1389-5575

Inc (ed.): New York, USA. pp. 349-370., ISBN: 978-1-61761-110-0

inflammation. *Gastroenterology.* Vol. 105. pp. 1681-1688, ISSN: 0016-5085

*of Clinical Investigation,* Vol. 29, pp. 643-649, ISSN: 0014-2972


## **Lipid Oxidation in Homogeneous and Micro-Heterogeneous Media in Presence of Prooxidants, Antioxidants and Surfactants**

Vessela D. Kancheva and Olga T. Kasaikina

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46021

### **1. Introduction**

30 Lipid Peroxidation

Uchida, K. (2003) 4-Hydroxy-2-nonenal: A product and mediator of oxidative stress. *Progress* 

Yin, H.; Xu, L. & Porter, N.A. (2011) Free radical lipid peroxidation: mechanisms and

*in Lipid Research*. Vol. 42, pp. 318–343. ISSN: 0163-7827

analysis. *Chemical Reviews.* 111: 5944-5972. ISSN: 0009-2665

The human body is constantly subjected to a significant oxidative stress as a result of the misbalance between antioxidative protective systems and the formation of strong oxidizing substances, including free radicals. The stress can damage DNA, proteins, lipids and carbohydrates and could cause negative effect to intracellular signal transmission. Antioxidants could be promising agents for management of oxidative stress-related diseases. Oxygen is essential for all living organisms, but at the same time it is a source of constant aggression for them. In its ground triplet state (3O2) oxygen has weak reactivity, but it can produce strongly aggressive and reactive particles such as singlet state oxygen (1O2), hydroperoxides (H2O2), superoxide anion (O2- ), hydroxylic radical (OH) and various peroxide (LO2) and alkoxy radicals (LO). It is well known that the latter lead to an oxidative degradation of biological macromolecules, changing their properties and thus the cell structure and functionality. The free radicals formation in the hydrophobic parts of the biological membranes initiates radical disintegration of the hydrocarbon "tails" of the lipids. This process is known as lipid peroxidation (Figure 1) [1-3].

**Figure 1.** Erosion of cell membrane, antioxidant neutralizes free radicals and lipid peroxidation

© 2012 Kancheva and Kasaikina, 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. © 2012 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.

In recent decades, many communications have been devoted to the significant role of physical factors, which control the structure of nutrition systems, in the chemistry of lipid oxidation in oil/water (O/W) emulsions [4,5]. In this case, the rate of lipid oxidation strongly depends on the physical properties of interfaces, because they affect the character of the interaction between water-soluble compounds of transition metals and hydroperoxides located inside and on the surface of emulsion droplets. For example, positively charged and high viscosity interfaces that hinder the contact between iron ions and hydroperoxides inhibit oxidation of fat emulsions [6,7]. Another example of the influence of physical factors on the oxidation is the antioxidant polar paradox [6-9], which is based on the fact that nonpolar antioxidants are efficient in O/W emulsions, because they are located in emulsion droplets together with oxidizable lipids. Polar antioxidants are more efficient in W/O emulsions because they are concentrated at the interfaces [10-12]. In the frame of this chapter, the main features of lipid oxidation in homogeneous and micro - heterogeneous oil media, formed by surfactants (W/O microemulsions) will be discussed.

## **2. Kinetic model of homogeneous lipid oxidation**

The kinetic model is based (Table 1) on the reactions and corresponding rate constants known for the oxidation of methyl linoleate (MeLi), because linoleic esters are easily oxidizable components of many natural lipid systems and thus determines the oxidizability of the lipid substrates.

The kinetic scheme of liquid-phase (homogeneous) oxidation of lipids (LH) includes reactions 1-12.

In the presence of an initiator (I), i.e. initiated oxidation, the formation of radicals occurs with a constant initiation rate RIN=2k1[I]. Under autoxidation conditions ([I]=0), the rates of radical formation in the reactions of LH with O2 (reaction 6) and decomposition of lipid hydroperoxides (LOOH) (reactions 7 and 8) increase as LOOH is accumulated. The chain termination occurs due to recombination or disproportionation of radicals (reactions 9, 11 and 12). The scheme of inhibited oxidation includes reactions 13-19, known for the phenolic antioxidants, AH.

The calculations was performed for three groups of AH, which differ in their activity in reaction 13 with peroxyl radicals LOO: group I (of the type of alpha-tocopherol) with k13=1.5 106M-1s-1; group II (AH of type of unhindered phenols, e.g. hydroquinone), with k13=1.5 105M-1s-1; and group III (AH of the type of sterically hindered phenols, e.g. butylated hydroxyl toluene, BHT with k13=1.5 104M-1s-1. The effect of AH regeneration in reaction 16 was considered for rapidly (k16=4 107M-1s-1) and slowly (k16=3 103M-1s-1) reacting phenoxyl radicals A**·**. Reaction 19 is the chain transfer by the inhibitor radical. It can be a hydrogen abstraction from the substrate molecule with regeneration of the inhibitor. Chain transfer can occur as the addition of A· to unsaturated bonds of polyene compounds, in this case, the inhibitor is not regenerated [13]. We examined both cases, rate constants k19 were varied within the 0-100 M-1s-1 range (taking into account the published data). In this series of calculations, we accepted that reaction 16 occurs as disproportionation.


of the lipid substrates.

reactions 1-12.

antioxidants, AH.

In recent decades, many communications have been devoted to the significant role of physical factors, which control the structure of nutrition systems, in the chemistry of lipid oxidation in oil/water (O/W) emulsions [4,5]. In this case, the rate of lipid oxidation strongly depends on the physical properties of interfaces, because they affect the character of the interaction between water-soluble compounds of transition metals and hydroperoxides located inside and on the surface of emulsion droplets. For example, positively charged and high viscosity interfaces that hinder the contact between iron ions and hydroperoxides inhibit oxidation of fat emulsions [6,7]. Another example of the influence of physical factors on the oxidation is the antioxidant polar paradox [6-9], which is based on the fact that nonpolar antioxidants are efficient in O/W emulsions, because they are located in emulsion droplets together with oxidizable lipids. Polar antioxidants are more efficient in W/O emulsions because they are concentrated at the interfaces [10-12]. In the frame of this chapter, the main features of lipid oxidation in homogeneous and micro - heterogeneous oil

The kinetic model is based (Table 1) on the reactions and corresponding rate constants known for the oxidation of methyl linoleate (MeLi), because linoleic esters are easily oxidizable components of many natural lipid systems and thus determines the oxidizability

The kinetic scheme of liquid-phase (homogeneous) oxidation of lipids (LH) includes

In the presence of an initiator (I), i.e. initiated oxidation, the formation of radicals occurs with a constant initiation rate RIN=2k1[I]. Under autoxidation conditions ([I]=0), the rates of radical formation in the reactions of LH with O2 (reaction 6) and decomposition of lipid hydroperoxides (LOOH) (reactions 7 and 8) increase as LOOH is accumulated. The chain termination occurs due to recombination or disproportionation of radicals (reactions 9, 11 and 12). The scheme of inhibited oxidation includes reactions 13-19, known for the phenolic

The calculations was performed for three groups of AH, which differ in their activity in reaction 13 with peroxyl radicals LOO: group I (of the type of alpha-tocopherol) with k13=1.5 106M-1s-1; group II (AH of type of unhindered phenols, e.g. hydroquinone), with k13=1.5 105M-1s-1; and group III (AH of the type of sterically hindered phenols, e.g. butylated hydroxyl toluene, BHT with k13=1.5 104M-1s-1. The effect of AH regeneration in reaction 16 was considered for rapidly (k16=4 107M-1s-1) and slowly (k16=3 103M-1s-1) reacting phenoxyl radicals A**·**. Reaction 19 is the chain transfer by the inhibitor radical. It can be a hydrogen abstraction from the substrate molecule with regeneration of the inhibitor. Chain transfer can occur as the addition of A· to unsaturated bonds of polyene compounds, in this case, the inhibitor is not regenerated [13]. We examined both cases, rate constants k19 were varied within the 0-100 M-1s-1 range (taking into account the published data). In this series of

calculations, we accepted that reaction 16 occurs as disproportionation.

media, formed by surfactants (W/O microemulsions) will be discussed.

**2. Kinetic model of homogeneous lipid oxidation** 

Note: The rate constants (k0 correspond to the oxidation of MeLi at 60oC; in reaction 1, 2 and 4, k are presented in s-1. Initial concentrations: [LH]=2.9M, [LOOH]0=10-5M, [I]=4 10-3M, [AH]0=10-4M, [O2]=10-3M=const; oxidation usually occurs at a constant oxygen pressure, therefore [O2] is included in the corresponding rate constants: k2=k4=k6=ki[O2].

**Table 1.** The Approximate Rate Constants of the Different Reactions Involved in the Autoxidation of Methyl Linoleate [13] and Limonene [14] in initiated oxidation, autoxidation and inhibited oxidation (at 60oC).


**Table 2.** The main kinetic parameters of initiated oxidation and lipid autoxidation

Under other equivalent conditions, the bimolecular decay of 2A**·** by disproportionation in which AH is regenerated gives a considerable advantage in retardation effects as compared with the situation where no regeneration occurs (recombination of 2A**·**). The presence of the second hydroxyl group in the aromatic ring results in higher k16. In this case, an increase in the induction period related to AH regeneration is most pronounced.

Lipid oxidation is one of the important reactions in biology. Chemical reaction kinetics considers two aspects: the rate of reaction and effective factors – temperature concentration of reactants and products. This knowledge is an essential prerequisite for modeling the lipid oxidation, the shelf life of stored foods, durability of low density proteins, and so on.

## **3. Effect of pro-oxidants (ROH) leading to acceleration of lipid hydroperoxides (LOOH) decomposition**

### **3.1. Kinetic modeling of lipid oxidation for different mechanism of LOOH decomposition**

A kinetic analysis of non-inhibited lipid (LH) autoxidation for different mechanisms of hydroperoxides (LOOH) decay is proposed [22]. It is based on using of mathematical simulation methods of LH autoxidation kinetics. Kinetic schemes of LH autoxidation for some different ways of hydroperoxides decay - mono-molecular, pseudo-mono-molecular and/or bimolecular mechanism are presented. This analysis permits establishing the influence degree of different hydroperoxides decay mechanisms on the kinetic parameters, characterizing the substrate oxidizability. The proposed kinetic analysis has been applied to the methyl linoleate, MeLi) autoxidation at 60°C.

The kinetic model that describes the lipid hydroperoxides decomposition taking into account the possibility of monomolecular (LOOH), pseudo-monomolecular (LOOH + LH) and bimolecular (2 LOOH) mechanisms in both cases: in presence of an oxygen (O2) and in its absence, i.e. in an inert atmosphere (N2) is illustrated by Scheme 1. In these equations:

Lipid Oxidation in Homogeneous and Micro-Heterogeneous Media in Presence of Prooxidants, Antioxidants and Surfactants 35

1 2 2 1 1 4 1 – 1 2 (1 [ ] *K LH K T LOOH K K LH* 2 1 2 *<sup>i</sup> kp T To LH k t kt* 2 2 1 4 (1 ) *<sup>K</sup> <sup>C</sup> K LH* 3 1 31 1 1 [ ] 1 [] *o o i e k e k K LH d k K LH* 2 2 <sup>1</sup> H ( [ ] <sup>4</sup> L ) *p p i o i t t k k O k T t LH k t k k* 

LH: is linoleic acid with its allylic hydrogen LOO•: peroxide radical LOOH: lipid hydroperoxides K1 and K2: are the equilibrium constants for complexes Q and D, respectively [T]: summary concentration of LOOH k30, k31 and k32: are the corresponding rate constants e0, e1, e2: are the corresponding radicals yield

34 Lipid Peroxidation

(R0)

The main kinetic parameters Initiated oxidation Lipid autoxidation

kp = k5 ; kt = k9

Oxidizability parameter a = kp/(2kt)0.5 a = kp/(2kt)0.5 Inhibition degree (ID) ID = 0/A ID = R0/RA Induction period (IP) IP=n[AH]0/RIN IP=n[AH]0/RIN Antioxidant efficiency nkA PF=IPA/IP0 and

**Table 2.** The main kinetic parameters of initiated oxidation and lipid autoxidation

the induction period related to AH regeneration is most pronounced.

**hydroperoxides (LOOH) decomposition** 

the methyl linoleate, MeLi) autoxidation at 60°C.

**decomposition** 

controlled RIN=2k1[I]

R0=kp [LH](RIN/2kt)0.5 RA=kp[LH]RIN/nkA[AH]0

Under other equivalent conditions, the bimolecular decay of 2A**·** by disproportionation in which AH is regenerated gives a considerable advantage in retardation effects as compared with the situation where no regeneration occurs (recombination of 2A**·**). The presence of the second hydroxyl group in the aromatic ring results in higher k16. In this case, an increase in

Lipid oxidation is one of the important reactions in biology. Chemical reaction kinetics considers two aspects: the rate of reaction and effective factors – temperature concentration of reactants and products. This knowledge is an essential prerequisite for modeling the lipid

oxidation, the shelf life of stored foods, durability of low density proteins, and so on.

**3.1. Kinetic modeling of lipid oxidation for different mechanism of LOOH** 

A kinetic analysis of non-inhibited lipid (LH) autoxidation for different mechanisms of hydroperoxides (LOOH) decay is proposed [22]. It is based on using of mathematical simulation methods of LH autoxidation kinetics. Kinetic schemes of LH autoxidation for some different ways of hydroperoxides decay - mono-molecular, pseudo-mono-molecular and/or bimolecular mechanism are presented. This analysis permits establishing the influence degree of different hydroperoxides decay mechanisms on the kinetic parameters, characterizing the substrate oxidizability. The proposed kinetic analysis has been applied to

The kinetic model that describes the lipid hydroperoxides decomposition taking into account the possibility of monomolecular (LOOH), pseudo-monomolecular (LOOH + LH) and bimolecular (2 LOOH) mechanisms in both cases: in presence of an oxygen (O2) and in its absence, i.e. in an inert atmosphere (N2) is illustrated by Scheme 1. In these equations:

**3. Effect of pro-oxidants (ROH) leading to acceleration of lipid** 

RIN=2k6[LH][O2] +

R0=kp [LH](RIN/kt)0.5 RA=kp[LH]RIN/nkA[AH]0

RAE=(IPA-IP0)/IP0

2k7[LH][LOOH]+2k8[LOOH]2

Rate of initiation (RIN) Constant and well-

Rate of oxidation (R0) and (RA) Rate of non-inhibited oxidation

Rate of inhibited oxidation (RA)

**Scheme 1.** Kinetic scheme of lipid hydroperoxide decomposition reactions

The kinetic scheme 2 is significantly simplified and readily solved assuming a quasi-steadystate for LOO•, rapid achievement of equilibrium and neglected of the loss of Q and LOOH since their decomposition rate constants are low. There are marked: **C** - is the ratio between the equilibrium constants of bi- and pseudo-mono-molecular mechanisms of LOOH decomposition, needed to be marked for the solution of the equation.

RIN: the rate of chain generation, kp: rate constants of chain propagation, kt: rate constants of chain

**Scheme 2.** Kinetic scheme of lipid autoxidation by Kancheva and Belyakov [22]

Figures 2-5 presents kinetics of different mechanisms of lipid hydroperixides decomposition. In Figure 6 it is shown, that ki doesn't change with growing of MeLi concentration from 0.3 to 1.7 M, when the concentration of MeLi hydroperoxydes is smaller than 5 10–3 M. It is established, that MeLi hydroperoxides decay is in agreement with a first order reaction and pseudo-mono-molecular mechanism (a reaction between hydroperoxides and non-oxidized lipid substrate; LOOH + LH).

**Figure 2.** Influence of dimer formation equilibrium constant (K2) on the kinetics of MeLi autoxidation at 60oC, when e2k32 has a great value (e2k32=2 10-6), [T0]=10-4M, d=2 10-7 and kp/(kt)1/2 = 6 10-2

The kinetic scheme 2 is significantly simplified and readily solved assuming a quasi-steadystate for LOO•, rapid achievement of equilibrium and neglected of the loss of Q and LOOH since their decomposition rate constants are low. There are marked: **C** - is the ratio between the equilibrium constants of bi- and pseudo-mono-molecular mechanisms of LOOH

decomposition, needed to be marked for the solution of the equation.

RIN: the rate of chain generation, kp: rate constants of chain propagation, kt: rate constants of chain **Scheme 2.** Kinetic scheme of lipid autoxidation by Kancheva and Belyakov [22]

and non-oxidized lipid substrate; LOOH + LH).

Figures 2-5 presents kinetics of different mechanisms of lipid hydroperixides decomposition. In Figure 6 it is shown, that ki doesn't change with growing of MeLi concentration from 0.3 to 1.7 M, when the concentration of MeLi hydroperoxydes is smaller than 5 10–3 M. It is established, that MeLi hydroperoxides decay is in agreement with a first order reaction and pseudo-mono-molecular mechanism (a reaction between hydroperoxides

**Figure 2.** Influence of dimer formation equilibrium constant (K2) on the kinetics of MeLi autoxidation at

60oC, when e2k32 has a great value (e2k32=2 10-6), [T0]=10-4M, d=2 10-7 and kp/(kt)1/2 = 6 10-2

**Figure 3.** Influence of dimer formation equilibrium constant (K2) on the kinetics of MeLi autoxidation at 60oC, when e2k32 has a small value (e2k32= 10-10), [T0]=10-4M, d=2 10-7 and kp/(kt)1/2 = 6 10-2(i.e. very small value of e2)

**Figure 4.** Influence of the substrate (MeLi) diluting with an inert solvent (concentrations of 25, 50 and 100%) at 60oC (e2k32=2 10-6)

**Figure 5.** Kinetic curves of inhibited oxidation and autoxidation of MeLi at 60oC, when there is no dimerization of lipid hydroperoxides (K2=0)

**Figure 6.** Effect of ROH (0.1M, 1-Octadecanol, 1-OD) on the kinetics of hydroperoxide accumulation of MeLi at 60oC, at different MeLi concentrations (0.3, 1.0 and 1.7M) and dependence of the effective initiation rate constants ki on [LH] in the absence (1) and in presence of 0.1M 1-Octadecanol (2).

It is shown, that in presence of a lipid hydroxyl compound ki ROH is strongly growing with the decrease of MeLi (LH) concentration (Figure 6). This is explained with the competition of reactions (LOOH + LH and LOOH + ROH). Some different mechanisms, which are possible for reaction between LOOH and ROH, were discussed (Scheme 3).

Ks: is the equilibrium constant for complex S, initial rate of decomposition of complex S (esks) Lipid hydroxy compounds (LOH) from the oxidized lipid substrate (LH) is formed during the whole oxidation process:

LOH and H2O with the rate e0 k30 [LOOH] from the hydroperoxides of substrate LH

LOH and H2O with the rate e1 k31 [Q] from the Q| LOH and H2O with the rate e3 k33 [S] from the S

38 Lipid Peroxidation

**Figure 5.** Kinetic curves of inhibited oxidation and autoxidation of MeLi at 60oC, when there is no

**Figure 6.** Effect of ROH (0.1M, 1-Octadecanol, 1-OD) on the kinetics of hydroperoxide accumulation of MeLi at 60oC, at different MeLi concentrations (0.3, 1.0 and 1.7M) and dependence of the effective initiation rate constants ki on [LH] in the absence (1) and in presence of 0.1M 1-Octadecanol (2).

the decrease of MeLi (LH) concentration (Figure 6). This is explained with the competition of reactions (LOOH + LH and LOOH + ROH). Some different mechanisms, which are possible

ROH is strongly growing with

It is shown, that in presence of a lipid hydroxyl compound ki

for reaction between LOOH and ROH, were discussed (Scheme 3).

dimerization of lipid hydroperoxides (K2=0)

**Scheme 3.** Kinetic scheme of lipid hydroperoxides (LOOH) decay in presence of ROH

$$\begin{aligned} \text{A} &= \frac{1}{4} \left( \frac{k\_p}{\sqrt{k\_t}} \text{[LH]} \right)^2 k\_i \\\\ k\_i &= \frac{4A}{(\frac{k\_p}{\sqrt{k\_t}} \text{[LH]})^2} \\\\ B &= \frac{k\_p}{\sqrt{k\_t}} \sqrt{k\_i \text{[}^{T}\_{o}\text{]}} \\\\ \left[ \text{T}\_o \right] &= \frac{B}{\sqrt{k\_p} \text{[}^{\text{LH}}\text{]} \sqrt{k\_i}} \text{)^2} \end{aligned}$$

There are presented some different mechanisms of the interaction between lipid hydroperoxides (LOOH) and hydroxy compounds (ROH):

**Scheme 4.** Additional lipid hydroperoxides decomposition in presence of an antioxidant (AH)

$$\begin{aligned} \text{LCOOH} + \text{AH} &\xleftarrow{\text{K}\_{\text{P}}} \text{P} & \xrightarrow[\text{LH}, \text{O}\_{2}]{\text{K}\_{\text{IP}}} \text{LO}\_{2}^{\cdot} \\ \text{K}\_{\text{P}} &= \frac{[\text{P}]}{[\text{LCOOH}][\text{AH}]} \end{aligned}$$

KP: the equilibrium constant for complex P,

kiP-initiation rate constant of P decomposition

Total hydroperoxides concentration [T] in presence of ROH and AH

[T] = [LOOH]+[Q]+[S]+[P]= [LOOH](1+K1[LH]+KS[ROH]+KP[AH])

**Scheme 5.** Equilibrium constant of complex formation between an antioxidant (AH) and lipid hydroperoxides (ROH)

$$\begin{aligned} \text{[}\text{AH+ROH} \xrightarrow{\text{[}\text{K}\text{]}} \text{[}\text{AH...O}^{\text{[}\text{H}\text{]}}\text{]}\\ \text{K}\_{0} = \frac{[\text{AH}]\_{0} - [\text{AH}]}{[\text{AH}][\text{ROH}]} \end{aligned} $$

$$[\text{AH}] = \frac{[\text{AH}]\_{0}}{1 + \text{K}\_{0}[\text{ROH}]}$$

#### **Scheme 6.**

It has been proven [23,24] that fatty alcohols with different chain length, mono- and diacylglycerols increase the rate of LOOH decomposition into free radicals and thus accelerated lipid oxidation in absence of an antioxidant. In presence of phenolic antioxidants ROH make complexes basing on H bond formation and thus decrease the antioxidant efficiency of them [25,26]. DL-alpha –tocopherol and butylated hydroxyl toluene demonstrate the best antioxidant efficiency in presence of ROH [26]. Taking into account that ROH are formed during the proceeding, transportation and storage of lipids and lipid containing products as a result of hydrolysis, it is of importance to know how to improve their oxidative stability.

## **4. Antioxidants – Inhibitors of lipid oxidation**

40 Lipid Peroxidation

**Scheme 4.** Additional lipid hydroperoxides decomposition in presence of an antioxidant (AH)

**Scheme 5.** Equilibrium constant of complex formation between an antioxidant (AH) and lipid

AH+ROH AH...O

K0=

[AH]=

K0

[AH]0 - [AH] [AH][ROH]

It has been proven [23,24] that fatty alcohols with different chain length, mono- and diacylglycerols increase the rate of LOOH decomposition into free radicals and thus accelerated lipid oxidation in absence of an antioxidant. In presence of phenolic antioxidants ROH make complexes basing on H bond formation and thus decrease the antioxidant efficiency of them [25,26]. DL-alpha –tocopherol and butylated hydroxyl toluene demonstrate the best antioxidant efficiency in presence of ROH [26]. Taking into account that ROH are formed during the proceeding, transportation and storage of lipids and lipid containing products as a result of hydrolysis, it is of importance to know how to improve

[AH]0 1+K0[ROH]

KP

[P] [LOOH][AH]

LH, O2

kiP

LO2

H

R

LOOH + AH P

KP

Total hydroperoxides concentration [T] in presence of ROH and AH [T] = [LOOH]+[Q]+[S]+[P]= [LOOH](1+K1[LH]+KS[ROH]+KP[AH])

KP: the equilibrium constant for complex P, kiP-initiation rate constant of P decomposition

hydroperoxides (ROH)

**Scheme 6.**

their oxidative stability.

=

The introduction of antioxidants in the affected body normalizes not only the peroxide oxidation, but also the lipid content. Antioxidants used in oncology are effective in the first stages as mono-therapy with antioxidants at high concentrations and at the last stages mainly as additives in the complex tumor therapy - the antioxidant is in low concentrations. In this respect the medical treatment of most of diseases includes formulations based on a combination of traditional drugs with targeted functionality and different antioxidants [3,27].

The activity of antioxidants depends on complex factors including the nature of the antioxidants, the condition of oxidation, the properties of substrate, being oxidized and the stage of oxidation [2,3,27-33].

Capacity of antioxidants has at least two sides: the antioxidant potential, determined by its composition and properties of constituents and is the subject of food chemistry, and the biological effects, depending, among other things, on bioavailability of antioxidants, and is a medico-biological problem.

#### **4.1. Classification of antioxidants [2,16,30-36]**

Depending on their mechanism of action:

**Antioxidants, inhibiting lipid oxidation by trapping lipid peroxide radicals**– they are aromatic compounds with a weak О-Н, N-H bonds (phenols, amines, aminophenols, diamines etc.

**Antioxidants, inhibiting the oxidation process by trapping alkyl radicals** – they are quinones, methylene quinones, which are effective in low oxygen concentration.

**Hydroperoxide decomposers** – these compounds react with hydroperoxides without formation of free radicals.

**Metal chelators** – oxidation process can be inhibited by addition of compounds, forming complexes with metal ions and thus made them inactive towards hydroperoxides. In this groups are hydroxyl acids, flavonoids etc.

**Antioxidants with multistage action** – systems containing such kind of compounds (alcohols and amines) inhibitors can be regenerated during the oxidation process.

**Inhibitors with combined action** – inhibitor molecule has two or more functional groups, each of them react in different reactions.

Depending on their nature:

**Natural antioxidants** – usually with low toxicity (with some exception), wide spectrum of biological and antioxidant activities.

**Synthetic** - they are with a high antioxidant activity. However, antioxidants for application in foods and additives or supplements they must pass additional criteria (no toxicity, safety, healthy, low cost, etc.)

Depending of their biological activity:

**Bio-antioxidants** – compounds with both biological and antioxidant activities. Last decades there is a growing interest to the nature-like bio-antioxidants. The most important known bio-antioxidants are flavonoids and phenolic acids.

**Antioxidants without a biological activity** - some even natural antioxidants can show a toxic activity and for that reason they must be tested.

Depending on the number of phenolic groups:

**Monophenols** *–* the known compounds are butylated hydroxyl toluene (ВНТ), Tocopherol (ТОН), p-coumaric acid (p-CumA), ferulic acid (FA), sinapic acid (SA) etc.

**Biphenols** – the known compounds are caffeic acid (CA), hydroquinone (HQ), tertbutylated hydroquinone (TBHQ) etc.

**Polyphenols** –flavonoids: quercetin (Qu), rutin (Ru), luteolin (Lu), kampferol (Kf), isorhamnetin (Isorh) etc.

The inherent compositional and structural complexity of real foods and in vivo studied means that systematic studies of lipid oxidation must first be carried out in model systems. The following models were applied to explain the structure-activity relationship of different phenolic antioxidants: model 1, a DPPH assay used for the determination of the radical scavenging capacity (AH+DPPH•→A**·**+DPPH-H); model 2, chemiluminescence (CL) of a model substrate RH (cumene and diphenylmethane) used for determination of the rate constant of a reaction with model peroxyl radicals (AH+RO2·→A·+ROOH); model 3, lipid autoxidation (LAO) used for the determination of the chain-breaking antioxidant efficiency and reactivity (AH+LOO**·**→A**·**+LOOH; A**·**+LH(+O2)→AH+LOO**·**); and model 4, theoretical methods used for predicting the activity (predictable activity by statistical and/or quantumchemical calculations).

#### **4.2. Structure of the antioxidants**

By combination of different experimental methods: DPPH test, lipid autoxidation kinetics, chemiluminescence kinetics and quantum chemical calculations it has been proven that the prooxidant activity of chalcones is due to the possible reaction of phenoxyl radicals formed with oxygen and formation of dioxiethanes, [37]Vasil'ev *et al.*, 2009:

New bis-coumarins are found to have anti-HIV activity [38]. Together with their antioxidant capacity (Fig. 8C) they are one of the most important bio-antioxidants nowadays.

The studied simple dihydroxy-coumarins are natural (Cum0) and nature-like synthetic compounds with a wide range of biological activities against cancer, inflammatory, cardiovascular diseases, diabetes etc. Together with the strong antioxidant activity and synergistic effect with Tocopherol, they are very important for the practical application, [40]Kancheva *et al*., 2010a.


Depending of their biological activity:

bio-antioxidants are flavonoids and phenolic acids.

toxic activity and for that reason they must be tested.

Depending on the number of phenolic groups:

hydroquinone (TBHQ) etc.

isorhamnetin (Isorh) etc.

chemical calculations).

*al*., 2010a.

**4.2. Structure of the antioxidants** 

**Bio-antioxidants** – compounds with both biological and antioxidant activities. Last decades there is a growing interest to the nature-like bio-antioxidants. The most important known

**Antioxidants without a biological activity** - some even natural antioxidants can show a

**Monophenols** *–* the known compounds are butylated hydroxyl toluene (ВНТ), Tocopherol

**Biphenols** – the known compounds are caffeic acid (CA), hydroquinone (HQ), tertbutylated

**Polyphenols** –flavonoids: quercetin (Qu), rutin (Ru), luteolin (Lu), kampferol (Kf),

The inherent compositional and structural complexity of real foods and in vivo studied means that systematic studies of lipid oxidation must first be carried out in model systems. The following models were applied to explain the structure-activity relationship of different phenolic antioxidants: model 1, a DPPH assay used for the determination of the radical scavenging capacity (AH+DPPH•→A**·**+DPPH-H); model 2, chemiluminescence (CL) of a model substrate RH (cumene and diphenylmethane) used for determination of the rate constant of a reaction with model peroxyl radicals (AH+RO2·→A·+ROOH); model 3, lipid autoxidation (LAO) used for the determination of the chain-breaking antioxidant efficiency and reactivity (AH+LOO**·**→A**·**+LOOH; A**·**+LH(+O2)→AH+LOO**·**); and model 4, theoretical methods used for predicting the activity (predictable activity by statistical and/or quantum-

By combination of different experimental methods: DPPH test, lipid autoxidation kinetics, chemiluminescence kinetics and quantum chemical calculations it has been proven that the prooxidant activity of chalcones is due to the possible reaction of phenoxyl radicals formed

New bis-coumarins are found to have anti-HIV activity [38]. Together with their antioxidant

The studied simple dihydroxy-coumarins are natural (Cum0) and nature-like synthetic compounds with a wide range of biological activities against cancer, inflammatory, cardiovascular diseases, diabetes etc. Together with the strong antioxidant activity and synergistic effect with Tocopherol, they are very important for the practical application, [40]Kancheva *et* 

capacity (Fig. 8C) they are one of the most important bio-antioxidants nowadays.

with oxygen and formation of dioxiethanes, [37]Vasil'ev *et al.*, 2009:

(ТОН), p-coumaric acid (p-CumA), ferulic acid (FA), sinapic acid (SA) etc.

**Table 3.** The main structures and activities with methods for benzoic acids, cinnamic acids, N-cinnamic acids amides and chalcones

*Chalcone Ch3(∆Hf*

*D1*

*Dioxiethanes D1 and D2 ∆Hf 0(D1•) = -19. 9 kcal/mol, ∆Hf 0(D2•) = -27.1 kcal/mol*

(b)



**Table 4.** Simple and Bis-Coumarins

(c) [38]Kancheva et al, 2010b (d) [39]Kancheva et al, 2012

**E.Simple Coumarins** 

R8

R5 R6

HO

**F.Bis-Coumarins** 

> O O

> > R5

OH

HO

H C

R4 R3

O O

Bis-Cum1

Bis-Cum2

Bis-Cum3

Bis-Cum4

Bis-Cum5

**Table 4.** Simple and Bis-Coumarins

O O

R3

*Chalcone Ch3(∆Hf*

R4

*∆Hf*

**Figure 7.** a) Optimized structures of Chalcone Ch3 (7a) and its aryl radical (7b); b) Optimized structures

(b)

*0(D2•) = -27.1 kcal/mol*

*<sup>0</sup> = -58.3 kcal/mol) Radical formed from Ch 3* (*∆Hf*

(a)

**Abbr. R3 R4 R5 R6 R8 Activity Method**  Cum0 H H H OH H Strong LAO Cum1 H CH3 H OH H Strong LAO Cum2 H CH3 H H OH Strong LAO Cum3 EtCOOMe CH3 H H OH Strong LAO Cum4 MeCOOEt CH3 H H OH Strong LAO Cum5 H CH3 OH H H Weak LAO Cum6 H CH3 H H H Weak LAO Cum7 H OH H H H Weak LAO

*<sup>0</sup> = -29.0 kcal/mol)*

*D2*

**Abbr. R3 R4 R5 Activity Method Abbr. R3** 

OH OH H Strong LAO Bis-Cum1 OH

OCH3 OH OCH3 Moderate LAO Bis-Cum2 OCH3

OCH3 OH NO2 Weak LAO Bis-Cum3 OCH3

OCH3 OCH3 H Weak LAO Bis-Cum4 OCH3

OCH3 OCH3 OCH3 Weak LAO Bis-Cum5 OCH3

of Dioxiethanes D1 and D2 formed from Ch3 radical and oxygen.

*D1*

*0(D1•) = -19. 9 kcal/mol, ∆Hf*

*Dioxiethanes D1 and D2* 

**Figure 8.** The main kinetic parameters PF, RAE (antioxidant efficiency) and ID (inhibition degree) of lipid autoxidation in presence of different antioxidants (for abbreviation see corresponding tables)

**Figure 9.** Radical scavenging activity (%) of studied compounds at different [DPPH]/[AH] ratio

Nature-like neo- and xhanthene-lignans recently synthesized showed activity agains cardiovascular, inflammatory and cancer diseases. Together with their excellent capacity to scavenge free radicals and to inhibit lipid autoxidation these bio-antioxidants are of great importance for the practie, as individuals and in binary mixtures with TOH [39].

**Figure 10.** Structures of Xanthene (MF1, MF2) and neo-lignans (MF3, MF4)

The highest values of radical scavenging activity (%RSAmax) and largest rate constants for reaction with DPPH radical were obtained for xanthenes and neo-lignans (compounds 2 and 3, Fig. 7B). Comparison of %RSAmax with that of standard antioxidants DL-a-tocopherol (TOH), caffeic acid (CA) and butylated hydroxyl toluene (BHT) give the following new order of %RSA max: TOH(61.1%) > CA(58.6%) > 3(36.3%) > 2(28.1%) > 4(6.7%) > 1(3.6%) = BHT(3.6%). On the basis of a comparable kinetic analysis with standard antioxidants a new order of the antioxidant efficiency were obtained: **PF:** 2(7.2) TOH(7.0) > CA(6.7) > 1(3.1) > 3(2.2) > FA(1.5) > 4(0.6); and of the antioxidant reactivity: **ID:** 2(44.0) >> TOH(18.7) >> CA(9.3) >> 1(8.4) > 3(2.8) >FA(1.0) > 4(0.9) [36].


**Table 5.** Standard Antioxidants


Glu: D-glucoside; Rut: rutinoside; Glu-Com: p-coumaroyl-glucosides;

46 Lipid Peroxidation

Nature-like neo- and xhanthene-lignans recently synthesized showed activity agains cardiovascular, inflammatory and cancer diseases. Together with their excellent capacity to scavenge free radicals and to inhibit lipid autoxidation these bio-antioxidants are of great

The highest values of radical scavenging activity (%RSAmax) and largest rate constants for reaction with DPPH radical were obtained for xanthenes and neo-lignans (compounds 2 and 3, Fig. 7B). Comparison of %RSAmax with that of standard antioxidants DL-a-tocopherol (TOH), caffeic acid (CA) and butylated hydroxyl toluene (BHT) give the following new order of %RSA max: TOH(61.1%) > CA(58.6%) > 3(36.3%) > 2(28.1%) > 4(6.7%) > 1(3.6%) = BHT(3.6%). On the basis of a comparable kinetic analysis with standard antioxidants a new order of the antioxidant efficiency were obtained: **PF:** 2(7.2) TOH(7.0) > CA(6.7) > 1(3.1) > 3(2.2) > FA(1.5) > 4(0.6); and of the antioxidant reactivity: **ID:** 2(44.0) >> TOH(18.7) >>

(3)

O O

(2) OH

R

(1)

Ri OH

> OH C H3C CH3 CH3

R1

R2

*Hydroquinone (HQ)* 

*TBHQ, HQ -strong activity, BHT –weak/moderate activity; LAO, CL, DPPH,Theor* 

*R1=H; R2=OH –tert-butylated-hydroquinone (TBHQ) R1=t-But; R2=CH3 –tert-butyl-hydroxytoluene (BHT)* 

R R'

COOMe

(4) Me OME

OH

OH

importance for the practie, as individuals and in binary mixtures with TOH [39].

O

O O R

O R

**Figure 10.** Structures of Xanthene (MF1, MF2) and neo-lignans (MF3, MF4)

OMe O

OH OH

RO O

CA(9.3) >> 1(8.4) > 3(2.8) >FA(1.0) > 4(0.9) [36].

RO O

HO O

*R=CH3 2,2,5,7,8-pentamethyl-chroman-3-ol* 

*R=Phytyl; alpha-tocoperol (TOH)* 

*ChrC1 and TOH -Strong activity,* 

**Table 5.** Standard Antioxidants

O R CH3

*LAO, CL, DPPH,Theor* 

CH3

*(Chroman C1)* 

CH3

H3C

HO

%QSARtheor=3.954+75.950*.I*3',4'-di-OH or 3-OH + 8.499.*I*5-OH – by statistical analysis (QSAR) of Amic *et al* [43]  *(I=1 for 3',4'-di-OH and/or3-OH) and I=1 for 5-OH); %QSAR=3.95+8.5+75.95 (for Qu all derivatives, Kf, Isrh)* – 88.40; *%QSAR=3.95+8.5 (for Kf 3Oderivatives and Isrh 3Oderivatives*) – 12.75

**Table 6.** Substitution pattern of the series of flavonoids examined for their radical scavenging activity [41-43]

#### **4.3. Synergism, additivism and/or antagonism of binary mixtures of phenolic antioxidants [30,31,35-37,41-45]**

It is known that in the literature usually are published data about mixtures without or with synergism between the components. Separation of different effects of binary mixtures (synergism, additivism and/or antagonism) of different antioxidants was made for the first time by Denisov [32]. The latest gives possibility to make differences about different effects of binary mixtures, not only to be separated as mixtures without or with a synergism.

*Synergism –* is observed when the inhibiting effect of the binary mixtures (IP1+2) is higher than the sum of the induction periods of the individual phenolic antioxidants (IP1 + IP2) i.e. IP1+2 > IP1 + IP2. The percent of the synergism is presented by the following formulae *% Synergism = 100[IP1+2 – (IP1 + IP2)]/ (IP1 + IP2).* 

*Additivism -* is observed when the inhibiting effect of the binary mixtures (IP1+2) is equal to the sum of the induction periods of the phenolic antioxidants alone (IP1 + IP2) i.e. IP1+2 = IP1 + IP2.

*Antagonism -* is observed when the inhibiting effect of the binary mixtures (IP1+2) is lower than the sum of the induction periods of the individual phenolic antioxidants (IP1 + IP2) i.e. IP1+2 < IP1 + IP2.


**Table 7.** Effects of equimolar (1:1) binary mixtures of studied antioxidants without and with alphatocopherol (-TOH)

Syn*er*gism obtained for different binary mixtures are explained taking into account that the during the oxidation process the antioxidant molecules of both strong antioxidants may be regenerated, which leads to higher antioxidant efficiency of the mixture, than of the individual compounds. The regeneration of both antioxidant molecules of compounds with catecholic moiety, QH2 (4-hydroxy-bis-coumarin, caffeic acid (CA) and MF1-MF3) and of tocopherol (TOH) is possible as a result of the following possible reactions:


QH● + TOH → QH2 + TO● (regeneration of QH2 by H transfer)


TO● + QH2 → TOH + QH● (regeneration of TOH by H transfer)

b. homo-disproportionation reaction of two equal radicals:

```
2QH•  QH2 + Q regeneration of QH2 (Q is quinone)
```

```
2TO• TOH +T=O regeneration of TOH (T=O is tocopheryl quinone)
```

```
c. cross-disproportionation reaction of different radicals:
```

```
QH• + TO •  QH2 + T=O regeneration of QH2
```
TO• + QH• TOH + Q regeneration of TOH.

As a result the oxidation stability of lipid sample increases, because the both antioxidants with strong efficiency are regenerated during the oxidation process.

In case of binary mixture of TOH with monophenolic antioxidants (AH), predominantly TOH molecule will be regenerated during the following reactions:


```
TO• + AH → TOH + A•
```

```
```

```
2TO• TOH +T=O
```
48 Lipid Peroxidation

IP1+2 < IP1 + IP2.

**Binary mixtures (1:1)** 

Qu (1) + Lu(2) 0.1

Qu (1) + Ru(2) 0.1

Qu-7(1) + Lu-7(2) 0.5

#Myr (1) +-TOH(2)

BisCum1(1) +

BisCum3(1) +

tocopherol (-TOH)

*[AH] mM* 

0.5

0.5

1.0

0.1 0.3 0.6 *IP1+2 h* 

7.5±0.8 12.3±0.9

8.3±0.8 21.5±0.6

2.0±0.2 2.1±0.2

10.5±0.9 20.5±1.5 31.1±1.5

*Antagonism -* is observed when the inhibiting effect of the binary mixtures (IP1+2) is lower than the sum of the induction periods of the individual phenolic antioxidants (IP1 + IP2) i.e.

> *IP1 h*

9.9±0.9 24.5±0.6

9.9±0.9 24.5±0.6

1.5±0.2 1.0±0.2

4.7±0.3 8.9±0.9 16.3±0.9

TOH(2) 0.1 12.6±0.9 7.9±0.5 10.5±0.9 Antagonism 38

TOH(2) 0.1 6.1±0.5 2.2±0.2 10.5±0.9 Antagonism 38 MF1(1)+-TOH(2) 0.1 10.0±0.9 4.0±0.3 10.5±0.9 Antagonism 39 MF2(1)+-TOH(2) 0.1 15.0±0.9 9.2±0.9 10.5±0.9 Antagonism 39 MF3(1)+-TOH(2) 0.1 14.0±0.9 2.8±0.2 10.5±0.9 Synergism,5.3% 39 MF4(1)+-TOH(2) 0.1 13.8±0.9 0.75±0.05 10.5±0.9 Synergism,22% 39 #SA(1) + -TOH(2) 0.1 45.0±1.0 8.5±0.5 21.0±1.5 Synergism,52% 44 Lipid substrate oxidized TGSO, 80oC , only # TGL,100oC **Table 7.** Effects of equimolar (1:1) binary mixtures of studied antioxidants without and with alpha-

Syn*er*gism obtained for different binary mixtures are explained taking into account that the during the oxidation process the antioxidant molecules of both strong antioxidants may be regenerated, which leads to higher antioxidant efficiency of the mixture, than of the individual compounds. The regeneration of both antioxidant molecules of compounds with catecholic moiety, QH2 (4-hydroxy-bis-coumarin, caffeic acid (CA) and MF1-MF3) and of

tocopherol (TOH) is possible as a result of the following possible reactions:

Qu (1)+ -TOH(2) 0.1 29.7±1.5 9.9±0.9 10.5±0.9 Synergism,46% 41 Ru (1) + -TOH(2) 0.1 24.9±1.5 2.7±0.2 10.5±0.9 Synergism,87% 41

CA (1) + -TOH(2) 0.1 20.4±1.5 9.8±0.9 10.5±0.9 Additivism 3 SA(1) +- TOH(2) 0.1 16.1±0.9 5.3±0.5 10.5±0.9 Additivism 3 BHT(1) +- TOH(2) 0.1 21.5±1.5 7.5±0.5 10.5±0.9 Synergism,19% 3 TBHQ(1) + TOH(2) 0.1 26.1±1.5 7.9±0.5 10.5±0.9 Synergism,42% 3 Cum1(1) + TOH(2) 0.1 11.8±0.9 1.5±0.2 10.5±0.9 Additivism 40 Cum6 (1) + TOH(2) 0.1 14.2±0.9 2.0±0.2 10.5±0.9 Synergism,14% 40 Cum4 (1) + TOH(2) 0.1 12.7±0.9 7.1±0.5 10.5±0.9 Antagonism 40

*IP2 h* 

2.2±0.2 6.3±0.4

2.7±0.2 2.8±0.2

1.8±0.2 1.4±0.2

3.2±0.2 5.5±0.5 7.4±0.5 *Effects,* 

Antagonism Antagonism,

Antagonism Antagonism

Antagonism Additivism

Synergism,33% Synergism,42% Synergism,14%

*% Ref* 

41 41

41 41

41 41

> 3 3 3

```
```
A• + TO• → TOH+ A-H or A• + TO• → T=O+ AH (depending on AH structure)

These reactions demonstrate that during oxidation process initial molecules of individual antioxidants are regenerated by different mechanisms in the binary mixtures. Nevertheless both binary mixtures may be used as effective antioxidant compositions. It is proven that the positions of phenolic hydroxyl groups in 4-hydroxy-bis-coumarins are of significance for their antioxidant activity and mechanism of action. Comparable kinetic analysis showed that the antioxidant efficiency (PF) and reactivity (ID) depend significantly from the substitution of the phenolic ring.

#### **5. Surface-active compounds – surfactants (S)**

Surfactants (S) are amphiphilic substances which adsorb at interface and decrease an excess of free energy (surface tension, ) of interface. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents, and dispersants. A surfactant molecule contains both a water insoluble hydrophobic component and a water soluble hydrophilic component (polar head).

Surfactant solutions are one of the simplest examples of self-assembling soft nano-systems, whose micro-aggregates (micelles) are of 1–500 nm in size [10,11]. Micelles are prevalent in naturally occurring and biological catalytic reactions Micelles are formed by those surfactants which possess rather long bulky hydrophobic part along with strong hydrophilic head. Such surfactants form direct micelles in water and other polar solvent, and reverse micelles in organic solution.

**Figure 11.** Micelles formed by surfactants in polar and nonpolar media

The phenomenon of micellar catalysis has been known for a rather long time and applied in many processes, but the significant influence of surfactants on the lipid and hydrocarbon oxidation has been found and studied only in recent decades [6-8, 46-48]. Specific features of micellar catalysis for oxidation processes have two causes: (1) Hydroperoxides (LOOH), which are formed as the primary oxidation products, are amphiphilic and surface active, in contrast to initial oils; (2) There is spontaneous allocation of amphiphilic compounds in every heterogeneous and colloid system resulting in the reduction in the total free energy of a system, including the interface boundaries (the rule of polarity equalization). In the presence of surfactants in oxidized oil hydroperoxides and surfactants form mixed micelles {nLOOH...mS}(Fig. 11c). Using the measurement of the interphase tension [49], nuclear magnetic resonance (NMR), and dynamic light scattering [50], it was shown the association of LOOH and a surfactant in combined micelles in which LOOH plays the role of a cosurfactant. The average self-diffusion coefficient for hydroperoxide decreases with growth of the surfactant (CTAC) concentration up to the equalization with the surfactant diffusion coefficient, when all LOOH is bound in mixed micelles {nLOOH···mS}. The mixed micelle effective size calculated by the Stocks–Einstein equation was ~2 nm [50]. The size determined by DLS for mixture cumene hydroperoxide and CTAB are about 20 nm. Hydroperoxide facilitates the colloid dilution of CTAB in organic medium.

#### **5.1. Effect of surfactants on lipid oxidation.**

The comparison of the effects of different surfactants on lipid and hydrocarbon oxidation reveals that cationic surfactants (CS) promote hydroperoxide destruction resulting in the formation of free radicals [46,47] and the oxidation as a whole (Fig.12a,b).

By means of NMR and GC–MS, it is shown that in the presence of CS (CTAC and CTAB)) cumene hydroperoxide decomposes into dimethyl phenylcarbinol, acetophenon, and dicumylperoxide which are known as resulting from the radical decomposition of hydroperoxide. In the presence of anionic SDS, cumene hydroperoxide decomposes without radical formation into phenol and acetone [48].

The kinetics of oxidation of sunflower (TGSO) and olive (TGOO) oil triacylglycerols (Fig.12a) and natural olefin (limonene) (Fig.12b) in the presence of surfactants show that cationic surfactants (CS) promote the oxidation, whereas the anionic sodium dodecylsulfate (SDS) has no influence in the case

of limonene and SDS demonstrates a weak retardation in TGOO oxidation (Fig.12a.) The chain breaking inhibitor -tocopherol completely suppress the limonene oxidation accelerated by CTAC and CTAB (Fig.12b). Before and after the induction periods, the oxidation rate is described by the well known equation for the liquid-phase chain oxidation of hydrocarbons and lipids (see above):

$$\mathbf{R}\_{02} = a \mathbf{\bar{l}} \,\mathbf{L}\mathbf{H} \,\mathbf{\bar{j}} \,\mathbf{R}\_{\text{IN}} \,\text{\,^{0.5}\,\text{\,}}\tag{1}$$

where RIN is the radical initiation rate. Ri can be calculated from the duration of the induction period (), caused with -tocopherol (Fig.12) as follows: RIN = 2[InH]/ .

**Figure 12.** (a). Effect of surfactant on the LOOH formation during autoxidation of TGOO at 100oC and TGSO at 80oC: 1, 4- without additives; 3, 5 - 0.1M CTAB; 2,4- 0.1 M SDS; 6 - 0.04 M 1-OD [48]; (b) Effect of surfactant on the oxygen absorption during 1M limonene oxidation at 60oC; 1 – 1mM CTAC; 2 – 0,8 mM CTAB; 3 – 1mM SDS; 3- without additives; [LOOH]=22mM; Arrows show the moments of introducing -tocopherol: 1-0,8 mM; 2 – 0,2 mM [51].

The rate constants for the propagation (kp) and termination (kt ) of the oxidation chain for limonene and its oxidizability parameter *a* = kp/(2kt)0.5 are known at the temperatures 30- 80oC [52]. So, the radical initiation rate can be calculated on the base of the measured value of oxygen uptake rate: Ri = (R02/*a*[LH])2. In the case of limonene both formulas give very close Ri values. These data show that the mechanism of the catalytic action of CS on the oxidation processes consists in the increase of the chain initiation rate, caused by acceleration of hydroperoxides decomposition into free radicals.

To estimate the mixed micelles {nLOOH… mS} as a free radical initiator quantitatively, cumene hydroperoxide and hydrogen peroxide, which are produced in industrial scale, and limonene hydroperoxide were taken, and natural polyphenol quercetin was used as a radical acceptor. The interaction of LOOH with quercetin (Qu) in the presence of surfactants (S) is described by reactions:

nLOOH + mS � {nLOOH … mS} LO2· Qu + LO2· products,

50 Lipid Peroxidation

(a) Direct micelle in water

**Figure 11.** Micelles formed by surfactants in polar and nonpolar media

Hydroperoxide facilitates the colloid dilution of CTAB in organic medium.

formation of free radicals [46,47] and the oxidation as a whole (Fig.12a,b).

The comparison of the effects of different surfactants on lipid and hydrocarbon oxidation reveals that cationic surfactants (CS) promote hydroperoxide destruction resulting in the

By means of NMR and GC–MS, it is shown that in the presence of CS (CTAC and CTAB)) cumene hydroperoxide decomposes into dimethyl phenylcarbinol, acetophenon, and dicumylperoxide which are known as resulting from the radical decomposition of hydroperoxide. In the presence of anionic SDS, cumene hydroperoxide decomposes without

The kinetics of oxidation of sunflower (TGSO) and olive (TGOO) oil triacylglycerols (Fig.12a) and natural olefin (limonene) (Fig.12b) in the presence of surfactants show that cationic surfactants (CS) promote the oxidation, whereas the anionic sodium dodecylsulfate

**5.1. Effect of surfactants on lipid oxidation.** 

radical formation into phenol and acetone [48].

(SDS) has no influence in the case

The phenomenon of micellar catalysis has been known for a rather long time and applied in many processes, but the significant influence of surfactants on the lipid and hydrocarbon oxidation has been found and studied only in recent decades [6-8, 46-48]. Specific features of micellar catalysis for oxidation processes have two causes: (1) Hydroperoxides (LOOH), which are formed as the primary oxidation products, are amphiphilic and surface active, in contrast to initial oils; (2) There is spontaneous allocation of amphiphilic compounds in every heterogeneous and colloid system resulting in the reduction in the total free energy of a system, including the interface boundaries (the rule of polarity equalization). In the presence of surfactants in oxidized oil hydroperoxides and surfactants form mixed micelles {nLOOH...mS}(Fig. 11c). Using the measurement of the interphase tension [49], nuclear magnetic resonance (NMR), and dynamic light scattering [50], it was shown the association of LOOH and a surfactant in combined micelles in which LOOH plays the role of a cosurfactant. The average self-diffusion coefficient for hydroperoxide decreases with growth of the surfactant (CTAC) concentration up to the equalization with the surfactant diffusion coefficient, when all LOOH is bound in mixed micelles {nLOOH···mS}. The mixed micelle effective size calculated by the Stocks–Einstein equation was ~2 nm [50]. The size determined by DLS for mixture cumene hydroperoxide and CTAB are about 20 nm.

(b) Reverse micelle in oil

(c) Mixed micelle {nROOH…mS} The initiation rate is equal to: Ri = -2d[Qu]/dt. Quercetin is characterized by an intense absorption band in a visible region of the electronic spectrum, it is soluble in water and organic solvents and readily reacts with free radicals; i.e. it is a convenient kinetic probe to study the processes of radical generation in various media. The generated peroxyl radicals come into the volume and can initiate chain oxidation, polymerization, or other radical processes. In the presence of oxygen the concentrations of hydroperoxide and other polar products increase during the accelerated oxidation, and this, in its turn, influences the structure and properties of micelles. The instability of micelles and also the varied composition of their polar cores do not allow applying the known and frequently used pseudophase approach to the analysis of the lipid oxidation kinetics in the presence of surfactants. The comparison of the activities of LOOH and cationic surfactants in the generation of radicals can be conducted on the basis of the specific rates of radical initiation i = Ri / ([LOOH]·[S]). Data in the Table 6 show that cationic surfactants catalyze free radical initiation from hydroperoxide both in water and in organic solutions that is both in direct and reverse mixed micelles.

It means that the CS–hydroperoxide system can be used both as a lipophilic and a hydrophilic initiator. Small amounts of LOOH and CS provide significant radical generation rates (10–8–10–7 Ms–1), which are inaccessible at low temperatures for the known azoinitiators. By their activity in the generation of radicals in organic media the surfactants can be arranged in the following order, which indicates the essential role of counter ions in the catalytic action of CS:


#### CTAC ≈ TDTAC > CTAB ≈ CPB > DCDMAB > CTAHS

**Table 8.** Specific rates of radical initiation in the system: cationic surfactant + hydroperoxide in chlorbenzene and in water solution [47]

Along with hydroperoxide, water, and other polar oxidation products, catalytic and inhibiting components can be concentrated in mixed micelles {nLOOH·mS}. The combination of cationic surfactants with transition metal compounds known as homogeneous catalysts of the hydrocarbon oxidation was found to demonstrate synergism, i.e., for the mixture of components the oxidation rate (RΣ) exceeds the sum of the rates in the experiments with separately used components (RMe and RS): β = RΣ/(RMe + RS) > 1. The ethylbenzene is oxidized selectively into acetophenon and water catalyzed with the combination of CTAB and cobalt acetylacetonate [53]. Under similar conditions limonene is oxidized with the primary formation of a carbonyl compound (carvon) [51].

Let us look on the mixed micelles of hydroperoxide and cationic surfactant once more. In mixed micelle, peroxide bond is localized in the interphase which has very strong intensity of electric field, about 5·105V/m. It affects peroxide bond, weakens it and facilitates decomposition into free radicals. Apparent activation energies of hydroperoxide decay decrease to 50-60 kJ/mol in mixed micelles from ~ 100 kJ/mol for thermal decay [51].

In the case of anionic surfactant the direction of electric field is different and decomposition into radicals is not facilitated. On the contrary, alkali metal alkyl sulfates [47,48] and alkyl phosphates [54,55] act as antioxidants to retard or completely suppress the oxidation process.

Nonionic surfactants form neutral micelles which have no electric field. May be, by that reason nonionic surfactants do not affect free radical formation in hydroperoxide decay, although they form mixed micelles {LOOH…S} with nonionic surfactant as well.

#### **5.2. Phospholipid oxidation.**

52 Lipid Peroxidation

and reverse mixed micelles.

the catalytic action of CS:

Surfactant

CTAC ≈ TDTAC > CTAB ≈ CPB > DCDMAB > CTAHS

Cumene hydroperoxide, 37оC

> i, (M·s)-1 Water

i, (M·s)-1 Organics

chlorbenzene and in water solution [47]

The initiation rate is equal to: Ri = -2d[Qu]/dt. Quercetin is characterized by an intense absorption band in a visible region of the electronic spectrum, it is soluble in water and organic solvents and readily reacts with free radicals; i.e. it is a convenient kinetic probe to study the processes of radical generation in various media. The generated peroxyl radicals come into the volume and can initiate chain oxidation, polymerization, or other radical processes. In the presence of oxygen the concentrations of hydroperoxide and other polar products increase during the accelerated oxidation, and this, in its turn, influences the structure and properties of micelles. The instability of micelles and also the varied composition of their polar cores do not allow applying the known and frequently used pseudophase approach to the analysis of the lipid oxidation kinetics in the presence of surfactants. The comparison of the activities of LOOH and cationic surfactants in the generation of radicals can be conducted on the basis of the specific rates of radical initiation i = Ri / ([LOOH]·[S]). Data in the Table 6 show that cationic surfactants catalyze free radical initiation from hydroperoxide both in water and in organic solutions that is both in direct

It means that the CS–hydroperoxide system can be used both as a lipophilic and a hydrophilic initiator. Small amounts of LOOH and CS provide significant radical generation rates (10–8–10–7 Ms–1), which are inaccessible at low temperatures for the known azoinitiators. By their activity in the generation of radicals in organic media the surfactants can be arranged in the following order, which indicates the essential role of counter ions in

> Hydrogen peroxide, 37oC

> > i, (M·s)-1 Water

i, (M·s)-1 Organics

CTAC 2,1·10-3 3,7·10-4 0,67·10-3 0,14·10-3 27·10-3 CTAB 1,9·10-3 1,9·10-4 0,2·10-3 0,14·10-3 3,6·10-3 CTAHS 0,17·10-3 1,1·10-4 0 0 2,5·10-3 DCDMAB 1,5·10-3 2,8·10-4 2,1·10-3 0,37·10-3 3,6·10-3 CPB 1,9·10-3 3,3·10-4 0,2·10-3 0,14·10-3 3,6·10-3 TDTAC 2,1·10-3 3,7·10-4 0,47·10-3 0,13·10-3 - SDS 0 0 0 0 0 Lecithin 0 0 0 0 0 **Table 8.** Specific rates of radical initiation in the system: cationic surfactant + hydroperoxide in

Along with hydroperoxide, water, and other polar oxidation products, catalytic and inhibiting components can be concentrated in mixed micelles {nLOOH·mS}. The combination of cationic surfactants with transition metal compounds known

Limonene hydroperoxide, 60 oC

> i, (M·s)-1 Organics

Phospholipids (PL) are natural surfactants, which are widely used in the production of food, drug, and cosmetics. PL are the basic lipid components of plasmatic cell membranes and membranes of subcellular organelles of animals, plants, and microorganisms. Phosphatidylcholines (1,2-diacyl-sn-glycero-3-phosphocholines, lecithins) are the most widely used; they are present in large amounts in myocardium, liver, kidneys, and egg yolk [56]. In lecithin molecules, anionic phosphate and cationic choline (tetraalkylammonium) groups are connected via a zwitterionic bond to form a neutral polar head. Hydrocarbon moiety represents residues of fatty acids, whose composition depends on the type of PLs (egg, soybean, fish, etc.). Lecithins have a zwitterionic structure in a wide pH range.

Unsaturated fatty acid residues of PL are readily oxidized with atmospheric oxygen as well as nonpolar unsaturated lipids. The primary products of PL oxidation are mainly isomeric hydroperoxides [9,12,57-59]. Lecithins are easily dissolved in organic solvents to yield compact reverse micelles [60,61]. In aqueous solutions, lecithin forms multilamellar liposomes or vesicles under the action of ultrasound dispersion [12,56,62]. Using the DLS method, it was found that, at egg lecithin concentrations 10–90 mg/mL, the size of microaggregates observed are equal to 5-6 nm in organic solvents and in water, liposomes are formed with a wide size distribution of 60–1000 nm.

PC oxidation in the presence of azoinitiators or transition metals occurs via free radical chain mechanism. The formation of micro-aggregates both in organic and water media results in a nonlinear dependence of the rate of oxygen absorption on substrate concentration (at constant initiation rate). The deviations from the linearity were observed at concentrations of egg lecithin above 5 mg/mL, corresponding to the formation of microaggregates. The rate increment caused by a further increase in the concentration markedly decreases [60,61]. It is possible that a partial shielding of active C–H bonds, which interact with peroxyl radicals from an initiator in solvent bulk results in a relative decrease in the oxidation rate.

**Figure 13.** a) Dependences of lecithin (45mg/mL) oxidation rates on the initiator concentration in logarithmic coordinates: 1 – in n-decane solution, 60oC, I – azobisisobutyronitrile, AIBN; 2 – in water, 37oC, I – azodiiso-butyramidine-dihydrochloride (AAPH); b) Temperature dependence of the rate of lecithin (45mg/mL) oxidation plotted in Arrhenius coordinates: 1 – in chlorbenzene, [AIBN]= 5mM; 2 – in water, [AAPH]=55mM.

It was shown in [60,61] that the dependences of PC oxidation rates on initiation rates differ in organic and water solutions. In organic solvents, RO2 is proportional square root of Ri, whereas in water, RO2 ~ Ri. It can be seen from Fig.13a, where the dependences of egg lecithin oxidation rates on the corresponding initiator concentration (it means RO2 – RINn, because RIN = ki[initiator]) are presented: in organics (chlorobenzene) n = 0,5 and n ≈ 1 in water media.

So, the rate of PL oxidation in an aqueous medium cannot be described by Eq. (1), common for lipid and hydrocarbon oxidation. Therefore, even in a narrow concentration range, it is unreasonable to compare the oxidizability of PL in water and an organic medium using parameter *a* = kp/(2kt)0.5. Nevertheless, in many studies devoted to the oxidation of phospholipids in various media, Eq. (1) was applied to describe the rate of oxidation (absorption of oxygen [63-66] or accumulation of hydroperoxides [9,58,67]) and to determine the oxidizability of PLs or individual phosphatidylcholines [68]. The majority of these works was carried out at the physiological temperature (37°С). The measurements were performed in different ranges of the overall concentrations of PL and with different initiators and inhibitors used to determine the initiation rates; therefore, the conclusions were very different right up to the opposite ones.

According to [58,68] the oxidizability of PLs in aqueous dispersions is lower than that in organic solvents by an order of magnitude; it is higher in reverse micelles than in molecular alcohol solutions [58]. In [65,68], it was assumed that the micro-heterogeneity of PL solutions and the dispersity of colloidal solutions do not influence the oxidizability of unsaturated lipids in both aqueous and organic media.

54 Lipid Peroxidation

oxidation rate.

3,4 lnRO2 - ln10<sup>7</sup>

2,2 2,4 2,6 2,8 3,0 3,2

in water, [AAPH]=55mM.

1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0

a

1

different right up to the opposite ones.

water media.

PC oxidation in the presence of azoinitiators or transition metals occurs via free radical chain mechanism. The formation of micro-aggregates both in organic and water media results in a nonlinear dependence of the rate of oxygen absorption on substrate concentration (at constant initiation rate). The deviations from the linearity were observed at concentrations of egg lecithin above 5 mg/mL, corresponding to the formation of microaggregates. The rate increment caused by a further increase in the concentration markedly decreases [60,61]. It is possible that a partial shielding of active C–H bonds, which interact with peroxyl radicals from an initiator in solvent bulk results in a relative decrease in the

**Figure 13.** a) Dependences of lecithin (45mg/mL) oxidation rates on the initiator concentration in logarithmic coordinates: 1 – in n-decane solution, 60oC, I – azobisisobutyronitrile, AIBN; 2 – in water, 37oC, I – azodiiso-butyramidine-dihydrochloride (AAPH); b) Temperature dependence of the rate of lecithin (45mg/mL) oxidation plotted in Arrhenius coordinates: 1 – in chlorbenzene, [AIBN]= 5mM; 2 –

1,5 2,0 2,5 3,0 3,5

4,0 lnRO2 - ln107

1

b

2,8 2,9 3,0 3,1 3,2 3,3 3,4

2

1000/T, K

ln [I]+ln 10-3

2

It was shown in [60,61] that the dependences of PC oxidation rates on initiation rates differ in organic and water solutions. In organic solvents, RO2 is proportional square root of Ri, whereas in water, RO2 ~ Ri. It can be seen from Fig.13a, where the dependences of egg lecithin oxidation rates on the corresponding initiator concentration (it means RO2 – RINn, because RIN = ki[initiator]) are presented: in organics (chlorobenzene) n = 0,5 and n ≈ 1 in

So, the rate of PL oxidation in an aqueous medium cannot be described by Eq. (1), common for lipid and hydrocarbon oxidation. Therefore, even in a narrow concentration range, it is unreasonable to compare the oxidizability of PL in water and an organic medium using parameter *a* = kp/(2kt)0.5. Nevertheless, in many studies devoted to the oxidation of phospholipids in various media, Eq. (1) was applied to describe the rate of oxidation (absorption of oxygen [63-66] or accumulation of hydroperoxides [9,58,67]) and to determine the oxidizability of PLs or individual phosphatidylcholines [68]. The majority of these works was carried out at the physiological temperature (37°С). The measurements were performed in different ranges of the overall concentrations of PL and with different initiators and inhibitors used to determine the initiation rates; therefore, the conclusions were very A comparison of the experimentally measured rate of O2 absorption during PL oxidation in aqueous solutions with the corresponding values obtained in an organic solvent at the same temperature, mass concentration of PL, and the initiation rate, which is governed by the contents of water- (AAPH) and oi-soluble (AIBN) azo-initiators, respectively, in the volumes of the solvents demonstrates the following [61]. At a temperature of 45°С, radical initiation rate of 22.5·10–8 M/s, and PL concentration of 45 mg/mL, the rates of oxygen absorption in water and chlorobenzene are 3.5·10–6 and 2.1·10–6 M/s, respectively. A comparison suggests that, in the presence of a source of radicals, PL organized into multilamellar liposomes is ~1.5\_fold faster oxidized in water than in the organic solution of reverse micelles. In order to explain this result, for micro-heterogeneous systems, one must introduce the concept of the effective (apparent) concentration of an oxidized substrate. In a system of multilamellar liposomes, the effective concentration of the oxidized substrate is higher than that in a system of reversed micelles occurring in an organic medium; therefore, a higher oxidation rate is observed at the same temperature and the rate of radical initiation. The rates of PL oxidation both in organics and in water, initiated by corresponding initiator, increase with temperature according to the Arrhenius equation (Fig.10b). The effective activation energy of AAPH-initiated PL oxidation in an aqueous solution (74 kJ/mol) is lower than the activation energy of AAPH decomposition (112 kJ/mol). Hence, a radical chain mechanism of PL oxidation in water is more complex than described above mechanism of model oil oxidation (Table 1). In a micro-heterogeneous medium, in addition to individual radicals and molecules, reagents that are included into microaggregates (liposomes) and characterized by reactivity different from that of molecular\_dispersed particles in corresponding reactions are involved in the stages of chain initiation, propagation, and termination. Crossdisproportionation reactions of different radicals occur to result in the imitation of a linear chain termination.


It turns out that catecholamines dopamine, adrenaline and noradrenaline are much more strong and effective inhibitors for PC oxidation than -tocopherol (Compare Fig.14 and Fig.15). Evidently, the positive charge of catecholamines at neutral pH facilitates their adsorption and protective action on the surface of negative charged liposomes [61].

**Figure 14.** Kinetic curves for O2 absorption during AAPH- initiated (55 mM) oxidation of PL (45 mg/ml) in water at 37°C in the presence of (1) 0.33 and (2) 0.05 mM α-tocopherol incorporated upon preparation of liposomes and (3)0.05 mM α-tocopherol introduced directly into solution

**Figure 15.** Effect of 0.1 mM (1) adrenalin, (2) dopamine, (3) noradrenalin on AAPH-initiated (55 mM) oxidation of PL (45 mg/ml) at 37°C in (a) water and (b) in phosphate buffer with pH 7.4; (4) no additives.

It must be noted that in the phosphate buffer, induction periods τ are nearly equal for all of the catecholamines (Fig.15b), while, in an aqueous solution, adrenalin provides a longer inhibition of the oxidation than dopamine and noradrenalin do (Fig.15a). The analysis of the ratios between the rates of radical initiation and the durations of the induction periods testified that, for all catecholamines in the buffer solution, the stoichiometry of inhibition, which is numerically equal to the number of radicals corresponding to one acceptor molecule, is n =(RIN·τ)/[CA]0 = 2, which is characteristic of catechols. In an aqueous and a physiological solution (0.9% NaCl), dopamine and noradrenalin exhibit n= 2, while for adrenaline, n= 4. Moreover, the adrenalin-containing mixture acquires a pink color in water.

Figure 16a illustrates variations in the optical absorption spectra of adrenalin solutions in water and a physiological solution (0.9% NaCl) during its free-radical oxidation initiated by AAPH. It can be seen that the oxidation results in the formation of a colored product with an absorption maximum at 480 nm. In the phosphate buffer of pH 7.4, adrenalin also undergoes transformations (Fig. 16b); however, they yield no colored product. The spectral characteristics of the pink product (ε = 4.02 × 103 М–1 cm–1 at 480 nm) correspond to adrenochrome (3-hydroxy-1-methyl-2,3-dihydro-1H-indole-5,6-dion), which is formed via the abstraction of four hydrogen atoms from adrenaline [68].

**Figure 16.** Variations in UV spectrum of 0.1 mM adrenalin solution in the process of its oxidation at 37°C (a) in water and 0.9% NaCl solution and (b) in phosphate buffer with pH 7.2.

**Figure 17.** Structures of adrenalin and adrenochrome

It is interesting that, in an aqueous solution, under the conditions of AAPH-initiated freeradical oxidation, adrenalin is quantitatively transformed into adrenochrome. However, in the phosphate buffer solution adrenochrome is not formed.

#### **6. Concluding remarks**

56 Lipid Peroxidation

additives.

[O2 ]\*10<sup>3</sup> , M 0 20 40 60 80 100 120 140

**Figure 14.** Kinetic curves for O2 absorption during AAPH- initiated (55 mM) oxidation of PL (45 mg/ml) in water at 37°C in the presence of (1) 0.33 and (2) 0.05 mM α-tocopherol incorporated upon

**Figure 15.** Effect of 0.1 mM (1) adrenalin, (2) dopamine, (3) noradrenalin on AAPH-initiated (55 mM) oxidation of PL (45 mg/ml) at 37°C in (a) water and (b) in phosphate buffer with pH 7.4; (4) no

It must be noted that in the phosphate buffer, induction periods τ are nearly equal for all of the catecholamines (Fig.15b), while, in an aqueous solution, adrenalin provides a longer inhibition of the oxidation than dopamine and noradrenalin do (Fig.15a). The analysis of the ratios between the rates of radical initiation and the durations of the induction periods testified that, for all catecholamines in the buffer solution, the stoichiometry of inhibition, which is numerically equal to the number of radicals corresponding to one acceptor molecule, is n =(RIN·τ)/[CA]0 = 2, which is characteristic of catechols. In an aqueous and a physiological solution (0.9% NaCl), dopamine and noradrenalin exhibit n= 2, while for adrenaline, n= 4. Moreover, the adrenalin-containing mixture acquires a pink color in water. Figure 16a illustrates variations in the optical absorption spectra of adrenalin solutions in water and a physiological solution (0.9% NaCl) during its free-radical oxidation initiated by AAPH. It can be seen that the oxidation results in the formation of a colored product with an

preparation of liposomes and (3)0.05 mM α-tocopherol introduced directly into solution

t, min

[O2 ]\*103 , M

0 10 20 30 40 50 60 70 80 90 100

b

4

t, min

3 2 1

1

3

2

4

a

t, min

3 2 1

0 20 40 60 80 100

[O2 ]\*103 , M

> Radical scavenging activity towards DPPH radical gives information only about the Hdonating capacity of the studied compounds and some preliminary information for their possibility to be used as antioxidants. Antioxidant activity is capacity of the compound to shorten the oxidation chain length as a result of its reaction with peroxyl radicals. For that reason we mean as antioxidant activity the chain-breaking activity of the compounds. This comparable study showed a good correlation between experimental antioxidant activity of compounds under study and their predictable activity by using TLC DPPH radical test.

> It has been demonstrated that phenolic compounds with catecholic moiety are the most powerful scavengers of free radicals and they may be used as effective chain-breaking antioxidants. The highest antiradical and antioxidant activity of phenolic antioxidants with

catecholic moiety is explained by possible mechanism of homo-disproportionation of their semiquinone radicals formed.

Thus regeneration of the antioxidant molecule during the oxidation process is possible. It has been found for the first time that only substitution in the aromatic nucleus of the studied bis-coumarins and xanthenes-lignans is responsible for their antioxidant activity.

It must be noted that antioxidants' activity depends significantly not only on their structural characteristics, but also on the properties of the substrate being oxidized and the experimental conditions applied. Structural characteristics of the complex system: oxidizing substrate - antioxidant must be considered. On the basis of this comparable analysis, the most effective individual antioxidants and binary mixtures were proposed for highest and optimal lipid oxidation stability.

## **Author details**

Vessela D. Kancheva *Lipid Chemistry Department, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria* 

Olga T. Kasaikina *N.N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow, Russia* 

### **Acknowledgement**

The financial support of Project No BG051PO001/3.3-05-0001 "Science and Business" - Ministry of Education and Sciences in Bulgaria for publishing this chapter in the Lipid Peroxidation monograph open access is gratefully acknowledged.

#### **7. References**


[5] Huang S W and Frankel EN (1997) Antioxidant activity of tea catechins in different lipid systems, *J. Agric. Food Chem*. 45:3033–30383.

58 Lipid Peroxidation

semiquinone radicals formed.

optimal lipid oxidation stability.

**Author details** 

Olga T. Kasaikina

**7. References** 

pp 233-238.

**Acknowledgement** 

Bulgaria, Chapter 1, pp 56-72.

USA, Ed. A.A.Farooqui, Chapter I, pp 1-45.

water emulsions, *J.Agric. Food Chem*. 44: 444–452.

Vessela D. Kancheva

catecholic moiety is explained by possible mechanism of homo-disproportionation of their

Thus regeneration of the antioxidant molecule during the oxidation process is possible. It has been found for the first time that only substitution in the aromatic nucleus of the studied

It must be noted that antioxidants' activity depends significantly not only on their structural characteristics, but also on the properties of the substrate being oxidized and the experimental conditions applied. Structural characteristics of the complex system: oxidizing substrate - antioxidant must be considered. On the basis of this comparable analysis, the most effective individual antioxidants and binary mixtures were proposed for highest and

bis-coumarins and xanthenes-lignans is responsible for their antioxidant activity.

*N.N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow, Russia* 

The financial support of Project No BG051PO001/3.3-05-0001 "Science and Business" - Ministry of Education and Sciences in Bulgaria for publishing this chapter in the Lipid

[1] Kancheva V (2010a) Antioxidants. Structure - activity relationship*.* In: "Antioxidants - Prevention and Healthy Aging", Ed. by F. Ribarova, SIMELPRESS Publ., Sofia,

[2] Kancheva V (2010b) Oxidative stress and lipid oxidation. In: "Antioxidants - Prevention and Healthy Aging", Ed. by F.Ribarova, SIMELPRESS Publ., Sofia, Bulgaria, Chapter 3

[3] Kancheva VD (2012) Phenolic antioxidants of natural origin – structure activity relationship and their beneficial effect on human health. In: "Phytochemicals and Human Health: Pharmacological and Molecular Aspects"*,* Nova Science Publishers Inc.,

[4] Huang SW, Frankel EN, Schwarz K, Aeschbach R, German JB (1996) Antioxidant activity of alpha-tocopherol and Trolox in different lipid substrates: Bulk oils and oil-in-

*Lipid Chemistry Department, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria* 

Peroxidation monograph open access is gratefully acknowledged.



[39] Kancheva VD, Saso L, Angelova SA, Foti MC, Slavova-Kazakova A, Draquino C, Enchev E, Firuzi O, Nechev J (2012) Antiradical and antioxidant activities of new bioantioxidants. Biochemie 94: 403-415.

60 Lipid Peroxidation

Catalysis, 33: 617-622;

*Sci. Food Agric.* 68:117-126.

33: 756-771.

888-890.

(in Russian).

of AbstractsIL-50.

Nederlands, pp 267.

1-11.

York, Washington, D.C. 2001.

[23] Belyakov VA, Kortenska VD, Rafikova VS, Yanishlieva NV (1992a) Kinetics of the inhibited oxidation of model lipid systems. Role of fatty alcohols. Kinetics and

[24] Belyakov VA, Kortenska VD, Rafikova VS, Yanishlieva NV (1992b) Mechanism of lipid autoxidation in presence of fatty alcohols and partial glycerols*.* Kinetics and Catalysis

[25] Kortenska VD, Yanishlieva NV, Roginskii VA (1991) Kinetics of inhibited oxidation of lipids in the presence of 1-Octadecanol and 1-Palmitoylglycerol, *J. Am. Oil Chem. Soc.* 68:

[26] Kortenska VD, Yanishlieva NV (1995) Effect of the phenol antioxidant type on the kinetics and mechanism of inhibited lipid oxidation in the presence of fatty alcohols, *J.* 

[27] Kortenska VD, Yanishlieva NV, Kasaikina OT, Totzeva IR, Boneva MI, Rusina IF (2002) Phenol antioxidant efficiency in presence of lipid hydroxy compounds in various lipid

[28] Burlakova E B (2007) Bioantioxidants. Molecular Cell Biophysics. Russ.Chem. J. 51:3-12

[29] Pobedimskiy DG, Burlakova EB (1993) Mechanism of Antioxidant Action in Living Organisms in Atmospheric Oxidation and Antioxidants. Ed. J. Scott. 2: 223-235. [30] Kancheva VD (2009) Phenolic antioxidants – radical scavenging and chain breaking

[31] Kancheva VD (2010) New bioantioxidants strategy. T3D-2010 International Symposium on Trends in Drug DSiscivery and Development 5th-8th January 2010 Delhi (India), Book

[32] Denisov ET, Denisova TG: Handbook of antioxidants. Bond dissociation energies, rate constasnts, activation energies and enthalpies of reactions. Second Ed. CRS Press, New

[33] Shahidi F: Natural antioxidants, a review in: Natural antioxidants. Chemistry, Health effects and applications, Shahidi, F. (ed) AOCS, Press, Champaign, Illinois, USA, 1999,

[34] Vermeris W, Nikolson R. Phenolic compound biochemistry. Springer 2006, The

[37] Vasilev RF, Kancheva VD, Fedorova GF, Batovska DI, Trofimov AV (2010) Antioxidant activity of chalcones. The chemiluminescence determination of the reactivity and quantum – chemical calculation of the energies and structures of rеagents and

[38] Kancheva VD, Boranova PV, Nechev JT, Manolov II (2010) Structure-activity relationship of new 4-hydroxy-bis-coumarins as radical scavengers and chain-breaking

[35] Frankel E N (1998) Lipid oxidation, The Oily Press, Dundee (Scotland) 1998, pp. 303. [36] Kamal-Eldin A, Makinen M, Lampi A-M (2003) The Challenging Contribution of Hydroperoxides to the Lipid Oxidation Mechanism, in Lipid Oxidation Pathways

(Kamal-Eldin, A., ed.), Chapter 1, AOCS Press, Champaign, II pp 1-36.

intermediates*.* Kinetics and Catalysis 51: 507-515.

antioxidants. Biochimie 92:1138-1146.

activities. Comparative study. Eur. J. Lipid Sci. Technol. 111: 1072-1089.

systems, *European J. Lipid Science and Technology* 104: 513-519.



## **Lipid Peroxidation End-Products as a Key of Oxidative Stress: Effect of Antioxidant on Their Production and Transfer of Free Radicals**

Hanaa Ali Hassan Mostafa Abd El-Aal

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/45944

## **1. Introduction**

62 Lipid Peroxidation

41-46.

13044.

765-768.

*Chem.* 220: 237—255.

23: 9416-9422.

[53] Bakunin VN, Popova ZV, Oganesova EYu , Parenago (2001) The change of hydrocarbon medium structure in the course of liquid phase oxidation, *Petr. Chem. (Engl. Transl.)* 41:

[54] Yoshimoto M, Miyazaki Y, Umemoto A, Walde P, Kuboi R, Nakao K (2007) Phosphatidylcholine vesicle-mediated decomposition of hydrogen peroxide, *Langmuir* 

[56] Xu L, Davis TA, Porter NA (2009) Rate Constants for Peroxidation of Polyunsaturated Fatty Acids and Sterols in Solution and in Liposomes, *J. Am. Chem. Soc*. 131: 13037–

[57] Wang X, Ushio H, Ohshima T (2003). Distributions of Hydroperoxide Positional Isomers Generated by Oxidation of 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-

[58] Gupta R, Muralidhara HS, Davis HT (2001) Structure and phase behavior of phospholipids-based micelles in nonaqueous media, *Langmuir* 17: 5176-5183. [59] Mengele EA, Kartasheva ZS, Plashchina IG, Kasaikina O.T*.* (2008) Specific features of

[60] Mengele E.A., Plashchina I.G., Kasaikina O.T. (2011) Kinetics of lecithin oxidation in

[61] Barclay LRC, MacNeil J M, VanKessel JA, Forrest BJ, Porter NA, Lehman LS, Smith IKJ, Ellington JC (1984) Autoxidation and aggregation of phospholipids in organic solvents

[62] Burton GW, Doba T, Gabe EJ, Hughes L, Lee FL, Prasad L, Ingold KU (1985) Autoxidation of biological molecules. 4. Maximizing the antioxidant activity of phenols,

[63] Roginsky V (2003) Chain breaking antioxidant activity of natural polyphenols as determined during the chain oxidation of methyl linoleate in Triton X-100 micelles,

[64] Roginsky V, Tikhonov I. (2010) Natural polyphenols as chain-breaking antioxidants

[65] Nakamura T., Maeda H.A. (1991) Simple assay for lipid hydroperoxides based on triphenylphosphine oxidation and high performance liquid chromatography, *Lipids* 26:

[66] Barclay LRC, Ingold KU (1981) Autoxidation of Biological Molecule. 2. The Autoxidation of a Model Membrane. A Comparison of the Autoxidation of Egg Lecithin Phospha-tidylcholine in Water and in Chlorobenzene *J. Am. Chem. Soc*. 103: 6478-6485. [67] Roginsky V, Lissi EA (2005) Review of methods to determine chain-breaking

[68] Green, S. (1956) Mechanism of the catalytic oxidation of adrenaline by ferritin*. J. Biol.* 

[55] Gregor Cevc. *Phospholipids Handbook*, Marvel Dekker Inc., New York, 1993.

phosphocholine in Liposome and in Methanol Solution, *J. Lipids* 38: 65-72.

lecithin oxidation in organic solvents, *Colloid J*. 70: 753-758.

during lipid peroxidation, *Chem. Phys. Lipids* 163: 127133.

antioxidant activity in food *Food Chem* 92: 235-254.

liposome water solutions, *Colloid J.* 73: 701-707.

*J. Am. Chem. Sос*. 106: 6740-6747.

*J. Am. Chem. Soc.* 107:7053-7065

*Arch. Biochem. Biophysics* 414: 261-267.

#### **1.1. Oxidative stress**

The term oxidative stress; is a state of unbalanced tissue oxidation refers to a condition in which cells are subjected to excessive levels of molecular oxygen or its chemical derivatives called reactive oxygen species (ROS).Under physiological conditions, the molecular oxygen undergoes a series of reactions that ultimately lead to the generation of superoxide anion (O2-), hydrogen peroxide (H2O2) and H2O. Peroxynitrite (OONO-), hypochlorus acid (HOCl), the hydroxyl radical (OH.), reactive aldehydes, lipid peroxides and nitrogen oxides are considered among the other oxidants that have relevance to vascular biology.

Oxygen is the primary oxidant in metabolic reactions designed to obtain energy from the oxidation of a variety of organic molecules. Oxidative stress results from the metabolic reactions that use oxygen, and it has been defined as a disturbance in the equilibrium status of pro-oxidant/anti-oxidant systems in intact cells. This definition of oxidative stress implies that cells have intact pro-oxidant/anti-oxidant systems that continuously generate and detoxify oxidants during normal aerobic metabolism. When additional oxidative events occur, the pro-oxidant systems outbalance the anti-oxidant, potentially producing oxidative damage to lipids, proteins, carbohydrates, and nucleic acids, ultimately leading to cell death in severe oxidative stress. Mild, chronic oxidative stress may alter the anti-oxidant systems by inducing or repressing proteins that participate in these systems, and by depleting cellular stores of anti-oxidant materials such as glutathione and vitamin E (Laval, 1996). Free radicals and other reactive species are thought to play an important role oxidative stress resulting in many human diseases. Establishing their precise role requires the ability to measure them and the oxidative damage that they cause (Halliwell and Whiteman, 2004).

© 2012 El-Aal, 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. © 2012 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.

Oxidative stress is involved in the process of aging (Kregel and Zhang 2007) and various chronic diseases such as atherosclerosis (Fearon and Faux 2009), diabetes (Ceriello and Motz, 2004) and eye disease (Li et al. 2009), whereas fruit and vegetable diets rich in antioxidants such as polyphenols, vitamin C, and carotenoids are correlated with a reduced risk of such chronic diseases (Dherani et al. 2008). An excessive amount of reactive oxygen/nitrogen species (ROS/RNS) leading to an imbalance between antioxidants and oxidants can cause oxidative damage in vulnerable targets such as unsaturated fatty acyl chains in membranes, thiol groups in proteins, and nucleic acid bases in DNA (Ceconi et al. 2003). Several assays to measure " total " antioxidant capacity of biological systems have been developed to investigate the involvement of oxidative stress in pathological conditions or to evaluate the functional bioavailability of dietary antioxidants. Conventional assays to determine antioxidant capacity primarily measure the antioxidant capacity in the aqueous compartment of plasma. Consequently, water soluble antioxidants such as ascorbic acid, uric acid, and protein thiols mainly influence these assays, whereas fat - soluble antioxidants such as tocopherols and carotenoids show little inf uence over the many results. However, there are new approaches to define the total antioxidant capacity of plasma, which reflect the antioxidant network between water - and fat - soluble antioxidants. Revelation of the mechanism of action of antioxidants and their true antioxidant potential can lead to identifying proper strategies to optimize the antioxidant defense systems in the body.

#### **1.2. Measurement of oxidative damage**

A basic approach to study oxidative stress would be to measure some products such as (i) free radicals; (ii) radical-mediated damages on lipids, proteins or DNA molecules; and iii) antioxidant enzymatic activity or concentration.

#### *1.2.1. Free radicals*

Free radicals are reactive compounds that are naturally produced in the human body. They can exert positive effects (e.g. on the immune system) or negative effects (e.g. lipids, proteins or DNA oxidation). Free radicals are normally present in the body in minute concentrations. Biochemical processes naturally lead to the formation of free radicals, and under normal circumstances the body can keep them in check. If there is excessive free radical formation, however, damage to cells and tissue can occur (Wilson, 1997). Free radicals are toxic molecules, may be derived from oxygen, which are persistently produced and incessantly attack and damage molecules within cells; most frequently, this damage is measured as peroxidized lipid products, protein carbonyl, and DNA breakage or fragmentation. Collectively, the process of free radical damage to molecules is referred to as oxidative stress (Reiter et al., 1997).To limit these harmful effects, an organism requires complex protection – the antioxidant system. This system consists of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase) and non-enzymatic antioxidants (e.g. vitamin E [tocopherol], vitamin A [retinol], vitamin C [ascorbic acid], glutathione and uric acid). An imbalance between free radical production and antioxidant defence leads to an oxidative stress state, which may be involved in aging processes and even in some pathology (e.g. cancer and Parkinson's disease).

#### *1.2.2. Formation of free radicals*

64 Lipid Peroxidation

antioxidant defense systems in the body.

**1.2. Measurement of oxidative damage** 

antioxidant enzymatic activity or concentration.

*1.2.1. Free radicals* 

Oxidative stress is involved in the process of aging (Kregel and Zhang 2007) and various chronic diseases such as atherosclerosis (Fearon and Faux 2009), diabetes (Ceriello and Motz, 2004) and eye disease (Li et al. 2009), whereas fruit and vegetable diets rich in antioxidants such as polyphenols, vitamin C, and carotenoids are correlated with a reduced risk of such chronic diseases (Dherani et al. 2008). An excessive amount of reactive oxygen/nitrogen species (ROS/RNS) leading to an imbalance between antioxidants and oxidants can cause oxidative damage in vulnerable targets such as unsaturated fatty acyl chains in membranes, thiol groups in proteins, and nucleic acid bases in DNA (Ceconi et al. 2003). Several assays to measure " total " antioxidant capacity of biological systems have been developed to investigate the involvement of oxidative stress in pathological conditions or to evaluate the functional bioavailability of dietary antioxidants. Conventional assays to determine antioxidant capacity primarily measure the antioxidant capacity in the aqueous compartment of plasma. Consequently, water soluble antioxidants such as ascorbic acid, uric acid, and protein thiols mainly influence these assays, whereas fat - soluble antioxidants such as tocopherols and carotenoids show little inf uence over the many results. However, there are new approaches to define the total antioxidant capacity of plasma, which reflect the antioxidant network between water - and fat - soluble antioxidants. Revelation of the mechanism of action of antioxidants and their true antioxidant potential can lead to identifying proper strategies to optimize the

A basic approach to study oxidative stress would be to measure some products such as (i) free radicals; (ii) radical-mediated damages on lipids, proteins or DNA molecules; and iii)

Free radicals are reactive compounds that are naturally produced in the human body. They can exert positive effects (e.g. on the immune system) or negative effects (e.g. lipids, proteins or DNA oxidation). Free radicals are normally present in the body in minute concentrations. Biochemical processes naturally lead to the formation of free radicals, and under normal circumstances the body can keep them in check. If there is excessive free radical formation, however, damage to cells and tissue can occur (Wilson, 1997). Free radicals are toxic molecules, may be derived from oxygen, which are persistently produced and incessantly attack and damage molecules within cells; most frequently, this damage is measured as peroxidized lipid products, protein carbonyl, and DNA breakage or fragmentation. Collectively, the process of free radical damage to molecules is referred to as oxidative stress (Reiter et al., 1997).To limit these harmful effects, an organism requires complex protection – the antioxidant system. This system consists of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase) and non-enzymatic antioxidants (e.g. vitamin E [tocopherol], vitamin A [retinol], vitamin C [ascorbic acid], glutathione and uric acid). An Normally, bonds don't split in a way that leaves a molecule with an odd, unpaired electron. But when weak bonds split, free radicals are formed. Free radicals are very unstable and react quickly with other compounds, trying to capture the needed electron to gain stability. Generally, free radicals attack the nearest stable molecule, gaining its electron. When the "attacked" molecule loses its electron, it becomes a free radical itself, beginning a chain reaction. Once the process is started, it can cascade, finally resulting in the disruption of a living cell. Some free radicals arise normally during metabolism. Sometimes the body's immune system's cells purposefully create them to neutralize viruses and bacteria. However, environmental factors such as pollution, radiation, and toxins can also spawn free radicals. Normally, the body can handle free radicals, but if antioxidants are unavailable, or if the free-radical production becomes excessive, damage can occur. Of particular importance is that free radical damage accumulates with age (Packer, 1994).

#### *1.2.3. Sources of free radicals*

Free radicals have two principle sources: endogenous sources and exogenous sources. Endogenous sources of free radicals include those that are generated intracellularly, acting within the cell, and those that are formed within the cell, but are released into the surrounding area. These intracellular free radicals result from auto-oxidation and consequent inactivation of small molecules such as reduced thiols and flavins. They may also occur as a result of the activity of certain oxidases, lipoxygenases, cyclo-oxygenases, dehydrogenases and peroxidases. Electron transfer from metals such as iron to oxygencontaining molecules can also initiate free radical reactions paradoxically; antioxidants may also produce free radicals (Weir et al., 1996). A wide range of free radical molecular species are endogenous. The singlet oxygen is not a free radical but is nevertheless a reactive oxygen species and capable of causing tissue damage (Zebger et al., 2004). Exogenous sources of free radicals are environmental sources. Environmental sources of free radicals include exposure to ionizing radiation (from industry, sun exposure, cosmic rays, and medical X-rays), ozone and nitrous oxide (primarily from automobile exhaust), heavy metals (such as mercury, cadmium, and lead), cigarette smoke (both active and passive), alcohol, unsaturated fat, and other chemicals and compounds from food, water, and air. The exogenous sources of free radicals resulting from ionizing radiation play a major role in free radical production. The energy transferred into water from ionizing particles ionizes the water molecule. The water ions produced dissociate yielding free radicals (Valencia and Moran, 2004).

There are two enzymes including Aldehyd oxidase (AO) and xanthine oxidase (XO), they have a very close evolutionary relationship, based on the recent cloning of the gens and they show a high degree of amino acid sequence homology (Terao et al., 2000). They have been

suggested to be relevant to the pathophysiology of a number of clinical disorders (Wright et al., 1995). **Aldehyd oxidase (AO)** commonly exists in vertebrates. Although the liver is the main site for aldehyde oxidase this enzyme has also been reported in kidney, lung, muscle, spleen, stomach, heart and brain (Beedham, 2002). The enzyme in liver of various species catalyzes the oxidation of a number of aldehydes and nitrogenous and also catalyzes the metabolism of physiological compounds such as retinaldehyde and monoamine neurotransimeters (Huang and Ichikawa, 1994). Reduction of oxygen during substrate turnover, leads to the formation of superoxide anion and hydrogen peroxide as ROS. This capacity has attracted attention to the possible role of aldehyde oxidase as a source of ROS. In vivo, it seems that aldehyde oxidase together with cytochrome P450 are quantitative, the most important cellular sources for ROS (Al-Omar et al., 2004). Additionally, the most likely sources of free radicals are **xanthine oxidase (XO)** (McCord, 1985). This enzyme is high particularly in liver and intestine. Although XO generates ROS and evidence has been presented for its role in the development of ischaemic intestinal, hepatic and renal damage (Cohen, 1992). It may also contribute to the development of lung and myocardial reperfusion injury after ischaemic episodes.

#### *1.2.4. Production of free radicals*

Free radicals are produced in a number of ways in biological systems (Halliwell and Whiteman, 2004):


**Figure 1.** The active oxygen system. Molecular oxygen is reduced to water in four single-electron steps. Reduction of non-radical forms of oxygen is a "forbidden" process and thus usually involves spin-orbit coupling by a heavy metal or a halide or excitation to singlet state. An example is Fenton's reaction, the reduction of peroxide to water and hydroxyl radical by ferrous iron. Hydroxyl radical is one of the most powerful oxidizing agents known.

## **2. Oxidative damage to lipids (Lipid peroxidation)**

66 Lipid Peroxidation

reperfusion injury after ischaemic episodes.

interfere with normal cellular metabolism.

of their catalytic function.

overtraining.

*1.2.4. Production of free radicals* 

Whiteman, 2004):

suggested to be relevant to the pathophysiology of a number of clinical disorders (Wright et al., 1995). **Aldehyd oxidase (AO)** commonly exists in vertebrates. Although the liver is the main site for aldehyde oxidase this enzyme has also been reported in kidney, lung, muscle, spleen, stomach, heart and brain (Beedham, 2002). The enzyme in liver of various species catalyzes the oxidation of a number of aldehydes and nitrogenous and also catalyzes the metabolism of physiological compounds such as retinaldehyde and monoamine neurotransimeters (Huang and Ichikawa, 1994). Reduction of oxygen during substrate turnover, leads to the formation of superoxide anion and hydrogen peroxide as ROS. This capacity has attracted attention to the possible role of aldehyde oxidase as a source of ROS. In vivo, it seems that aldehyde oxidase together with cytochrome P450 are quantitative, the most important cellular sources for ROS (Al-Omar et al., 2004). Additionally, the most likely sources of free radicals are **xanthine oxidase (XO)** (McCord, 1985). This enzyme is high particularly in liver and intestine. Although XO generates ROS and evidence has been presented for its role in the development of ischaemic intestinal, hepatic and renal damage (Cohen, 1992). It may also contribute to the development of lung and myocardial

Free radicals are produced in a number of ways in biological systems (Halliwell and

a. Exposure to ionizing radiation is a major cause of free radical production. When irradiated water is ionized, and electron is removed from the molecule, leaving behind an ionized water molecule. The damaging species resulting from the radiolysis of water are the free radicals •H and •OH and hydrated electrons. They are highly reactive and have a lifetime on the order of 10 -9 to 10 -11 seconds. The hydroxyl radical is extremely reactive and is carcinogenic. Since water presents the largest number of target molecules in a cell, most of the energy transfer goes on in water when a cell is irradiated, rather then the solute consisting of protein, carbohydrate, nucleic acid, and bioinorganic molecules. Oxygen is an excellent electron acceptor and can combine with the hydrogen radical to form a peroxyl radical. Hydrogen peroxide is toxic and when present in sufficient quantities can

b. Enzymes and transport molecules also generate free radicals as a normal consequence

c. Auto-oxidation reactions produce free radicals from the spontaneous oxidation of

d. Physical exercise also increases oxidative stress and causes disruptions of the homeostasis. Training can have positive or negative effects on oxidative stress depending on training load, training specificity and the basal level of training. Moreover, oxidative stress seems to be involved in muscular fatigue and may lead to

biological molecules involved in non-enzymatic electron transfers.

The peroxidation of lipids is basically damaging because the formation of lipid peroxidation products leads to spread of free radicals reactions. The important role of lipids in cellular components emphasizes the significance of understanding the mechanisms and consequences of lipid peroxidation in biological systems. Polyunsaturated fatty acids (PUFAs) serve as excellent substrates for lipid peroxidation because of the presence of active bis-allylic methylene groups. The carbon-hydrogen bonds on these activated methylene units have lower bond dissociation energies, making these hydrogen atoms more easily abstracted in radical reactions (Davies et al., 1981). The susceptibility of a particular PUFA toward peroxidation increases with an increase in the number of unsaturated sites in the lipid chain (Nagaoka et al., 1990).

Lipid hydroperoxides are non-radical intermediates derived from unsaturated fatty acids, phospholipids, glycolipids, cholesterol esters and cholesterol itself. Their formation occur in enzymatic or non-enzymatic reactions involving activated chemical species known as "reactive oxygen species" (ROS) which are responsible for toxic effects in the body via various tissue damages. These ROS include among others hydroxyl radicals, lipid oxyl or peroxyl radicals, singlet oxygen, and peroxinitrite formed from nitrogen oxide (NO), all these groups of atoms behave as a unit and are now named "free radical". These chemical forms are defined as any species capable of independent existence that contains one or more unpaired electrons (those which occupy an atomic or molecular orbital by themselves). They are formed either by the loss of a single electron from a non-radical or by the gain of a single electron by a non-radical. They can easily be formed when a covalent bond is broken if one electron from each of the pair shared remains with each atom, this mechanism is known as homolytic fission. In water, this process generates the most reactive species, hydroxyl radical OH.. Chemists know well that combustion which is able at high temperature to rupture C-C, C-H or C-O bonds is a free-radical process. The opposite of this mechanism is the heterolytic fission in which, after a covalent break, one atom receives both electrons (this gives a negative charge) while the other remains with a positive charge.Lipid peroxidation leads to the breakdown of lipids and to the formation of a wide array of primary oxidation products such as conju- dienes or lipid hydroperoxides, and secondary products including MDA, F2-isoprostane or expired pentane, ethane or hexane. Measurement of conjugated dienes is interesting because it detects molecular reorganisation of poly- unsaturated fatty acids during the initial phase lipid peroxidation. Lipid hydroperoxide is another marker of the initial.(reaction of FR and is a specific marker of cellular damage. Other products are often used to measure oxida- stress but have the disadvantage of being secon- dary oxidation products. One of them, MDA, is during fatty acid auto-oxidation. This sub- is most commonly measured by its reaction with thiobarbituric acid, which generates thiobarbi- turic acid reactive substances (TBARS). Although of the results.MDA overestimation), this method is accepted as a the general marker of lipid peroxidation but results are subject to caution (Sies, 1997). In addition, some studcable ies tend to show that MDA is not an adapted method ucts used for such methods.

The peroxidation of lipids involves three distinct steps: initiation, propagation and termination. The initiation phase of lipid peroxidation may proceed by the reaction of an activated oxygen species such as singlet oxygen (1O2), O2-, or HO· with a lipid substrate or by the breakdown of preexisting lipid hydroperoxides by transition metals. In the former case, peroxidation occurs by abstraction of a hydrogen atom from a methylene carbon in the lipid substrate (LH) to generate a highly reactive carbon-centered lipid radical (L·) (Kelly et al., 1998). In the propagation phase of lipid peroxidation, molecular oxygen adds rapidly to L at a diffusion controlled rate to produce the lipid peroxyl radical (LOO·). The peroxyl radical can abstract a hydrogen atom from a number of in vivo sources, such as DNA and proteins, to form the primary oxidation product, a lipid hydroperoxide (LOOH). Alternatively, antioxidants such as α -tocopherol (α -TOH) can act as excellent hydrogen atom donors , generating LOOH and the relatively inert α-tocopherol phenoxyl radical (α - TO·). In the absence of antioxidants or other inhibitors, LOO· can abstract a hydrogen from another lipid molecule (LH), producing another highly reactive carbon centered radical (L·), which then propagates the radical chain as presented in Figure 2 (Waldeck and Stocke , 1996). The lipid hydroperoxide (ROOH) is unstable in the presence of iron or other metal catalysts because ROOH will participate in a Fenton reaction leading to the formation of reactive alkoxy radicals. Therefore, in the presence of irron, the chain reactions are not only propagated but amplified. Among the degradation products of ROOH are aldehydes, such as malondialdehyde, and hydrocarbons, such as ethane and ethylene, which are commonly measured end products of lipid peroxidation (Sener et al., 2004).

Abbreviations: NRP, nonradical product; LOOH, lipid hydroperoxide; α-TOH, α -tocopherol; α -TO· , α -TOH radical; LH, lipid substrate; LOO· , lipid peroxyl radical. Adapted from Waldeck and Stocke(1996)

**Figure 2.** Overview of lipid peroxidation.

During peroxidation pathway via reactive intermediates, several end products are formed such as aldehyde [malondialdehyde + 4-hydroxynonenal], pentane and ethane, 2,3 transconjugated diens, isoprostains and chlesteroloxides. The biological activities of MDA and other aldehydes include cross-linking with DNA and proteins, which alters the function/activity of these molecules. MDA + 4HNE have shown tissue toxicity. MDA can react with amino and thiol groups, the aldehydes are more diffusible than free radicals, which means damage is exported to distance sites. Aldehydes are quickly removed from cells as several enzymes control their metabolism (Ustinova and Riabinin 2003).

#### **3. Antioxidants**

68 Lipid Peroxidation

are formed either by the loss of a single electron from a non-radical or by the gain of a single electron by a non-radical. They can easily be formed when a covalent bond is broken if one electron from each of the pair shared remains with each atom, this mechanism is known as homolytic fission. In water, this process generates the most reactive species, hydroxyl radical OH.. Chemists know well that combustion which is able at high temperature to rupture C-C, C-H or C-O bonds is a free-radical process. The opposite of this mechanism is the heterolytic fission in which, after a covalent break, one atom receives both electrons (this gives a negative charge) while the other remains with a positive charge.Lipid peroxidation leads to the breakdown of lipids and to the formation of a wide array of primary oxidation products such as conju- dienes or lipid hydroperoxides, and secondary products including MDA, F2-isoprostane or expired pentane, ethane or hexane. Measurement of conjugated dienes is interesting because it detects molecular reorganisation of poly- unsaturated fatty acids during the initial phase lipid peroxidation. Lipid hydroperoxide is another marker of the initial.(reaction of FR and is a specific marker of cellular damage. Other products are often used to measure oxida- stress but have the disadvantage of being secon- dary oxidation products. One of them, MDA, is during fatty acid auto-oxidation. This sub- is most commonly measured by its reaction with thiobarbituric acid, which generates thiobarbi- turic acid reactive substances (TBARS). Although of the results.MDA overestimation), this method is accepted as a the general marker of lipid peroxidation but results are subject to caution (Sies, 1997). In addition, some studcable ies tend to show that

The peroxidation of lipids involves three distinct steps: initiation, propagation and termination. The initiation phase of lipid peroxidation may proceed by the reaction of an activated oxygen species such as singlet oxygen (1O2), O2-, or HO· with a lipid substrate or by the breakdown of preexisting lipid hydroperoxides by transition metals. In the former case, peroxidation occurs by abstraction of a hydrogen atom from a methylene carbon in the lipid substrate (LH) to generate a highly reactive carbon-centered lipid radical (L·) (Kelly et al., 1998). In the propagation phase of lipid peroxidation, molecular oxygen adds rapidly to L at a diffusion controlled rate to produce the lipid peroxyl radical (LOO·). The peroxyl radical can abstract a hydrogen atom from a number of in vivo sources, such as DNA and proteins, to form the primary oxidation product, a lipid hydroperoxide (LOOH). Alternatively, antioxidants such as α -tocopherol (α -TOH) can act as excellent hydrogen atom donors , generating LOOH and the relatively inert α-tocopherol phenoxyl radical (α - TO·). In the absence of antioxidants or other inhibitors, LOO· can abstract a hydrogen from another lipid molecule (LH), producing another highly reactive carbon centered radical (L·), which then propagates the radical chain as presented in Figure 2 (Waldeck and Stocke , 1996). The lipid hydroperoxide (ROOH) is unstable in the presence of iron or other metal catalysts because ROOH will participate in a Fenton reaction leading to the formation of reactive alkoxy radicals. Therefore, in the presence of irron, the chain reactions are not only propagated but amplified. Among the degradation products of ROOH are aldehydes, such as malondialdehyde, and hydrocarbons, such as ethane and ethylene, which are commonly

MDA is not an adapted method ucts used for such methods.

measured end products of lipid peroxidation (Sener et al., 2004).

To minimize the negative effects of ROS generated by any pro-oxidant, endogenous defensive mechanisms called antioxidant defense (AD) system, which utilizes enzymatic and non-enzymatic mechanisms. Antioxidants are naturally occurring substances that combat oxidative damage in biological entities. An antioxidant achieves this by slowing or preventing the oxidation process that can damage cells in the body. This it does by getting oxidized itself in place of the cells. Thus an antioxidant can also be termed as a reducing agent. Antioxidants are considered as important in the fight against the damage that can be done by free radicals produced due to oxidative stress. Although the human body has its own defenses against oxidative stress, these become weak with age or in the case of an illness. Although, antioxidants are sold in various forms as dietary supplements there is no clinching clinical evidence in favor of antioxidants as beneficial in maintaining health and preventing disease. However, there is a lot of anecdotal evidence that those who partake of antioxidant-rich food are better protected against problems such as heart disease, macular degeneration, diabetes, and cancer. Antioxidants are either hydrophilic or hydrophobic. Water soluble or hydrophilic antioxidants are active in the blood plasma while the water insoluble antioxidants protect the cell membranes. How do antioxidants work? Antioxidants work by bringing under control the rogue and unstable oxygen molecules that have an odd number of electrons. These oxygen molecules known as free radicals are highly reactive; they attack cells, DNA, and protein thereby accelerating the aging process. The antioxidants work in harmony and the efficacy of one antioxidant depends upon the availability and concentration of another. Essentially, antioxidants work by donating an electron to the unstable free radical. This stabilizes the free radical and converts it into a harmless compound that may safely be removed from the body. Antioxidants are segregated into two classes based on their mode of operating. They can either be chainbreaking or preventive. Chain-breaking antioxidants such as vitamins E and C halt the process of radical formation by stabilizing free radical molecules so that the chain-like process of radical formation is arrested. Preventive antioxidants such as superoxide dismutase and catalase prevent chain initiations by scavenging for initiator radicals and stabilizing them. They also stabilize transition metal radicals like iron and copper. These metals work as catalysts in the production of free radicals. Antioxidants and their various forms. Antioxidants are chiefly available to us through vitamins, enzymes, and minerals. Vitamin E is actually a group of eight tocopherols. Alpha-tocopherol is the most widely available tocopherol and also the most potent in terms of its effect on the body. Vitamin E is fat-soluble and protects cell membranes that are mainly composed of fatty acids. Vitamin C or ascorbic acid is water-soluble and it scavenges for free radicals that are present in aqueous environments within the human body. Beta carotene is also water soluble and is particularly effective in tackling free radicals in areas of low oxygen concentration. Selenium, manganese and zinc are trace elements that are important components of several antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase. Enzymes work as both primary and secondary antioxidants and help repair oxidized DNA and target lipids that are oxidized. Other substances that are now being considered for their antioxidant properties include uric acid and phytochemicals found in plants.

Fruits and vegetables that have been identified as sources of powerful antioxidants help people counter the risk of heart ailments and different types of cancers. However, there is a possibility that these benefits obtained from fruits and vegetables could be a result of not just antioxidants but a mix that includes flavonoids as well. Although, clinical trials have not put forth conclusive evidence in favor of antioxidants as being helpful to our health the vast number of observational studies and anecdotal evidence offers a very strong suggestion that antioxidants are indeed of much use in keeping the body healthy. It is only a matter of time before scientists unravel the exact mechanism that governs the working of antioxidants in the body. Most nutritionists agree that the best source of antioxidants is natural food. One should try and avoid supplements if possible. It is also important to keep in mind that a high dosage of antioxidant supplements can have a detrimental effect on the body. Excessive vitamin E can lead to blood hemorrhage. Vitamin C in large amounts can cause diarrhea and also atherosclerosis. High amounts of selenium can cause hair loss and rashes on the skin. Antioxidants are thought to protect the body against the destructive effects of free radicals. Antioxidants neutralize free radicals by donating one of their own electrons, ending the electron- gain reaction. The antioxidant nutrients themselves don't become free radicals by donating an electron because they are stable in either form. They act as scavengers, helping to prevent cell and tissue damage that could lead to cellular damage and disease (Reiter, 2003; Reiter et al., 2004).

A rangeor of antioxidants are active in the body including enzymatic and non-enzymatic. All of them can to redox status. Redox status is directly linked and be intracellular or extracellular antioxidants . The body produces several enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPX), and glutathione reductase (GR) that neutralize many types of free radicals. Supplements of these enzymes are available for oral administration. However, their absorption is probably minimal at best. Supplementing with the "building blocks" the body requires to make SOD, catalase, and glutathione peroxidase may be more effective. These building block nutrients include the minerals manganese, zinc, and copper for SOD and selenium for GSHPX. While the nonenzymatic defense consists of substances of low molecular weight such as reduced glutathione vitamin C, vitamin E, beta-carotene, lutein, lycopene, vitamin B2, coenzyme Q10, and cysteine (an amino acid). Herbs, such as bilberry, turmeric (curcumin), grape seed or pine bark extracts, and ginkgo can also provide powerful antioxidant protection for the body. Melatonin is a hormone secreted by pineal gland and proves to be powerful antioxidant and free radical scavenger (Yang et al.,2002 and Koc et al., 2003).

#### **3.1. Enzymatic antioxidants**

70 Lipid Peroxidation

plants.

combat oxidative damage in biological entities. An antioxidant achieves this by slowing or preventing the oxidation process that can damage cells in the body. This it does by getting oxidized itself in place of the cells. Thus an antioxidant can also be termed as a reducing agent. Antioxidants are considered as important in the fight against the damage that can be done by free radicals produced due to oxidative stress. Although the human body has its own defenses against oxidative stress, these become weak with age or in the case of an illness. Although, antioxidants are sold in various forms as dietary supplements there is no clinching clinical evidence in favor of antioxidants as beneficial in maintaining health and preventing disease. However, there is a lot of anecdotal evidence that those who partake of antioxidant-rich food are better protected against problems such as heart disease, macular degeneration, diabetes, and cancer. Antioxidants are either hydrophilic or hydrophobic. Water soluble or hydrophilic antioxidants are active in the blood plasma while the water insoluble antioxidants protect the cell membranes. How do antioxidants work? Antioxidants work by bringing under control the rogue and unstable oxygen molecules that have an odd number of electrons. These oxygen molecules known as free radicals are highly reactive; they attack cells, DNA, and protein thereby accelerating the aging process. The antioxidants work in harmony and the efficacy of one antioxidant depends upon the availability and concentration of another. Essentially, antioxidants work by donating an electron to the unstable free radical. This stabilizes the free radical and converts it into a harmless compound that may safely be removed from the body. Antioxidants are segregated into two classes based on their mode of operating. They can either be chainbreaking or preventive. Chain-breaking antioxidants such as vitamins E and C halt the process of radical formation by stabilizing free radical molecules so that the chain-like process of radical formation is arrested. Preventive antioxidants such as superoxide dismutase and catalase prevent chain initiations by scavenging for initiator radicals and stabilizing them. They also stabilize transition metal radicals like iron and copper. These metals work as catalysts in the production of free radicals. Antioxidants and their various forms. Antioxidants are chiefly available to us through vitamins, enzymes, and minerals. Vitamin E is actually a group of eight tocopherols. Alpha-tocopherol is the most widely available tocopherol and also the most potent in terms of its effect on the body. Vitamin E is fat-soluble and protects cell membranes that are mainly composed of fatty acids. Vitamin C or ascorbic acid is water-soluble and it scavenges for free radicals that are present in aqueous environments within the human body. Beta carotene is also water soluble and is particularly effective in tackling free radicals in areas of low oxygen concentration. Selenium, manganese and zinc are trace elements that are important components of several antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase. Enzymes work as both primary and secondary antioxidants and help repair oxidized DNA and target lipids that are oxidized. Other substances that are now being considered for their antioxidant properties include uric acid and phytochemicals found in

Fruits and vegetables that have been identified as sources of powerful antioxidants help people counter the risk of heart ailments and different types of cancers. However, there is Antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPX), and glutathione reductase (GR). Non-en-zymatic antioxidants include a variety of FR quenchers such as vitamin A (retinol), vitamin C - (ascorbic acid), vitamin E (tocopherol), flavonoids, thiols (including glutathione [GSH], ubidecarenone uric acid, bilirubin, ferritin) and micronutrients (iron, copper, zinc, selenium, mangawhich which act as enzymatic cofactors. The antioxidant system efficiency depends on nutritional ineccentric

(vitamins and micronutrients) and on endoge nous antioxidant enzyme production, which can be modified by exercise, training, nutrition and agdative Moreover, the antioxidant system efficiency is important in sport physiology because exercise increases the production of FR.

#### *3.1.1. Superoxide dismutase (SOD, EC 1.15.1.1)*

Superoxide dismutase (SOD) is the major defence upon superoxide radicals and is the first defence line against oxidative stress. SOD represents a group of enzymes that catalyse the dismutation of O2– and the formation of H2O2. SOD is an enzyme (EC 1.15.1.1) discovered by McCord and Fridovich, which plays an important role in the defense mechanism of biological cells exposed to oxygen (McCord and Fridovich 1969). SOD catalyzes the dismutation of superoxide anion radical (O 2 • −) into an oxygen molecule and a hydrogen peroxide. This reaction is recognized as an antioxidant system that protects cells from superoxide toxicity. There are several types of SOD, depending on the type of metal ion. Three major isoforms of mammalian SOD have been identif ed with different tissue distributions (Zelko 2002). Cu/Zn - SOD (SOD1) exists in the cytoplasm, lysosomes, and nuclear compartments of mammalian cells (Bannister and et al., 1987; Zelko et al., 2002). In humans, the liver has a relatively high amount and activity of SOD1 (Nozik-Grayck et al., 2005). Human SOD1 is a homodimer containing one copper ion and one zinc ion in each 16 - kDa subunit which consists of 153 amino acids. The copper ion is held by interaction with imidazolate ligands of the histidine residues in SOD1 in the enzymatic active site. The zinc ion (Zn 2 +) contributes to the stabilization of the enzyme (Johnson and Giulivi , 2005).

#### *3.1.2. Catalase (CAT, EC 1.11.1.6)*

Catalse (CAT) is one of the major antioxidant enzymes (Scandalios et al., 1997). It is one of the first enzymes to be purified and crystallized and has gained a lot of attention in recent years because of its link to cancer, diabetes and aging in humans and animals (Preston et al., 2001). It is present in every cell and in particular in cell structures that use oxygen in order to detoxify toxic substances and produce H2O2. Catalase converts H2O2 into water andoxygen (Greenwald, 1990 ;Yasminch and Theologides, 1993). Catalase can also use H2O2 in order to detoxify some toxic substances via a peroxidase reaction (Mayo et al., 2003). There are many evidences that the changes of catalase activity as well as the mechanisms of its regulation are essential in the response to stress situations which catalyzes the dismutation of H2O2, forming O2 and H2O resulting good protection the cells from the toxic effects of hydrogen peroxide (Brioukhanov and Netrusor, 2004).

#### *3.1.3. Glutathione peroxidase (GPX, EC 1.11.1.9)*

GPX was discovered in 1957 ny Mills. It exists in cell cytosol and mitochondria and has the ability to transform H2O2 into waterThis reaction uses GSH and transforms it into oxidised glutathione (GSSG). GPX and CAT have the same action upon H2O2, debut GPX is more efficient with high ROS concentra-tion and CAT has an important action with lower H2O2 concentration. GPx is a glycoprotein containing a single selenocysteine residue at the active center of each subunit. To protect biological organisms from oxidative damage, GPx catalyses the reduction of hydrogen peroxide and lipid hydroperoxides to water and their corresponding alcohols, respectively, as follows (Antunes et al., 2002): ROOH + 2GSH→ROH +GSSG + H2O2 where reduced monomeric glutathione (GSH) is essential as a hydrogen donor, and GSH is oxidized to glutathione disulf de (GSSG). There are five main mammalian isozymes, which vary in the structure (amino acid sequence and subunit), tissue distribution (liver, kidney,erythrocyte, blood plasma, among others), location (cytoplasm, intestine, extracellular f uid), and substrate specif city (hydrogen peroxide and lipid hydroperoxides) (Dudek et al., 2002). GXP is a selenium dependent enzyme that is ubiquitously expressed and protects cells against oxidative damage by reducing hydrogen peroxide and a wide range of organic peroxides with reducing glutathione (Arthur, 2000). It has been suggested that GPX has anti - inflammatory activity in the cardiovascular system. An increase in cytosolic GPx is linked to a lower risk of cardiovascular disease (Blankenberg et al. 2003).

#### *3.1.4. Glutathione reductase (GR, EC 1.6.4.2)*

72 Lipid Peroxidation

*3.1.1. Superoxide dismutase (SOD, EC 1.15.1.1)* 

(Johnson and Giulivi , 2005).

*3.1.2. Catalase (CAT, EC 1.11.1.6)* 

(vitamins and micronutrients) and on endoge nous antioxidant enzyme production, which can be modified by exercise, training, nutrition and agdative Moreover, the antioxidant system efficiency is important in sport physiology because exercise increases the production of FR.

Superoxide dismutase (SOD) is the major defence upon superoxide radicals and is the first defence line against oxidative stress. SOD represents a group of enzymes that catalyse the dismutation of O2– and the formation of H2O2. SOD is an enzyme (EC 1.15.1.1) discovered by McCord and Fridovich, which plays an important role in the defense mechanism of biological cells exposed to oxygen (McCord and Fridovich 1969). SOD catalyzes the dismutation of superoxide anion radical (O 2 • −) into an oxygen molecule and a hydrogen peroxide. This reaction is recognized as an antioxidant system that protects cells from superoxide toxicity. There are several types of SOD, depending on the type of metal ion. Three major isoforms of mammalian SOD have been identif ed with different tissue distributions (Zelko 2002). Cu/Zn - SOD (SOD1) exists in the cytoplasm, lysosomes, and nuclear compartments of mammalian cells (Bannister and et al., 1987; Zelko et al., 2002). In humans, the liver has a relatively high amount and activity of SOD1 (Nozik-Grayck et al., 2005). Human SOD1 is a homodimer containing one copper ion and one zinc ion in each 16 - kDa subunit which consists of 153 amino acids. The copper ion is held by interaction with imidazolate ligands of the histidine residues in SOD1 in the enzymatic active site. The zinc ion (Zn 2 +) contributes to the stabilization of the enzyme

Catalse (CAT) is one of the major antioxidant enzymes (Scandalios et al., 1997). It is one of the first enzymes to be purified and crystallized and has gained a lot of attention in recent years because of its link to cancer, diabetes and aging in humans and animals (Preston et al., 2001). It is present in every cell and in particular in cell structures that use oxygen in order to detoxify toxic substances and produce H2O2. Catalase converts H2O2 into water andoxygen (Greenwald, 1990 ;Yasminch and Theologides, 1993). Catalase can also use H2O2 in order to detoxify some toxic substances via a peroxidase reaction (Mayo et al., 2003). There are many evidences that the changes of catalase activity as well as the mechanisms of its regulation are essential in the response to stress situations which catalyzes the dismutation of H2O2, forming O2 and H2O resulting good protection the cells from the

GPX was discovered in 1957 ny Mills. It exists in cell cytosol and mitochondria and has the ability to transform H2O2 into waterThis reaction uses GSH and transforms it into oxidised glutathione (GSSG). GPX and CAT have the same action upon H2O2, debut GPX is more

toxic effects of hydrogen peroxide (Brioukhanov and Netrusor, 2004).

*3.1.3. Glutathione peroxidase (GPX, EC 1.11.1.9)* 

Glutathione Reductase (GR) is a key enzyme of glutathione metabolism and is widespread in all tissues and blood cells. It a flavin enzyme involved in the defense of the erythrocyte against hemolysis. This enzyme catalyses reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) in the presence of NADPH and maintains a high intracellular GSH/GSSG ratio of about 500 in red blood cells (Kondo et al., 1980). GR is important not only for the maintaining thr required GSH level but also for reducing protein thiols to their native state.This enzyme is conserved between all kingdoms. In bacteria, yeasts and animals, one GR gene is found, however in plant genomes two GR genes are enclosed. Under normal conditions, GSH and GR are involved in the detoxification of H2O2 generated in the light by the Mehler reaction in chloroplasts. Disturbances GH level have been correlated with oxidative stress induced by various factors including toxicity, pollutants, inflammation and different diseases particularly red blood cell defects.

#### *3.1.5. Glutathione-S-transferase (GST, EC 2.5.1.18)*

Glutathione-S-Transferase (GST) catalyzes the conjugation with glutathione of a number of electrophilic xenobiotics, including several carcinogens, mutagens and anticancer drugs (Hayes and Pulford, 1995). These electrophiles are made less reactive by conjugation with glutathione and the conjugates are thought to be less toxic to the cell. Consiquently, GSTs are believed to play an important role in the defense of cells against these zenobiotic toxins. Several antineoplastic drugs particularly the reactive electrophilic alkylating agents, can form conjugates with glutathione both spontaneously and in GST-catalyzed reactions (Awasthi et al., 1996). Morever, Some studies reported that there are a good association between cellular resistance to some anticancers drugs and expression of particular isozymes of GST (Hayes and Pulford, 1995).

#### *3.2.1. Glutathione (GSH)*

Gultathione (GSH) is a small molecule found in almost every cell (Anderson, 1997). It is the smallest intrecellular thiol (SH) molecule. Its high electron-donating capacity (high negative redox potential) combined with high intracellular concentration generate great reducing power (Kidd, 1997). Glutathione is a cysteine-containing peptide found in most forms of aerobic life. It is not required in the diet and is instead synthesized in cells from its constituent amino acids (Meister and Anderson, 1983; Meister, 1988). It can react non-enzymatically with ROS and GSH peroxidase catalyses the destruction of hydrogen peroxide and hydroperoxides resulting in its oxidation to the disulphide form (GSSG). Firstly, glutathione is the major antioxidant produced by the cell protecting it from free radicals as oxygen radicals which are highly reactive substances can damage or destroy key cell components. Its antioxidant properties result from the thiol group in its cysteine moiety is a reducing agent and can be reversibly oxidized and reduced. In cells, glutathione is maintained in the reduced form by the enzyme glutathione reductase and in turn reduces other metabolites and enzyme systems, such as ascorbate in the glutathione-ascorbate cycle, glutathione peroxidases and glutaredoxins, as well as reacting directly with oxidants. Due to its high concentration and its central role in maintaining the cell's redox state, glutathione is one of the most important cellular antioxidants. In some organisms glutathione is replaced by other thiols, such as by mycothiol in the Actinomycetes, bacillithiol in some Gram-positive bacteria, or by trypanothione in the Kinetoplastids (Meister and Larsson, 1995). Secondly, GSH is a very important detoxifing agent, enabling the body to get rid of undesirable toxins and pollutants. It forms a soluble compound with the toxin that can then be excreted through the urine or the gut. The liver and kidneys contain high levels of GSH as they have the greatest exposure to toxins. The lung are also rich in glutathione partly for the same reason. Thirdly, GSH plays a crucial role in maintaining a normal balance between oxidation and anti-oxidation. This in turn regulates many of the cell's vital functions such as the synthesis and repair of DNA, the synthesis of proteins and the activation, maintaining the essential thiol status of protein, immune function, regulate nitric oxide homeostasis, modulate the activity of neurotransmitter receptors and regulation of enzymes (Oja et al., 2000; Hogg, 2002). The lower level of GSH is related to different physiological and biochemical disturbances.

#### *3.2.2. Ascorbic acid*

Ascorbic acid or "vitamin C" is a monosaccharide oxidation-reduction (redox) catalyst found in both animals and plants (Peake , 2003). As one of the enzymes needed to make ascorbic acid has been lost by mutation during primate evolution, humans must obtain it from the diet; it is therefore a vitamin. Most other animals are able to produce this compound in their bodies and do not require it in their diets. Ascorbic acid is required for the conversion of the procollagen to collagen by oxidizing proline residues to hydroxyproline. In other cells, it is maintained in its reduced form by reaction with glutathione, which can be catalysed by protein disulfide isomerase and glutaredoxins. Ascorbic acid is redox catalyst which can reduce, and thereby neutralize, reactive oxygen species such as hydrogen peroxide. In addition to its direct antioxidant effects, ascorbic acid is also a substrate for the redox enzyme ascorbate peroxidase, a function that is particularly important in stress resistance in plants. Ascorbic acid is present at high levels in all parts of plants and can reach concentrations of 20 millimolar in chloroplasts. It act as a marked antioxidant that help in the treatement of different diseases such as cancer and cardiovascular (Coulter et al., 2006 ; Cook et al., 2007).

#### *3.2.3. Melatonin*

74 Lipid Peroxidation

*3.2.1. Glutathione (GSH)* 

Gultathione (GSH) is a small molecule found in almost every cell (Anderson, 1997). It is the smallest intrecellular thiol (SH) molecule. Its high electron-donating capacity (high negative redox potential) combined with high intracellular concentration generate great reducing power (Kidd, 1997). Glutathione is a cysteine-containing peptide found in most forms of aerobic life. It is not required in the diet and is instead synthesized in cells from its constituent amino acids (Meister and Anderson, 1983; Meister, 1988). It can react non-enzymatically with ROS and GSH peroxidase catalyses the destruction of hydrogen peroxide and hydroperoxides resulting in its oxidation to the disulphide form (GSSG). Firstly, glutathione is the major antioxidant produced by the cell protecting it from free radicals as oxygen radicals which are highly reactive substances can damage or destroy key cell components. Its antioxidant properties result from the thiol group in its cysteine moiety is a reducing agent and can be reversibly oxidized and reduced. In cells, glutathione is maintained in the reduced form by the enzyme glutathione reductase and in turn reduces other metabolites and enzyme systems, such as ascorbate in the glutathione-ascorbate cycle, glutathione peroxidases and glutaredoxins, as well as reacting directly with oxidants. Due to its high concentration and its central role in maintaining the cell's redox state, glutathione is one of the most important cellular antioxidants. In some organisms glutathione is replaced by other thiols, such as by mycothiol in the Actinomycetes, bacillithiol in some Gram-positive bacteria, or by trypanothione in the Kinetoplastids (Meister and Larsson, 1995). Secondly, GSH is a very important detoxifing agent, enabling the body to get rid of undesirable toxins and pollutants. It forms a soluble compound with the toxin that can then be excreted through the urine or the gut. The liver and kidneys contain high levels of GSH as they have the greatest exposure to toxins. The lung are also rich in glutathione partly for the same reason. Thirdly, GSH plays a crucial role in maintaining a normal balance between oxidation and anti-oxidation. This in turn regulates many of the cell's vital functions such as the synthesis and repair of DNA, the synthesis of proteins and the activation, maintaining the essential thiol status of protein, immune function, regulate nitric oxide homeostasis, modulate the activity of neurotransmitter receptors and regulation of enzymes (Oja et al., 2000; Hogg, 2002). The lower level of GSH is

related to different physiological and biochemical disturbances.

Ascorbic acid or "vitamin C" is a monosaccharide oxidation-reduction (redox) catalyst found in both animals and plants (Peake , 2003). As one of the enzymes needed to make ascorbic acid has been lost by mutation during primate evolution, humans must obtain it from the diet; it is therefore a vitamin. Most other animals are able to produce this compound in their bodies and do not require it in their diets. Ascorbic acid is required for the conversion of the procollagen to collagen by oxidizing proline residues to hydroxyproline. In other cells, it is maintained in its reduced form by reaction with glutathione, which can be catalysed by protein disulfide isomerase and glutaredoxins. Ascorbic acid is redox catalyst which can reduce, and thereby neutralize, reactive oxygen species such as hydrogen peroxide. In

*3.2.2. Ascorbic acid* 

Melatonin (N-acetyl-5-methoxytryptamine), is synthesized from serotonin in the pineal gland which contains all the enzymes necessary for the methoxylation and acetylation reactions. Melatonin is released in mammals during the dark-phase of the circadian cycle, and declines with age (Tan et al., 2001). It is able to reduce the free radical formation which follows the interaction between transition metal ions and amyliod-beta peptide (Zatta et al., 2003). As a free radical scavenger melatonin exhibits several important properties: It has both lipophilic and hydrophilic and it passes all bio-barriers, e.g. blood brain barrier and placenta (Wakatsuki et al., 1999).

Reiter (1995) reported that melatonin seems to be more effective than other antioxidants (e.g. mannitol, glutathione and vitamin E) in protecting against oxidative damage. Thus, it may provide protection against diseases that cause degenerative or proliferative changes by shielding macromolecules, particularly DNA from such injuries. Besides its direct free radical scavenging action, melatonin functions as an indirect antioxidant by stimulating the activities of antioxidiative enzymes in addition to protecting against lipid peroxidation (Undeger et al., 2004).

Melatonin has been found to be a direct free radical scavenger and an indirect antioxidant that, may have an active role in protection against genetic damage due to endogenously produced free radicals and it may be of use in reducing damage from physical and chemical mutagens and carcinogens that generate free radicals (Bandyopadhyay et al., 2000).

#### *3.2.4. Tocopherols and tocotrienols (vitamin E)*

Vitamin E is the collective name for a set of eight related tocopherols and tocotrienols, which are fat-soluble vitamins with antioxidant properties (Roberts et al., 2007). Of these, αtocopherol has been most studied as it has the highest bioavailability, with the body preferentially absorbing and metabolising this form. It has been claimed that the αtocopherol form is the most important lipid-soluble antioxidant, and that it protects membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction. This removes the free radical intermediates and prevents the propagation reaction from continuing (Cook et al., 2007). This reaction produces oxidised αtocopheroxyl radicals that can be recycled back to the active reduced form through reduction by other antioxidants, such as ascorbate, retinol or ubiquinol.This is in line with findings showing that α-tocopherol, but not water-soluble antioxidants, efficiently protects glutathione peroxidase 4 (GPX4)-deficient cells from cell death. GPx4 is the only known enzyme that efficiently reduces lipid-hydroperoxides within biological membranes. However, the roles and importance of the various forms of vitamin E are presently unclear, and it has even been suggested that the most important function of α-tocopherol is as a signaling molecule, with this molecule having no significant role in antioxidant metabolism. The functions of the other forms of vitamin E are even less well-understood, although γtocopherol is a nucleophile that may react with electrophilic mutagens, and tocotrienols may be important in protecting neurons from damage. However it has a protective action against different diseases including cancer (Coulter et al., 2006).

#### **3.3. Total antioxidant capacity**

Epidemiologic studies have demonstrated an inverse association between consumption of fruits and vegetables and morbidity and mortality from degenerative diseases. The antioxidant content of fruits and vegetables may contribute to the protection they offer from disease. Because plant foods contain many different classes and types of antioxidants, knowledge of their total antioxidant capacity (TAC), which is the cumulative capacity of food components to scavenge free radicals, would be useful for epidemiologic purposes. To accomplish this, a variety of foods commonly consumed in Italy, including 34 vegetables, 30 fruits, 34 beverages and 6 vegetable oils, were analyzed using three different assays, i.e., Trolox equivalent antioxidant capacity (TEAC), total radical-trapping antioxidant parameter (TRAP) and ferric reducing-antioxidant power (FRAP). These assays, based on different chemical mechanisms, were selected to take into account the wide variety and range of action of antioxidant compounds present in actual foods. Among vegetables, spinach had the highest antioxidant capacity in the TEAC and FRAP assays followed by peppers, whereas asparagus had the greatest antioxidant capacity in the TRAP assay. Among fruits, the highest antioxidant activities were found in berries (i.e., blackberry, redcurrant and raspberry) regardless of the assay used. Among beverages, coffee had the greatest TAC, regardless of the method of preparation or analysis, followed by citrus juices, which exhibited the highest value among soft beverages. Finally, of the oils, soybean oil had the highest antioxidant capacity, followed by extra virgin olive oil, whereas peanut oil was less effective. Such data, coupled with an appropriate questionnaire to estimate antioxidant intake, will allow the investigation of the relation between dietary antioxidants and oxidative stress-induced diseases (Pellegrini et al., 2003 ; Puchau et al., 2009 ; Dilis and Trichopoulou, 2010).

#### **4. Nutritional therapy with natural antioxidants**

Antioxidants have been the focus of research on the relationship between The role of dietary factors in protecting against the change from native to oxidized LDL has received considerable attention. An overview of epidemiological research suggests that individuals with the highest intakes of antioxidant vitamins, whether through diet or supplements, tend to lower of various disease. Research examining the effects of a diet rich in fruits and vegetables on disease has been carried out using several types of study.There is strong scientific evidence to support an increase in intakes of vegetables and fruit in the prevention of disease. Further research is required to clarify which particular components of fruit and vegetables are responsible for their protective effects. Numerous epidemiological studies have indicated that diets rich in fruits and vegetables are correlated with a reduced risk of chronic diseases (German, 1999; Benzie, 2003; Hassan, 2005; Hassan and Yosef, 2009; Hassan et al., 2010). It is probable that antioxidants, present in the fruits and vegetables such as polyphenols, carotenoids, and vitamin C, prevent damage from harmful reactive oxygen species, which either are continuously produced in the body during normal cellular functioning or are derived from exogenous sources (Gate et al. 1999). The possible protective effect of antioxidants in fruits and vegetables against ROS has led people to consume antioxidant supplements against chronic diseases.

76 Lipid Peroxidation

glutathione peroxidase 4 (GPX4)-deficient cells from cell death. GPx4 is the only known enzyme that efficiently reduces lipid-hydroperoxides within biological membranes. However, the roles and importance of the various forms of vitamin E are presently unclear, and it has even been suggested that the most important function of α-tocopherol is as a signaling molecule, with this molecule having no significant role in antioxidant metabolism. The functions of the other forms of vitamin E are even less well-understood, although γtocopherol is a nucleophile that may react with electrophilic mutagens, and tocotrienols may be important in protecting neurons from damage. However it has a protective action against

Epidemiologic studies have demonstrated an inverse association between consumption of fruits and vegetables and morbidity and mortality from degenerative diseases. The antioxidant content of fruits and vegetables may contribute to the protection they offer from disease. Because plant foods contain many different classes and types of antioxidants, knowledge of their total antioxidant capacity (TAC), which is the cumulative capacity of food components to scavenge free radicals, would be useful for epidemiologic purposes. To accomplish this, a variety of foods commonly consumed in Italy, including 34 vegetables, 30 fruits, 34 beverages and 6 vegetable oils, were analyzed using three different assays, i.e., Trolox equivalent antioxidant capacity (TEAC), total radical-trapping antioxidant parameter (TRAP) and ferric reducing-antioxidant power (FRAP). These assays, based on different chemical mechanisms, were selected to take into account the wide variety and range of action of antioxidant compounds present in actual foods. Among vegetables, spinach had the highest antioxidant capacity in the TEAC and FRAP assays followed by peppers, whereas asparagus had the greatest antioxidant capacity in the TRAP assay. Among fruits, the highest antioxidant activities were found in berries (i.e., blackberry, redcurrant and raspberry) regardless of the assay used. Among beverages, coffee had the greatest TAC, regardless of the method of preparation or analysis, followed by citrus juices, which exhibited the highest value among soft beverages. Finally, of the oils, soybean oil had the highest antioxidant capacity, followed by extra virgin olive oil, whereas peanut oil was less effective. Such data, coupled with an appropriate questionnaire to estimate antioxidant intake, will allow the investigation of the relation between dietary antioxidants and oxidative stress-induced diseases (Pellegrini et al.,

Antioxidants have been the focus of research on the relationship between The role of dietary factors in protecting against the change from native to oxidized LDL has received considerable attention. An overview of epidemiological research suggests that individuals with the highest intakes of antioxidant vitamins, whether through diet or supplements, tend to lower of various disease. Research examining the effects of a diet rich in fruits and vegetables on disease has been carried out using several types of study.There is strong

different diseases including cancer (Coulter et al., 2006).

2003 ; Puchau et al., 2009 ; Dilis and Trichopoulou, 2010).

**4. Nutritional therapy with natural antioxidants** 

**3.3. Total antioxidant capacity** 

## **5. Nutritional factors as natural antioxidants agents in alleviating the oxidative stress induced by environmental pollutents: Some experimental studies for the author**

### **5.1. Mitigating effects of antioxidant properties of black berry juice on sodium fluoride induced hepatotoxicity and oxidative stress in rats**

Fluorosis is a serious public health problem in many parts of the world. As in the case of many chronic degenerative diseases, increased production of reactive oxygen species has been considered to play an important role, even in the pathogenesis of chronic fluoride toxicity. Black berry is closely linked to its protective properties against free radical attack. Therefore, the aim of this study was to demonstrate the role of black berry juice (BBJ) in decreasing the hepatotoxicity and oxidative stress of sodium fluoride )NaF). Results showed that NaF caused elevation in liver TBARS and nitric oxide (NO), and reduction in superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (TAC) and glutathione (GSH.( Plasma transaminases (AST and ALT), creatine kinase (CK), lactate dehydrogenase (LDH), total lipids )TL), cholesterol, triglycerides (TG), and low density lipoprotein–cholesterol (LDL–c) were increased, while high density lipoprotein–cholesterol (HDL–c) was decreased. On the other hand, BBJ reduced NaF-induced TBARS, NO, TL, cholesterol, TG, LDL–c, AST, ALT, CK and LD. Moreover, it ameliorated NaFinduced decrease in SOD, CAT, GSH, TAC and HDL–c. Therefore, the present results revealed that BBJ has a protective effect against NaF-induced hepatotoxicity by antagonizing the free radicals generation and enhancement of the antioxidant defence mechanisms. (Hassan and Yousef, 2009).

#### **5.2. Evaluation of free radical-scavenging and antioxidant properties of black berry against fluoride toxicity in rats**

Oxidative damage to cellular components such as lipids and cell membranes by free radicals and other reactive oxygen species is believed to be associated with the development of degenerative diseases. Fluoride intoxication is associated with oxidative stress and altered anti-oxidant defense mechanism. So the present study was extended to investigate black berry anti-oxidant capacity towards superoxide anion radicals, hydroxyl radicals and nitrite in different organs of fluoride-intoxicated rats. The data indicated that sodium fluoride (10.3 mg/kg bw) administration induced oxidative stress as evidenced by elevated levels of lipid peroxidation and nitric oxide in red blood cells, kidney, testis and brain tissues. Moreover, significantly decreased glutathione level, total anti-oxidant capacity and superoxide dismutase activitywere observed in the examined tissues. On the other hand, the induced oxidative stress and the alterations in anti-oxidant system were normalized by the oral administration of black berry juice (1.6 g/ kg bw). Therefore it can be concluded that black berry administration could minimize the toxic effects of fluoride indicating its free radicalscavenging and potent anti-oxidant activities. (Hassan and Fattoh, 2010)

## **5.3. Garlic oil as a modulating agent for oxidative stress and neurotoxicity induced by sodium nitrite in male albino rats**

In the present study, we investigated the neurobiochemical alterations and oxidative stress induced by food preservative; sodium nitrite (NaNO2) as well as the role of the garlic oil in amelioration of the neurotoxicity in male albino rats. Serum and brain homogenates of the rats received NaNO2 (80 mg/kg body weight) for 3 months exhibited significant decrease in acetylcholine esterase (AChE) activity as well as the levels of phospholipids, total protein and the endogenous antioxidant system (glutathione; GSH and superoxide dismutase; SOD). In contrast, lactic dehydrogenase (LDH) activity, brain thiobarbituric acid reactive substances (TBARS) and nitric oxide (NO) levels were significantly increased. On the other hand, the oral administration of garlic oil (5 ml/kg body weight) daily for 3 months significantly improved the neurobiochemical disorders and inhibited the oxidative stress induced by NaNO2 ingestion. So, this study reveals the neural toxic effects of NaNO2 by exerting oxidative stress and retrograde the endogenous antioxidant system. However, garlic oil has a promising role in attenuating the obtained hazard effects of sodium nitrite by its high antioxidant properties which may eventually be related with the preservation of SOD activity and primary mitochondrial role against nitrite-induced neurotoxicity in rats . (Hassan et al., 2010).

#### **5.4.** *In vivo* **evidence of hepato-and-reno-protective effect of garlic oil against sodium nitrite-induced oxidative stress**

Sodium nitrite (NaNO2), a food color fixative and preservative, contributes to carcinogenesis. We investigated the protective role of garlic oil against NaNO2-induced abnormalities in metabolic biochemical parameters and oxidative status in male albino rats. NaNO2 treatment for a period of three months induced a significant increase in serum levels of glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin, urea and creatinine as well as hepatic AST and ALT. However, significant decrease was recorded in liver ALP activity, glycogen content, and renal urea and creatinine levels. In parallel, a significant increase in lipid peroxidation, and a decrease in glutathione content and catalase activity were observed in the liver and the kidney. However, garlic oil supplementation showed a remarkable amelioration of these abnormalities. Our data indicate that garlic is a phytoantioxidant with powerful chemopreventive properties against chemically-induced oxidative stress (Hassan et al., 2009).

## **5.5. Ameliorating effect of chicory (Cichorium intybus L)-supplemented diet against nitrosamine precursors-induced liver injury and oxidative stress in male rats**

The current study was carried out to elucidate the modulating effect of chicory (*Cichorium intybus L.)-*supplemente diet against nitrosamnine-induced oxidative stress and hepatotoxicity in male rats. Rats were divided into four groups and treated for 8 weeks as follow: group 1 served as control; group 2 fed on chicory-supplemented diet (10% w/w); group 3 received simultaneously nitrosamine precursors [sodium nitrite (0.05% in drinking water) plus chlorpromazine (1.7 mg/kg body weight)] and group 4 received nitrosamine precursors and fed on chicory-supplemented diet. The obtained results revealed that rats received nitrosamine precursors showed a significant increase in liver TBARS and total lipids, total cholesterol, bilirubin, and enzymes activity (AST, ALT, ALP and GGT) in both serum and liver. While a significant decrease in the levels of GSH, GSH-Rx, SOD, catalase, total protein and albumin was recorded. On the other hand, chicory-supplemented diet succeeded to modulate these observed abnormalities resulting from nitrosamine compounds as indicated by the reduction of TBARS and the pronounced improvement of the investigated biochemical and antioxidant parameters. So, it could be concluded that chicory has a promising role and it worth to be considered as a natural substance for ameliorating the oxidative stress and hepatic injury induced by nitrosamine compounds **(Hassan and Yousef, 2010).** 

#### **6. Summary**

78 Lipid Peroxidation

(Hassan et al., 2010).

**sodium nitrite-induced oxidative stress** 

degenerative diseases. Fluoride intoxication is associated with oxidative stress and altered anti-oxidant defense mechanism. So the present study was extended to investigate black berry anti-oxidant capacity towards superoxide anion radicals, hydroxyl radicals and nitrite in different organs of fluoride-intoxicated rats. The data indicated that sodium fluoride (10.3 mg/kg bw) administration induced oxidative stress as evidenced by elevated levels of lipid peroxidation and nitric oxide in red blood cells, kidney, testis and brain tissues. Moreover, significantly decreased glutathione level, total anti-oxidant capacity and superoxide dismutase activitywere observed in the examined tissues. On the other hand, the induced oxidative stress and the alterations in anti-oxidant system were normalized by the oral administration of black berry juice (1.6 g/ kg bw). Therefore it can be concluded that black berry administration could minimize the toxic effects of fluoride indicating its free radical-

scavenging and potent anti-oxidant activities. (Hassan and Fattoh, 2010)

**induced by sodium nitrite in male albino rats** 

**5.3. Garlic oil as a modulating agent for oxidative stress and neurotoxicity** 

In the present study, we investigated the neurobiochemical alterations and oxidative stress induced by food preservative; sodium nitrite (NaNO2) as well as the role of the garlic oil in amelioration of the neurotoxicity in male albino rats. Serum and brain homogenates of the rats received NaNO2 (80 mg/kg body weight) for 3 months exhibited significant decrease in acetylcholine esterase (AChE) activity as well as the levels of phospholipids, total protein and the endogenous antioxidant system (glutathione; GSH and superoxide dismutase; SOD). In contrast, lactic dehydrogenase (LDH) activity, brain thiobarbituric acid reactive substances (TBARS) and nitric oxide (NO) levels were significantly increased. On the other hand, the oral administration of garlic oil (5 ml/kg body weight) daily for 3 months significantly improved the neurobiochemical disorders and inhibited the oxidative stress induced by NaNO2 ingestion. So, this study reveals the neural toxic effects of NaNO2 by exerting oxidative stress and retrograde the endogenous antioxidant system. However, garlic oil has a promising role in attenuating the obtained hazard effects of sodium nitrite by its high antioxidant properties which may eventually be related with the preservation of SOD activity and primary mitochondrial role against nitrite-induced neurotoxicity in rats .

**5.4.** *In vivo* **evidence of hepato-and-reno-protective effect of garlic oil against** 

Sodium nitrite (NaNO2), a food color fixative and preservative, contributes to carcinogenesis. We investigated the protective role of garlic oil against NaNO2-induced abnormalities in metabolic biochemical parameters and oxidative status in male albino rats. NaNO2 treatment for a period of three months induced a significant increase in serum levels of glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin, urea and creatinine as well as hepatic AST and ALT.

#### **6.1. Oxidative stress**

The term oxidative stress refers to a condition in which cells are subjected to excessive levels of molecular oxygen or its chemical derivatives called reactive oxygen species (ROS).Under physiological conditions, the molecular oxygen undergoes a series of reactions that ultimately lead to the generation of superoxide anion (O2-), hydrogen peroxide (H2O2) and H2O. Peroxynitrite (OONO-), hypochlorus acid (HOCl), the hydroxyl radical (OH.), reactive aldehydes, lipid peroxides and nitrogen oxides are considered among the other oxidants that have relevance to vascular biology.

Oxygen is the primary oxidant in metabolic reactions designed to obtain energy from the oxidation of a variety of organic molecules. Oxidative stress results from the metabolic reactions that use oxygen, and it has been defined as a disturbance in the equilibrium status of pro-oxidant/anti-oxidant systems in intact cells. This definition of oxidative stress implies that cells have intact pro-oxidant/anti-oxidant systems that continuously generate and detoxify oxidants during normal aerobic metabolism. When additional oxidative events occur, the pro-oxidant systems outbalance the anti-oxidant, potentially producing oxidative damage to lipids, proteins, carbohydrates, and nucleic acids, ultimately leading to cell death in severe oxidative stress. Mild, chronic oxidative stress may alter the anti-oxidant systems by inducing or repressing proteins that participate in these systems, and by depleting cellular stores of anti-oxidant materials such as glutathione and vitamin E (Laval, 1996). Free radicals and other reactive species are thought to play an important role oxidative stress resulting in many human diseases. Establishing their precise role requires the ability to measure them and the oxidative damage that they cause (Halliwell and Whiteman, 2004).

A basic approach to study oxidative stress would be to measure some products such as (i) free radicals; (ii) radical-mediated damages on lipids, proteins or DNA molecules; and iii) antioxidant enzymatic activity or concentration.

#### **6.2. Free radicals**

Free radicals are reactive compounds that are naturally produced in the human body. They can exert positive effects (e.g. on the immune system) or negative effects (e.g. lipids, proteins or DNA oxidation). Free radicals are normally present in the body in minute concentrations. Biochemical processes naturally lead to the formation of free radicals, and under normal circumstances the body can keep them in check. If there is excessive free radical formation, however, damage to cells and tissue can occur (Wilson, 1997). Free radicals are toxic molecules, may be derived from oxygen, which are persistently produced and incessantly attack and damage molecules within cells; most frequently, this damage is measured as peroxidized lipid products, protein carbonyl, and DNA breakage or fragmentation. Collectively, the process of free radical damage to molecules is referred to as oxidative stress (Reiter et al., 1997).To limit these harmful effects, an organism requires complex protection – the antioxidant system. This system consists of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase) and non-enzymatic antioxidants (e.g. vitamin E [tocopherol], vitamin A [retinol], vitamin C [ascorbic acid], glutathione and uric acid). An imbalance between free radical production and antioxidant defence leads to an oxidative stress state, which may be involved in aging processes and even in some pathology (e.g. cancer and Parkinson's disease).

#### **6.3. Oxidative damage to lipids (Lipid peroxidation)**

The peroxidation of lipids involves three distinct steps: initiation, propagation and termination. The initiation reaction between an unsaturated fatty acid and the hydroxyl radical involves the abstraction of a H atom from the methylvinyl group on the fatty acid. The remaining carbon-centred radical, forms a resonance structure sharing this unpaired electron among carbons 9 to 13. In the propagation reactions, this resonance structure reacts with triplet oxygen, which is a biradical having two unpaired electrons and therefore reacts readily with other radicals. This reaction forms a peroxy radical. The peroxy radical then abstracts a H atom from a second fatty acid forming a lipid hydroperoxide and leaving another carbon centered free radical that can participate in a second H abstraction. Therefore, once one hydroxyl radical initiates the peroxidation reaction by abstracting a single H atom, it creates a carbon radical product that is capable of reacting with ground state oxygen in a chain reaction. The role of the hydroxyl radical is analogous to a "spark" that starts a fire. The basis for the hydroxyl radical's extreme reactivity in lipid systems is that at very low concentrations it initiates a chain reaction involving triplet oxygen, the most abundant form of oxygen in the cell (Benderitter et al., 2003).

The lipid hydroperoxide (ROOH) is unstable in the presence of irron or other metal catalysts because ROOH will participate in a Fenton reaction leading to the formation of reactive alkoxy radicals. Therefore, in the presence of irron, the chain reactions are not only propagated but amplified. Among the degradation products of ROOH are aldehydes, such as malondialdehyde, and hydrocarbons, such as ethane and ethylene, which are commonly measured end products of lipid peroxidation (Sener et al., 2004). During peroxidation pathway via reactive intermediates, several end products are formed such as aldehyde [malondialdehyde + 4-hydroxynonenal], pentane and ethane, 2,3 transconjugated diens, isoprostains and chlesteroloxides. The biological activities of MDA and other aldehydes include cross-linking with DNA and proteins, which alters the function/activity of these molecules. MDA + 4HNE have shown tissue toxicity. MDA can react with amino and thiol groups, the aldehydes are more diffusible than free radicals, which means damage is exported to distance sites. Aldehydes are quickly removed from cells as several enzymes control their metabolism (Ustinova and Riabinin 2003).

#### **6.4. Antioxidants**

80 Lipid Peroxidation

**6.2. Free radicals** 

cancer and Parkinson's disease).

**6.3. Oxidative damage to lipids (Lipid peroxidation)** 

reactions that use oxygen, and it has been defined as a disturbance in the equilibrium status of pro-oxidant/anti-oxidant systems in intact cells. This definition of oxidative stress implies that cells have intact pro-oxidant/anti-oxidant systems that continuously generate and detoxify oxidants during normal aerobic metabolism. When additional oxidative events occur, the pro-oxidant systems outbalance the anti-oxidant, potentially producing oxidative damage to lipids, proteins, carbohydrates, and nucleic acids, ultimately leading to cell death in severe oxidative stress. Mild, chronic oxidative stress may alter the anti-oxidant systems by inducing or repressing proteins that participate in these systems, and by depleting cellular stores of anti-oxidant materials such as glutathione and vitamin E (Laval, 1996). Free radicals and other reactive species are thought to play an important role oxidative stress resulting in many human diseases. Establishing their precise role requires the ability to measure them and the oxidative damage that they cause (Halliwell and Whiteman, 2004).

A basic approach to study oxidative stress would be to measure some products such as (i) free radicals; (ii) radical-mediated damages on lipids, proteins or DNA molecules; and iii)

Free radicals are reactive compounds that are naturally produced in the human body. They can exert positive effects (e.g. on the immune system) or negative effects (e.g. lipids, proteins or DNA oxidation). Free radicals are normally present in the body in minute concentrations. Biochemical processes naturally lead to the formation of free radicals, and under normal circumstances the body can keep them in check. If there is excessive free radical formation, however, damage to cells and tissue can occur (Wilson, 1997). Free radicals are toxic molecules, may be derived from oxygen, which are persistently produced and incessantly attack and damage molecules within cells; most frequently, this damage is measured as peroxidized lipid products, protein carbonyl, and DNA breakage or fragmentation. Collectively, the process of free radical damage to molecules is referred to as oxidative stress (Reiter et al., 1997).To limit these harmful effects, an organism requires complex protection – the antioxidant system. This system consists of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase) and non-enzymatic antioxidants (e.g. vitamin E [tocopherol], vitamin A [retinol], vitamin C [ascorbic acid], glutathione and uric acid). An imbalance between free radical production and antioxidant defence leads to an oxidative stress state, which may be involved in aging processes and even in some pathology (e.g.

The peroxidation of lipids involves three distinct steps: initiation, propagation and termination. The initiation reaction between an unsaturated fatty acid and the hydroxyl radical involves the abstraction of a H atom from the methylvinyl group on the fatty acid. The remaining carbon-centred radical, forms a resonance structure sharing this unpaired electron among carbons 9 to 13. In the propagation reactions, this resonance structure reacts

antioxidant enzymatic activity or concentration.

Antioxidants are thought to protect the body against the destructive effects of free radicals. Antioxidants neutralize free radicals by donating one of their own electrons, ending the electron- gain reaction. The antioxidant nutrients themselves don't become free radicals by donating an electron because they are stable in either form. They act as scavengers, helping to prevent cell and tissue damage that could lead to cellular damage and disease (Reiter, 2003).

The body produces several enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSHPX), that neutralize many types of free radicals. Supplements of these enzymes are available for oral administration. However, their absorption is probably minimal at best. Supplementing with the "building blocks" the body requires to make SOD, catalase, and glutathione peroxidase may be more effective. These building block nutrients include the minerals manganese, zinc, and copper for SOD and selenium for GSHPX.

In addition to enzymes, many vitamins, minerals and hormones act as antioxidants in their own right, such as vitamin C, vitamin E, beta-carotene, lutein, lycopene, vitamin B2, coenzyme Q10, and cysteine (an amino acid). Herbs, such as bilberry, turmeric (curcumin),

grape seed or pine bark extracts, and ginkgo can also provide powerful antioxidant protection for the body. Melatonin is a hormone secreted by pineal gland and proves to be powerful antioxidant and free radical scavenger (Yang et al.,2002 and Koc et al., 2003).

#### **6.5. Nutritional therapy with natural antioxidants**

Antioxidants have been the focus of research on the relationship between The role of dietary factors in protecting against the change from native to oxidized LDL has received considerable attention. An overview of epidemiological research suggests that individuals with the highest intakes of antioxidant vitamins, whether through diet or supplements, tend to experience 20–40% lower risk of coronary heart disease (CHD) than those with the lowest intake or blood levels (diet and disease. Research examining the effects of a diet rich in fruits and vegetables on disease has been carried out using several types of study.

There is strong scientific evidence to support an increase in intakes of vegetables and fruit in the prevention of disease. Further research is required to clarify which particular components of fruit and vegetables are responsible for their protective effects.

Numerous epidemiological studies have indicated that diets rich in fruits and vegetables are correlated with a reduced risk of chronic diseases (Banerjee and Maulik, 2002 ; Sesso et al., 2003). It is probable that antioxidants, present in the fruits and vegetables such as polyphenols, carotenoids, and vitamin C, prevent damage from harmful reactive oxygen species, which either are continuously produced in the body during normal cellular functioning or are derived from exogenous sources (Gate et al. 1999). The possible protective effect of antioxidants in fruits and vegetables against ROS has led people to consume antioxidant supplements against chronic diseases.

## **6.6. Some experimental studies for the author about food factors as a nutritional antioxidants agents in alleviating the oxidative stress induced by environmental pollutents**


#### **Author details**

82 Lipid Peroxidation

**pollutents** 

2010)

grape seed or pine bark extracts, and ginkgo can also provide powerful antioxidant protection for the body. Melatonin is a hormone secreted by pineal gland and proves to be powerful antioxidant and free radical scavenger (Yang et al.,2002 and Koc et al., 2003).

Antioxidants have been the focus of research on the relationship between The role of dietary factors in protecting against the change from native to oxidized LDL has received considerable attention. An overview of epidemiological research suggests that individuals with the highest intakes of antioxidant vitamins, whether through diet or supplements, tend to experience 20–40% lower risk of coronary heart disease (CHD) than those with the lowest intake or blood levels (diet and disease. Research examining the effects of a diet rich in fruits

There is strong scientific evidence to support an increase in intakes of vegetables and fruit in the prevention of disease. Further research is required to clarify which particular

Numerous epidemiological studies have indicated that diets rich in fruits and vegetables are correlated with a reduced risk of chronic diseases (Banerjee and Maulik, 2002 ; Sesso et al., 2003). It is probable that antioxidants, present in the fruits and vegetables such as polyphenols, carotenoids, and vitamin C, prevent damage from harmful reactive oxygen species, which either are continuously produced in the body during normal cellular functioning or are derived from exogenous sources (Gate et al. 1999). The possible protective effect of antioxidants in fruits and vegetables against ROS has led people to consume

**6.6. Some experimental studies for the author about food factors as a nutritional antioxidants agents in alleviating the oxidative stress induced by environmental** 

1. In vivo evidence of hepato-and-reno-protective effect of garlic oil against sodium nitrite-induced oxidative stress Int. Int. J. Biol. Sci. (5)3: 249-255. (Hassan et al., 2009) 2. Mitigating effects of antioxidant properties of black berry juice on sodium fluoride induced hepatotoxicity and oxidative stress in rats. Food Chem.Toxicol., 47 2332–2337.

3. Evaluation of free radical-scavenging and antioxidant properties of black berry against fluoride toxicity in rats. Food Chem.Toxicol., 48: 1999-2004. (Hassan and Fattoh, 2010). 4. Garlic oil as a modulating agent for oxidative stress and neurotoxicity induced by sodium nitrite in male albino rats. Food Chem. Toxicol., 48: 1980-1985. (Hassan et al.,

5. Ameliorating effect of chicory (Cichorium intybus L)-supplemented diet against nitrosamine precursors-induced liver injury and oxidative stress in male rats. Food

Chem. Toxicol., 48: 2163-2169. (Hassan and Yousef, 2010).

and vegetables on disease has been carried out using several types of study.

components of fruit and vegetables are responsible for their protective effects.

**6.5. Nutritional therapy with natural antioxidants** 

antioxidant supplements against chronic diseases.

(Hassan and Yousef, 2010).

Hanaa Ali Hassan Mostafa Abd El-Aal

*Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt* 

#### **7. References**


women: results from the Women's Antioxidant Cardiovascular Study. Arch. Int. Med., 167 (15): 1610–1618.


Hassan, H. A and Yousef M. I. (2009): Mitigating effects of antioxidant properties of black berry juice on sodium fluoride induced hepatotoxicity and oxidative stress in rats. Food Chem.Toxicol., 47 2332–2337

84 Lipid Peroxidation

744

3328 – 3335 .

J. Nutr., 140: 1274-1279.

Pharmacol.., 142(2):231-255.

Chem. Toxicol., 48: 1980-1985.

2004.

167 (15): 1610–1618.

women: results from the Women's Antioxidant Cardiovascular Study. Arch. Int. Med.,

Coulter, I., Hardy, M., Morton, S., Hilton, L., Tu, W., Valentine, D. and Shekelle, P. (2006). Antioxidants vitamin C and vitamin e for the prevention and treatment of cancer. J. f general int. medici.: official j. Soc. Res. Educ. in Primary Care Int. Medici., 21 (7): 735–

Davies, AG., Griller, D., Ingold, KU., Lindsay, DA. and Walton, JC. (1981). An electron spin resonance study of pentadienyl and related radicals: homolytic fission of cyclobut-2-

Dherani, M. , Murthy, GV., Gupta, SK., Young, IS. and Maraini, G, Dherani, M, Murthy, GV, Gupta, SK, Young, IS, Maraini, G, Camparini, M, Price, GM, John, N, Chakravarthy, U, Fletcher, AE. (2008). Blood levels of vitamin C,carotenoids and retinol are inversely associated with cataract in a North Indian population .Invest. Ophthalmol. Vis. Sci., 49:

Dilis, V. and Trichopoulou, A. (2010). Antioxidant intakes and food sources in greek adults

Dudek, H., Farbiszewski, R., Michno, T., Łebkowski, W. J. and Kozłowski, A. (2002). Activity of glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R) and superoxide

Fearon, IM. and Faux, SP. (2009) . Oxidative stress and cardiovascular disease: novel tools

Gate L, Paul J, Ba GN, Tew KD and Tapiero H. (1999): Oxidative stress induced in

German, JB. (1999). Food processing and lipid oxidation". Advances in experimental

Greenwald, R. A. (1990). Superoxide dismutase and catalase as therapeutic agents for

Halliwell, B. and Whiteman, M. (2004): Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?. Br. J.

Hassan, H. A. (2005). The ameliorating effect of black grape juice on fluoride induced

Hassan, H. A ; El-Agmy Sh. M; Gaur, R. L; Fernando, A. Raj, M., HG and Ouhtit A. (2009): In vivo evidence of hepato-and-reno-protective effect of garlic oil against sodium nitrite-

Hassan, H. A and A. M. Fattoh (2010): Evaluation of free radical-scavenging and antioxidant properties of black berry against fluoride toxicity in rats. Food Chem.Toxicol.,48:1999-

Hassan H. A. Hafez H. S. and Zeghebar F. E. (2010): Garlic oil as a modulating agent for oxidative stress and neurotoxicity induced by sodium nitrite in male albino rats. Food

pathologies: the role of antioxidants. Biomed Pharmacother,53: 169 -180.

human diseases. A critical review. Free Radic. Boil. Med., 8(2): 210-219.

medicine and biology. Advanc. Experim. Medici. Biol., 459: 23–50.

testicular toxicity in adult albino rats. Egypt. J. Zool., 44:507–525.

induced oxidative stress Int. Int. J. Biol. Sci. (5)3: 249-255.

enylmethyl radicals. J. Chem. Soc. Perkin. Trans., II:633-641.

give (free)radical insight . J. Mol. Cell Cardiol., 47 : 372 – 381 .

dismutase in the brain tumors 55, 5-6: 252-256.


Metabolic and molecular bases of inherited diseases. New York: Mc Graw- Hill, 1461- 1477.


1477.

Academic Press, 1994.

(2):273–285.

1393.

Nutr. Exerc. Metab., 13 (2): 125–151.

young adults J. Am. Coll. Nutr., 28: 648-656.

importance of melatonin. Aging (Milano), 7: 340-351.

molecular biology of antioxidant defenses Pp 343-406.

organ damage in rats. J Pineal Res., 36(4):232-241.

Metabolic and molecular bases of inherited diseases. New York: Mc Graw- Hill, 1461-

Nagaoka, S., Okauchi, Y., Urano, S., Nagashima, U. and Mukai, K. (1990). Kinetic and ab initio study of the prooxidant effect of vitamin E: hydrogen abstraction from fatty acid

Nozik-Grayck, E., Suliman, H. and Piantadosi, C. (2005). Extracellular superoxide

Oja, S. S., Janaky, R., Varga, V. and Saranasaari, P. (2000): Modulation of glutamate receptor

Packer, L. (1994). Methods in Enzymology; Oxygen Radicals in Biological Systems, Part C,

Peake, J. (2003). Vitamin C: Effects of exercise and requirements with training. Int. J. Sport

Pellegrini, N. Serafini, M., Colombi, B., Del Rio, D., Salvatore, S., Bianchi, M. and Brighenti, F. (2003): Total antioxidant capacity of plant foods, beverages and oils consumed in italy

Preston, T.J., Muller. W.J. and Singh, G. (2001): Scavenging of extracellular H2O2 by catalase inhibits the proliferation of HER-2/Neu-transformed Rat-1 fibroblasts through the

Puchau, B., Ángeles, M., Zulet, de Echávarri, A. G., Hermsdorff, H. H. M., and Martínez, J. A., (2009). Dietary total antioxidant capacity: A novel indicator of diet quality in healthy

Reiter, R. J. (1995). Oxygen radical detoxification processes during aging: the functional

Reiter, R. J. (2003). Melatonin: clinical relevance. Best Pract. Res. Clinic. Endocr. Metabol., 17

Reiter, R. J.; Carneiro, R. C. and Oh, C. S. (1997): Melatonin in relation to cellular

Reiter, R. J.,Tan, D. X., Gitto, E., Sainz, R. M., Mayo, J.C., Leon, J., Manchester, L. C.,Vijayalaxm, Kilic, E. and Kilic, U. (2004). Pharmacological utility of melatonin in reducing oxidative cellular and molecular damage. Pol. J. Pharmacol.,56(2):159-170. Roberts, LJ., Oates, JA. and Linton, MF. (2007). The relationship between dose of vitamin E and suppression of oxidative stress in humans. Free Radic. Biol. Med., 43 (10): 1388–

Scandalios, J. G., Guan, l. and Polidoros, A. (1997): Catalase in plants.: Gene structure, properties, regulation and expression In J. G. Scandalios Ed., Oxidative stress and the

Sener, G.; Paskaloglu, K.; Toklu, H.; Kapucu, C.; Ayanoglu-Dulger, G.; Kacmaz, A. and Sakarcan, A. (2004): Melatonin ameliorates chronic renal failure-induced oxidative

esters and egg yolk lecithin. J. Am. Chem. Soc., 112:8921-8924.

assessed by three different in vitro assays. J. Nutr. 133:2812-2819.

induction of a stress response. J. Biological Chem., 276: 9558-9564.

antioxidiative defense mechanisms. Horm. Metab. Res., 29: 363-372.

dismutase. Int. J. Biochem. Cell Biol., 37 (12): 2466–2471.

functions by glutathione. Neurochem. Int., 37: 299-306.


Zelko, I., Mariani, T. and Folz, R. (2002). Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radic. Biol. Med., 33 (3): 337–349.

## **Iron Overload and Lipid Peroxidation in Biological Systems**

Paula M. González, Natacha E. Piloni and Susana Puntarulo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46181

### **1. Introduction**

88 Lipid Peroxidation

Zelko, I., Mariani, T. and Folz, R. (2002). Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene

structures, evolution, and expression. Free Radic. Biol. Med., 33 (3): 337–349.

Fe is an essential element for the growth and well-being of almost all living organisms, except for some strains of lactobacillus, where the role of Fe may be assumed by another metal [1]. It is involved in many biological functions since by varying the ligands to which it is coordinated, Fe has access to a wide range of redox potentials and can participate in many electron transfer reactions, spanning the standard redox potential range. It is also involved in O2 transport, activation, and detoxification, in N2 fixation and in several of the reactions of photosynthesis [2]. However, there are problems in the physiological management of Fe, since in spite of its overall abundance, usable Fe is in short supply because at physiological pH under oxidizing conditions, Fe is extremely insoluble. Anytime Fe exceeds the metabolic needs of the cell it may form a low molecular weight pool, referred to as the labile iron pool (LIP), which catalyzed the conversion of normal by-products of cell respiration, like superoxide anion (O2- ) and hydrogen peroxide (H2O2), into highly damaging hydroxyl radical (•OH) through the Fenton reaction (reaction 1) or by the Fe2+ catalyzed Haber-Weiss reaction (reaction 2), or into equally aggressive ferryl ions or oxygen-bridged Fe2+/Fe3+ complexes. Fe3+ can be reduced either by O2- (reaction 3) or by ascorbate leading to further radical production.

$$\mathrm{Fe}^{2+} + \mathrm{H}\_{2}\mathrm{O}\_{2} \xrightarrow[\text{(Fe)}]{} \mathrm{Fe}^{3+} + \mathrm{HO}^{-} + \bullet\mathrm{OH} \tag{1}$$

$$\rm O\_2^- + H\_2O\_2 \Rightarrow O\_2 + HO^- + \text{ }\bullet OH \tag{2}$$

$$\text{Fe}^{3+} + \text{O}\_2^{-} \Rightarrow \text{Fe}^{2+} + \text{O}\_2 \tag{3}$$

Defense against the toxic effect of Fe and O2 mixtures is provided by two specialized Febinding proteins: the extracellular transferrin (Tf) and the intracellular ferritin (Ft). Both

© 2012 González 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. © 2012 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.

retain Fe in the form of Fe3+ which unless mobilized will not be able to efficiently catalyze the production of free radicals. Fe is stored mainly intracellularly, where its potentially damaging effects are greatest.

The marine ecosystem can be seen as an integrative system with many factors that interact with the biota. Natural variables such as temperature, winds, precipitations, tide flows, currents, human activities, affect metal deposition into the sea. Once metals become bioavailable, they can enter the food web starting with the primary producers, and also in heterotrophic organisms at the bottom of the marine food chain, such as benthic filter feeders. Metals follow a bioaccumulation process inside the animals, depending on the animal's detoxification capacities and on exogenous Fe availability.

In plants, Fe concentrations increased during seed maturation, and by immunodetection experiments it was indicated that Ft concentration of seeds also increased with maturity, containing up to 1800 atoms of Fe per molecule [3]. This seed Fe could be stored for future use during seedling growth, as has been proposed by Hyde et al. [4], avoiding toxicity. Over growth, the oxidative stress depends upon a wide array of factors related to an enhanced radical production due to several metabolic pathways activated during the initial water uptake, including mitochondrial O2 consumption. On the other hand, excess Fe effects seem to be limited mostly to the hydrophobic domain of the cell following different profile than during physiological development.

In the last decade or so, important advances have been made in the knowledge of conditions that involve Fe-overload in humans. Those conditions would include short term processes, as organ or tissue ischemia-reperfusion and local inflammation, as well as progressive pathologies essentially affecting the central nervous system. In the first case, the decompartamentalization of Fe would lead to the expansion of the LIP and the increase of the oxidative damage. In the second case, it has been described an increase in Fe levels in the substantia nigra of Parkinsonian brains [5], Hallervorden-Spatz syndrome [6] and in mitochondria of Friedrich´s ataxia cerebella [7]. Hereditary hemochromatosis is a very common genetic defect in the Caucasian population, with an autosomal recessive inheritance. It is characterized by inappropriately increased Fe absorption from the duodena and upper intestine, with consequent deposition in various parenchymal organs, notably the liver, pancreas, heart, pituitary gland and skin [8]. Fe overload is characterized by the presence of several clinical manifestations such as: increased susceptibility to infections, hepatic dysfunction, tumors, joint diseases, myocardiopathy, and endocrine alterations. Fe overload has been also observed (a) if dietary Fe is excessive, such as in the severe Bantu siderosis, reported in the Bantu tribe of Africa who drink acidic beer out of Fe pots, (b) in other inherited diseases, such as congenital atransferrinemia (lacking circulating Tf), and (c) during the medical treatment of thalassemia. Moreover, clinical and epidemiologic observations indicated that increased Fe storage status is a risk factor in several diseases such as porphyria cutanea tarda and sudden infant death syndrome, among others.

Oxidative damage to lipids had been studied over several decades, and it had been characterized in terms of the nature of the oxidant, the type of lipid, and the severity of the oxidation. Many stable products are formed during the process and accordingly, the assays developed to assess these products to evaluate lipid peroxidation include many techniques. The most currently used assay is the determination of malondialdehyde (MDA) formation with the thiobarbituric acid reactive substances test (TBARS). However, electron paramagnetic resonance (EPR) spectroscopy has shown the capacity of detecting, in the presence of exogenous traps, the presence of the lipid radical formed during peroxidation, by yielding unique and stable products. EPR, also known as Electron Spin Resonance (ESR) is at present the only analytical approach that permits the direct detection of free radicals. This technique reports on the magnetic properties of unpaired electrons and their molecular environment [9].

This chapter will be dedicated to overview the Fe-related alterations in oxidative metabolism in photosynthetic and non-photosynthetic organisms after experimental exposure to excess Fe employing different protocols of administration. Data assessing lipid peroxidation post-treatment both, as TBARS generation and/or EPR detection of lipid radicals, are reviewed in a wide range of biological systems.

### **2. Fe overload in aquatic organisms**

90 Lipid Peroxidation

damaging effects are greatest.

during physiological development.

retain Fe in the form of Fe3+ which unless mobilized will not be able to efficiently catalyze the production of free radicals. Fe is stored mainly intracellularly, where its potentially

The marine ecosystem can be seen as an integrative system with many factors that interact with the biota. Natural variables such as temperature, winds, precipitations, tide flows, currents, human activities, affect metal deposition into the sea. Once metals become bioavailable, they can enter the food web starting with the primary producers, and also in heterotrophic organisms at the bottom of the marine food chain, such as benthic filter feeders. Metals follow a bioaccumulation process inside the animals, depending on the

In plants, Fe concentrations increased during seed maturation, and by immunodetection experiments it was indicated that Ft concentration of seeds also increased with maturity, containing up to 1800 atoms of Fe per molecule [3]. This seed Fe could be stored for future use during seedling growth, as has been proposed by Hyde et al. [4], avoiding toxicity. Over growth, the oxidative stress depends upon a wide array of factors related to an enhanced radical production due to several metabolic pathways activated during the initial water uptake, including mitochondrial O2 consumption. On the other hand, excess Fe effects seem to be limited mostly to the hydrophobic domain of the cell following different profile than

In the last decade or so, important advances have been made in the knowledge of conditions that involve Fe-overload in humans. Those conditions would include short term processes, as organ or tissue ischemia-reperfusion and local inflammation, as well as progressive pathologies essentially affecting the central nervous system. In the first case, the decompartamentalization of Fe would lead to the expansion of the LIP and the increase of the oxidative damage. In the second case, it has been described an increase in Fe levels in the substantia nigra of Parkinsonian brains [5], Hallervorden-Spatz syndrome [6] and in mitochondria of Friedrich´s ataxia cerebella [7]. Hereditary hemochromatosis is a very common genetic defect in the Caucasian population, with an autosomal recessive inheritance. It is characterized by inappropriately increased Fe absorption from the duodena and upper intestine, with consequent deposition in various parenchymal organs, notably the liver, pancreas, heart, pituitary gland and skin [8]. Fe overload is characterized by the presence of several clinical manifestations such as: increased susceptibility to infections, hepatic dysfunction, tumors, joint diseases, myocardiopathy, and endocrine alterations. Fe overload has been also observed (a) if dietary Fe is excessive, such as in the severe Bantu siderosis, reported in the Bantu tribe of Africa who drink acidic beer out of Fe pots, (b) in other inherited diseases, such as congenital atransferrinemia (lacking circulating Tf), and (c) during the medical treatment of thalassemia. Moreover, clinical and epidemiologic observations indicated that increased Fe storage status is a risk factor in several diseases

such as porphyria cutanea tarda and sudden infant death syndrome, among others.

Oxidative damage to lipids had been studied over several decades, and it had been characterized in terms of the nature of the oxidant, the type of lipid, and the severity of the

animal's detoxification capacities and on exogenous Fe availability.

Fe content in the upper earth's crust is around 6% [10]. The Fe concentration in sediments influences the Fe concentration in the associated surrounding seawater. However, the concentration of dissolved Fe (defined as Fe that can diffuse through a membrane of less than 0.45 μm) in open-oceanic waters is extremely low (< 56 ng/l) [11]. Natural parameters that augment the Fe levels in coastal and central oceanic areas are: aeolian deposition of dust, river discharge, washout of dust particles in the atmosphere by rainfall, ground water discharge, glacial melting, volcanic sediments, coastal erosion and up-welling of Fe-rich deep waters over hydrothermal vents [12]. Human activities also have a great impact on Fe levels, especially around coastal areas. Chemical and mining industries, disposal of waste metal, ports, aeolian deposition of atmospheric dust from polluted areas, are some of the human activities bringing Fe and other metals to the marine ecosystem. Therefore waters from different regions may have different Fe concentrations. Fe was recognized as a bioactive element [13] and a deficiency in Fe had been suggested to limit primary productivity in some ocean regions [14,15]. Fe uptake is strictly required for phytoplankton development since the photosynthetic apparatus contains numerous loci for Fe. Moreover, it was pointed out that it is critical to avoid Fe overload in water with low organic matter content under aquarium conditions to prevent Fe-dependent toxicity [16].

Over a decade ago, Estévez et al. [17] studied the effect of *in vivo* Fe supplementation to the green algae *Chlorella vulgaris* in terms of the establishment of oxidative stress conditions. Growth under laboratory conditions increased with Fe availability up to 90 M with increases in biomass, suggesting that Fe supply at concentrations lower than 90 M could be considered limiting for algal growth. However, Kolber et al. [18] pointed out that in their field experiments in the equatorial Pacific, 2 days following Fe enrichment, photosynthetic energy conversion efficiency began to decline. It was also indicated that some algal cultures showed deleterious effects if exceeding an Fe threshold (14-28 M) in unpolluted freshwater [16]. Between 90 and 200 M Fe in *C. vulgaris* cultures, there was no effect on growth with increased Fe additions and further increases on Fe availability led to a drastic decrease in the growth of the cultures (Table 1). The increase of Fe at the intracellular level showed a linear dependence with the concentration of added Fe below 200 M Fe, however concentrations between 200 and 500 M Fe added to the medium led to a less active increase in intracellular Fe (Table 1), suggesting an intracellular control for Fe uptake. Thus, the data presented by Estévez et al. [17] under laboratory conditions suggested the possibility that excess Fe could be responsible for the decrease in *C. vulgaris* growth by inducing oxidative stress. Accordingly, when *C. vulgaris* cells were incubated with an EPR-spin trapping for lipid radicals (-(4-pyridyl 1-oxide)-*N-t*-butyl nitrone, POBN), a POBN-spin adduct was observed. The spin adduct EPR spectra exhibit hyperfine splitting that were characteristic for POBN/lipid radicals, a*N* = 15.56 G and a*H* = 2.79 G, possibly generated from membrane lipids as a result of -scission of lipid-alkoxyl radicals [19,20]. Quantification of lipid radical EPR signals in algal cells indicated that Fe supplementation significantly increased radical content in the membranes supplemented with the higher Fe dose, as compared to cells supplemented with 90 M Fe (Table 1). These results indicate that lipid peroxidation was increased by Fe availability. In this context, even though an increased content of antioxidants has been detected in *C. vulgaris* cells exposed to increased Fe, the damaging potential of Fe excess in the cell did not seem to be efficiently controlled by the activity of the antioxidants [17].


1Taken from [17]. nd stands for not determinated.

*C. vulgaris* cultures were supplemented with up to 500 M Fe (EDTA:Fe, 2:1). Development was followed measuring chlorophyll content and each experimental value represents chlorophyll content of the cultures after 12 days of development. Intracellular Fe content as a function of the Fe addition to the incubation medium was spectrophotometrically measured. Data are expressed as means SE of 4-6 independent experiments, with two replicates in each experiment. Lipid radicals were detected and quantify by EPR. \*significantly different from value without Fe added, p 0.05. ANOVA.

\*\*significantly different from value in the presence of 90 M Fe added, p 0.05. ANOVA.

**Table 1.** Fe supplementation effect on *C. vulgaris* culture after 12 days of development1

It has been postulated that if as a result of ozone loss, UV-B flux at the surface of the earth increases, negative impacts on biological organisms will be inevitable since UV-B radiation causes a multitude of physiological and biochemical changes in photosynthetic organisms, probably related to oxidative stress [21,22]. Estévez el at. [17] exposed to 30 kJ/m2 UV-B *C.*  *vulgaris* cells grown at up to 500 M Fe. They observed that either 50 or 90 M Fe did not alter significantly cell morphology. However, 30 kJ/m2 UV-B exposure of algal cultures grown at 500 M Fe affected cellular internal structure and there were no signs of cellular division. Exposure of *C. vulgaris* cells to 30 kJ/m2 UV-B during lag phase did not significantly affect the content of lipid radicals in log phase of development under conditions of standard supplementation of Fe (90 M) (Figure 1). This parameter was significantly increased by the addition of 500 M Fe during development of the cultures in the absence of UV-B irradiation. Exposure of the cultures grown at 500 M Fe to 30 kJ/m2 UV-B during log phase led to a further increase in the content of lipid radicals in the membranes. In conclusion, even though exposure of *C. vulgaris* cells to UV-B under Fe standard concentration did not lead to cellular oxidative alterations, increase in Fe availability (500 M Fe) was responsible for a substantial increase in lipid deterioration in the membranes by oxidative stress. These data strongly suggest that oxidative stress triggered by an excess content of Fe could affect cellular growth and have a negative biogeoimpact to phytoplankton when exposed to other environmental conditions.

92 Lipid Peroxidation

the antioxidants [17].

Fe added (M)

1Taken from [17]. nd stands for not determinated.

Chlorophyll content (M)

Intracellular Fe (nmol (107 cell)-1)

0 1.2 4 1 nd 50 4.5\* 18 2\* nd 90 7.0\* 28 3\* 6 2 200 6.8\* 62 3\* nd 300 6.0\* 65 7\* nd 500 1.2 85 10\* 36 9\*\*

*C. vulgaris* cultures were supplemented with up to 500 M Fe (EDTA:Fe, 2:1). Development was followed measuring chlorophyll content and each experimental value represents chlorophyll content of the cultures after 12 days of

It has been postulated that if as a result of ozone loss, UV-B flux at the surface of the earth increases, negative impacts on biological organisms will be inevitable since UV-B radiation causes a multitude of physiological and biochemical changes in photosynthetic organisms, probably related to oxidative stress [21,22]. Estévez el at. [17] exposed to 30 kJ/m2 UV-B *C.* 

spectrophotometrically measured. Data are expressed as means SE of 4-6 independent experiments, with two

development. Intracellular Fe content as a function of the Fe addition to the incubation medium was

**Table 1.** Fe supplementation effect on *C. vulgaris* culture after 12 days of development1

replicates in each experiment. Lipid radicals were detected and quantify by EPR. \*significantly different from value without Fe added, p 0.05. ANOVA.

\*\*significantly different from value in the presence of 90 M Fe added, p 0.05. ANOVA.

Lipid radicals (pmol (107 cell)-1)

showed deleterious effects if exceeding an Fe threshold (14-28 M) in unpolluted freshwater [16]. Between 90 and 200 M Fe in *C. vulgaris* cultures, there was no effect on growth with increased Fe additions and further increases on Fe availability led to a drastic decrease in the growth of the cultures (Table 1). The increase of Fe at the intracellular level showed a linear dependence with the concentration of added Fe below 200 M Fe, however concentrations between 200 and 500 M Fe added to the medium led to a less active increase in intracellular Fe (Table 1), suggesting an intracellular control for Fe uptake. Thus, the data presented by Estévez et al. [17] under laboratory conditions suggested the possibility that excess Fe could be responsible for the decrease in *C. vulgaris* growth by inducing oxidative stress. Accordingly, when *C. vulgaris* cells were incubated with an EPR-spin trapping for lipid radicals (-(4-pyridyl 1-oxide)-*N-t*-butyl nitrone, POBN), a POBN-spin adduct was observed. The spin adduct EPR spectra exhibit hyperfine splitting that were characteristic for POBN/lipid radicals, a*N* = 15.56 G and a*H* = 2.79 G, possibly generated from membrane lipids as a result of -scission of lipid-alkoxyl radicals [19,20]. Quantification of lipid radical EPR signals in algal cells indicated that Fe supplementation significantly increased radical content in the membranes supplemented with the higher Fe dose, as compared to cells supplemented with 90 M Fe (Table 1). These results indicate that lipid peroxidation was increased by Fe availability. In this context, even though an increased content of antioxidants has been detected in *C. vulgaris* cells exposed to increased Fe, the damaging potential of Fe excess in the cell did not seem to be efficiently controlled by the activity of

Algal cultures (2×105 cells ml−1) were added with Fe starting day 0 of growth, and were assayed on day 12 (log phase). Data are expressed as means SE of 4-6 independent experiments, with 2 replicates in each experiment. \*significantly different from values for cells grown in the presence of 90 M Fe without exposure to UV-B, p 0.05. ANOVA.

\*\*significantly different from values for cells added with 500 M Fe without exposure to UV-B, p 0.05. ANOVA. (Statview SE+, v 1.03, Abacus Concepts Inc, Berkeley, CA).

Lipid radicals were detected and quantified by EPR and intracellular Fe by the use of acid solutions to digest the cells which were measured spectrophotometrically after reduction with thioglycolic acid followed by the addition of bathophenanthroline.

**Figure 1.** Effect of Fe addition on UV-B-dependent lipid radical (□) and intracellular Fe content ( ) in algae cells. Taken from [17].

Marine animals incorporate Fe bound to inorganic particles or to organic matter during food ingestion. Further, dissolved Fe is absorbed over the respiratory surfaces and mantle tissue in filter-feeding molluscs. The extrapallial water around these tissues is constantly exchanged with the surrounding seawater. Marine invertebrates are less tolerant of metal accumulation than vertebrates and can be affected at lower metal concentrations. Bivalves are widely used as sentinel organisms in marine pollution monitoring programs, due to their sessile and filtering habits, and their ability to bioaccumulate organic pollutants and metals in their tissues [23]. The exposure of marine molluscs to metals has been shown to induce oxidative stress through the formation of reactive O2 species (ROS) and reactive nitrogen species (RNS), leading to lipid peroxidation. Bivalves have also been used as models for the study of the effect of Fe supplementation. Viarengo et al. [24] treated the mussel *Mytilus galloprovincialis* with 300-600 g Fe/l (as FeCl3) and observed a significantly Fe accumulation in the digestive gland (DG) (190 25, 394 131 and 412 146 g Fe/l in 0, 300 and 600 g Fe/l supplemented animals, respectively). The TBARS content was measured in animals treated with 600 g Fe/l, and a significant increase was observed among control and treated mussels. Lately, Alves de Almeida et al. [25] exposed mussels from *Perna perna*  species to 500 g/l Fe (as FeSO4) and it was reported that mussels exposed to Fe for 12, 24 and 72 h presented increased phospholipid hydroperoxide glutathione peroxidase (PHGPx) activity, and no differences in MDA levels. However, at 120 h of Fe exposure both, MDA and PHGPx were significantly higher than control. Such increased MDA levels agree with previous findings by Viarengo et al. [24]. The negative correlation observed between PHGPx activity and MDA levels after Fe exposure, supports an interpretation that PHGPx protects tissues from lipid peroxidation. Thus, the exposure of mussels to Fe along with a concomitant increase in •OH formation would be involved in the modulation of PHGPx activity, however the precise mechanism remains unclear. Also, exposure of mussels to 500 g/l of Fe caused no changes in other antioxidant enzymes such as glutathione *S*-transferase and glutathione peroxidase. These data suggest that PHGPx have a role in the susceptibility of DG of mussels against lipid peroxidation, and that exposure to transition metals such as Fe could lead mussels to stimulate PHGPx in order to prevent lipid peroxidation. Thus, the authors postulated that the evaluation of MDA levels in parallel with antioxidant defenses, such as PHGPx, could be considered as a potential new biomarker of toxicity associated with contaminant exposure in marine organisms.

Recently, González et al. [26] investigated the oxidative effects produced by the *in vivo* Fe exposure of the bivalve *Mya arenaria.* The soft shell clams were collected on an intertidal sand flat near Bremerhaven, Germany, and the bivalves were placed in small aquaria containing 500 μM Fe (EDTA:Fe, 2:1). Exposure to 500 μM Fe in natural seawater resulted in a significant increase in DG total Fe content (Table 2). After 2 days of exposure to Fe, TBARS content showed a significant increase by approximately 3.8-fold as compared to control values. This increase was followed by a decrease to control values at treatment day 7 and afterwards TBARS concentration increased constantly until day 17 (Table 2). The LIP in DG tissue increased on day 7 of exposure to high dissolved Fe concentration. By day 9, the LIP increase was accompanied by a significant induction of the oxidative stress signals, ROS and ascorbyl radical content and correlated with the final increase of TBARS content in tissues. Once the LIP has increased, the catalytically active Fe is able to efficiently catalyze Fenton [27,28] and Haber-Weiss reactions [29,30] and consistently and drastically accelerated accumulation of TBARS. Contrary, oxidative stress effects measured on day 2 of treatment cannot be attributed to a significant increase of the LIP, since neither total Fe content nor the LIP were enhanced over the initial values in the 0 day exposure group. However, the H2O2 scavenging antioxidant, catalase (CAT), increased after 2 days of treatment compared to controls (day 0) but the activity went back to control level on day 7 of exposure. Catalase activity was, however, increased again on day 9 of exposure compared to controls [26]. It was postulated that the initial phase of elevated oxidative stress, occurring before significant Fe accumulation could be attributed to indirect effects under the experimental exposure conditions. Metabolic rates were not measured, but it is possible that Fe exposure triggers an initial stress response including accelerated respiration as the animals are pumping to rid themselves of the inflowing Fe enriched seawater. H2O2 is a good candidate for triggering cellular responses since it is a stable species [27]. H2O2 diffuses freely into the tissue and leads the oxidative stress, and further increases causes oxidative damage, assessed as TBARS content. H2O2 induced oxidative stress may have triggered the endogenous antioxidant system in such a manner that by day 7 of exposure to Fe excess the TBARS content was reduced to the starting values. Even though the superoxide dismutase (SOD) activity was not changed, induction of other protective mechanisms, such as metallothioneins, might act as effective transient control of heavy metal effects during the initial phase of exposure [24,25].


2Taken from [26].

94 Lipid Peroxidation

with contaminant exposure in marine organisms.

exchanged with the surrounding seawater. Marine invertebrates are less tolerant of metal accumulation than vertebrates and can be affected at lower metal concentrations. Bivalves are widely used as sentinel organisms in marine pollution monitoring programs, due to their sessile and filtering habits, and their ability to bioaccumulate organic pollutants and metals in their tissues [23]. The exposure of marine molluscs to metals has been shown to induce oxidative stress through the formation of reactive O2 species (ROS) and reactive nitrogen species (RNS), leading to lipid peroxidation. Bivalves have also been used as models for the study of the effect of Fe supplementation. Viarengo et al. [24] treated the mussel *Mytilus galloprovincialis* with 300-600 g Fe/l (as FeCl3) and observed a significantly Fe accumulation in the digestive gland (DG) (190 25, 394 131 and 412 146 g Fe/l in 0, 300 and 600 g Fe/l supplemented animals, respectively). The TBARS content was measured in animals treated with 600 g Fe/l, and a significant increase was observed among control and treated mussels. Lately, Alves de Almeida et al. [25] exposed mussels from *Perna perna*  species to 500 g/l Fe (as FeSO4) and it was reported that mussels exposed to Fe for 12, 24 and 72 h presented increased phospholipid hydroperoxide glutathione peroxidase (PHGPx) activity, and no differences in MDA levels. However, at 120 h of Fe exposure both, MDA and PHGPx were significantly higher than control. Such increased MDA levels agree with previous findings by Viarengo et al. [24]. The negative correlation observed between PHGPx activity and MDA levels after Fe exposure, supports an interpretation that PHGPx protects tissues from lipid peroxidation. Thus, the exposure of mussels to Fe along with a concomitant increase in •OH formation would be involved in the modulation of PHGPx activity, however the precise mechanism remains unclear. Also, exposure of mussels to 500 g/l of Fe caused no changes in other antioxidant enzymes such as glutathione *S*-transferase and glutathione peroxidase. These data suggest that PHGPx have a role in the susceptibility of DG of mussels against lipid peroxidation, and that exposure to transition metals such as Fe could lead mussels to stimulate PHGPx in order to prevent lipid peroxidation. Thus, the authors postulated that the evaluation of MDA levels in parallel with antioxidant defenses, such as PHGPx, could be considered as a potential new biomarker of toxicity associated

Recently, González et al. [26] investigated the oxidative effects produced by the *in vivo* Fe exposure of the bivalve *Mya arenaria.* The soft shell clams were collected on an intertidal sand flat near Bremerhaven, Germany, and the bivalves were placed in small aquaria containing 500 μM Fe (EDTA:Fe, 2:1). Exposure to 500 μM Fe in natural seawater resulted in a significant increase in DG total Fe content (Table 2). After 2 days of exposure to Fe, TBARS content showed a significant increase by approximately 3.8-fold as compared to control values. This increase was followed by a decrease to control values at treatment day 7 and afterwards TBARS concentration increased constantly until day 17 (Table 2). The LIP in DG tissue increased on day 7 of exposure to high dissolved Fe concentration. By day 9, the LIP increase was accompanied by a significant induction of the oxidative stress signals, ROS and ascorbyl radical content and correlated with the final increase of TBARS content in tissues. Once the LIP has increased, the catalytically active Fe is able to efficiently catalyze Fenton [27,28] and Haber-Weiss reactions [29,30] and consistently and drastically accelerated

\*significantly different from the value at day 0 with p < 0.05,

\*\*p < 0.01 and

\*\*\*p < 0.001. ANOVA.

Experimental bivalves were placed in small aquaria containing 13 l (1 l/animal) of natural seawater of 23-26‰ at 10°C, and 500 μM Fe (Fe:EDTA, 1:2).

**Table 2.** Fe supplementation effect on lipid peroxidation in *Mya arenaria*<sup>2</sup>

Other studies evaluate the impact of nutritional Fe on Fe level and concentrations of MDA in tissues. Baker et al. [31] analyzed the Fe in the diet of the African catfish, *Clurims gariepinus*. This fish model is of particular relevance when considering that *C. gariqinus* is typically cultured in earth-ponds, and these may be high in dissolved Fe content. Additionally, catfish may consume mud-burrowing organisms to supplement their diet, with incidental associated silt consumption, and therefore further metal loading. After 5 weeks of feeding the animals with a diet supplemented with Fe (6354.4 mg Fe/kg), the total Fe content was measured in muscle, liver and blood-plasma and no significant differences with control animals were found, suggesting the possibility of efficient regulation of Fe status by the fish. MDA determination in tissues revealed that there was significantly more MDA in livers and hearts of fish fed high Fe diets than in controls, and no significant difference was found in skeletal-muscle. Values of MDA concentration were higher in Festressed liver tissue comparative to other tissues, possible because hepatic tissue is lipid-rich making the liver a target organ for lipid peroxidation. The relative lack of response in skeletal muscle may have resulted from decreased abundance of polyunsaturated fatty acids within this tissue, and these findings are consistent with those of Desjardins et al. [32].

All together these data show that Fe in aquatic ecosystems could be a major stressor having a main role in lipid peroxidation not only in unicellular species, such as algae, but also in higher organisms, such as invertebrates and vertebrates. These kind on analyses should be performed before consider ecological strategies which may involved Fe fertilization in seawater [33-35], to increase primary production in the oceans as an answer to global temperature increments. These actions may drastically modify marine communities in ocean layers triggering oxidative reactions, which should be properly considered due to the fact that Fe may be profitable or unfavorable, depending of its usefulness as a micronutrient or as a catalyzer of free radical reactions.

#### **3. Fe overload in soybean seeds**

Plants have developed several mechanisms to maintain fairly constant internal concentrations of mineral nutrients over a wide range of external concentrations. To avoid Fe-dependent oxidative cellular damage, Fe2+ is either incorporated into the mineral core of Ft [36] which is located exclusively in the plastids [37] or reoxidized by O2 and chelated by organic acids [38]. Bienfait et al. [39] reported that plants grown on Fe-EDTA formed a substantial pool of free space Fe in the roots and that Fe could be mobilized upon Fe-free growth in order to be transferred to the leaves. During growth in water culture at pH 5 to 6, a free space pool of 500 to 1000 nmol/g FW was formed in roots of bean grown in the presence of Fe-EDTA 20 M and a pool of 20 to 50 nmol/g FW in roots without Fe supplementation. Like Ft in the cell, the free space Fe3+ precipitate is not only an immobile result of a defensive action against an excessive Fe supply; the plant may also use it as storage form of Fe that can be mobilized [39]. Even more, Caro and Puntarulo [40] indicated that O2 radical generation depends on total Fe content, however it could mostly reflect Fe content in the free space. In soybean, Fe3+ reduction is an obligatory step in Fe uptake, and this is probably true for all strategy I plants [41]. Both total Fe content and the *in vitro* rate of Fe reduction were higher in roots grown in the presence of exogenously added Fe (up to 500 M) than in roots grown in absence of supplemented Fe (Table 3). However, no visual differences (e.g. evidence of damage) between any of the roots or growth (assessed as the fresh weight of the roots, 0.21 ± 0.01 g/root) have been observed at the studied range of Fe supplementation. Total Fe content in soybean roots exposed to 50 and 500 M Fe-EDTA, was higher than in roots grown in absence of supplemented Fe (Table 3) and lipid oxidation, assessed as the content of TBARS, were not significantly affected by Fe supplementation up to 500 M, to the incubation medium (Table 3). However, Fe supplementation to the roots did affect -tocopherol content that was significantly decreased in the homogenates and the microsomes isolated from roots supplemented with Fe, as compared with values in roots developed in absence of Fe [40]. These data suggest that *in vivo* Fe supplementation could increase O2 radical generation in soybean roots that was adequately control.


\*significantly different from values without Fe addition, p 0.05. ANOVA.

96 Lipid Peroxidation

as a catalyzer of free radical reactions.

**3. Fe overload in soybean seeds** 

Fe content was measured in muscle, liver and blood-plasma and no significant differences with control animals were found, suggesting the possibility of efficient regulation of Fe status by the fish. MDA determination in tissues revealed that there was significantly more MDA in livers and hearts of fish fed high Fe diets than in controls, and no significant difference was found in skeletal-muscle. Values of MDA concentration were higher in Festressed liver tissue comparative to other tissues, possible because hepatic tissue is lipid-rich making the liver a target organ for lipid peroxidation. The relative lack of response in skeletal muscle may have resulted from decreased abundance of polyunsaturated fatty acids within this tissue, and these findings are consistent with those of Desjardins et al. [32].

All together these data show that Fe in aquatic ecosystems could be a major stressor having a main role in lipid peroxidation not only in unicellular species, such as algae, but also in higher organisms, such as invertebrates and vertebrates. These kind on analyses should be performed before consider ecological strategies which may involved Fe fertilization in seawater [33-35], to increase primary production in the oceans as an answer to global temperature increments. These actions may drastically modify marine communities in ocean layers triggering oxidative reactions, which should be properly considered due to the fact that Fe may be profitable or unfavorable, depending of its usefulness as a micronutrient or

Plants have developed several mechanisms to maintain fairly constant internal concentrations of mineral nutrients over a wide range of external concentrations. To avoid Fe-dependent oxidative cellular damage, Fe2+ is either incorporated into the mineral core of Ft [36] which is located exclusively in the plastids [37] or reoxidized by O2 and chelated by organic acids [38]. Bienfait et al. [39] reported that plants grown on Fe-EDTA formed a substantial pool of free space Fe in the roots and that Fe could be mobilized upon Fe-free growth in order to be transferred to the leaves. During growth in water culture at pH 5 to 6, a free space pool of 500 to 1000 nmol/g FW was formed in roots of bean grown in the presence of Fe-EDTA 20 M and a pool of 20 to 50 nmol/g FW in roots without Fe supplementation. Like Ft in the cell, the free space Fe3+ precipitate is not only an immobile result of a defensive action against an excessive Fe supply; the plant may also use it as storage form of Fe that can be mobilized [39]. Even more, Caro and Puntarulo [40] indicated that O2 radical generation depends on total Fe content, however it could mostly reflect Fe content in the free space. In soybean, Fe3+ reduction is an obligatory step in Fe uptake, and this is probably true for all strategy I plants [41]. Both total Fe content and the *in vitro* rate of Fe reduction were higher in roots grown in the presence of exogenously added Fe (up to 500 M) than in roots grown in absence of supplemented Fe (Table 3). However, no visual differences (e.g. evidence of damage) between any of the roots or growth (assessed as the fresh weight of the roots, 0.21 ± 0.01 g/root) have been observed at the studied range of Fe supplementation. Total Fe content in soybean roots exposed to 50 and 500 M Fe-EDTA, was higher than in roots grown in absence of supplemented Fe

**Table 3.** Fe supplementation effects in soybean after 24 h of incubation

Robello et al. [42] reported that total Fe content in soybean embryonic axes exposed to 500 uM Fe-EDTA was higher than in axes grown in absence of supplemented Fe after 24 h of incubation. However, neither Fe reduction rate nor growth assessed, either as the fresh weight or the dry weight of the embryonic axes, were significantly affected by Fe supplementation to the incubation medium. Membrane integrity was no affected by the supplementation with 50 and 500 M Fe:EDTA (1:2) since electrolyte leakage at 24 h and 48 h of imbibition was not significantly different from electrolyte leakage found in nonsupplemented Fe axes (15.3 0.7 and 8.0 0.3%, after 24 h and 48 h of incubation with 500 M Fe, as compared to 12.4 0.4 and 8.6 0.6%, after 24 h and 48 h of incubation in the absence of added Fe, respectively). Moreover, as it was previously reported in soybean roots [43], Fe accumulation was not followed by Ft accumulation in soybean embryonic axes upon growth. Without any significant change in the content of Ft in the embryonic axes incubated for 24 h upon Fe supplementation, a 53% decrease in the Fe content per molecule of Ft was observed in the presence of 500 M Fe (Table 3). These data differed from previous observations showing Fe induction of Ft synthesis and accumulation in soybean [44], however, the nature of the model employed by Lescure et al. [44], cells in suspension grown heterotropically, could alter the kinetic of the response. In this regard, it should not be discarded that a transient increase in Ft content could occur under these experimental conditions before 24 h of imbibition. The observed rapid decrease in Fe content per molecule of Ft, as compared to non-added Fe conditions, could reflect an early loosing of Ft molecules altered by free radicals, or a reduction of its capacity of binding Fe, or both. The increase in the protein sensitivity to proteases would lead to an early degradation, as compared to axes grown in a non-added Fe medium. The increased rate of ROS generation could be due to the significant increase in the LIP under conditions of Fe supplementation. However, it is important to point out that the substantial increase in the total Fe content in axes grown in the presence of 500 M Fe for 24 h, as compared to seeds grown in non-added Fe medium, could not be allocated as the measured increase in the LIP that would represent only the 10% of the increase in the total Fe content. Besides the LIP critical importance as initiator of free radical reactions and the decisive requirement of keeping Fe concentration as low as possible to minimize cellular deterioration, the role of other soluble and insoluble Fe-storage proteins, the formation and contribution of Fenitrosyl complexes, glutathione, nitric oxide, etc. should be considered among other nonprotein agents, as possible candidates to handle Fe transport and storage under stress conditions since TBARS content was not significantly affected in Fe overloaded soybean embryonic axes (Table 3). Beside the apoplastic space [45], Lanquar et al. [46] identified the vacuole as a major compartment for Fe storage in plant seeds and showed that retrieval of the Fe stored in vacuoles is an essential step for successful germination in a wide range of environments.

On the other hand, recently Simontacchi et al. [47] summarized assays performed to characterize lipid radical-dependent oxidation in photosynthetic organisms where EPR was successfully employed to evaluate not only lipidperoxidation but also to analyze the relative scavenging capacity of plant extracts, the effects of both, natural environmental challenges and oxidative stress situations, in several model and biological systems. Further studies should be oriented in this direction to explore the critical effect of Fe overload on radicaldependent pathways that play a major role in plant metabolism.

#### **4. Fe overload in mammals**

Fe overload in mammals has been often associated with injury, fibrosis, and cirrhosis in the liver followed by cardiac disease, endocrine abnormalities, arthropathy, osteoporosis and skin pigmentation [48]. Several mechanisms has been proposed whereby excess hepatic Fe causes cellular injury, but Fe-induced peroxidative injury to phospholipids of organelle membranes is a potential unifying mechanisms underlying the major theories of cellular injury in Fe overload [49]. With progressively increasing Fe deposition, the capacity to maintain Fe in storage forms is exceeded resulting in a transient increase in the hepatic LIP [50]. Moreover, Fe-catalyzed generation of ROS has been implicated in the pathogenesis of many disorders including atherosclerosis [51,52], cancer [53], ischaemia reperfusion injury [54,55] besides in Fe overload [56], such as haemochromatosis [57].

98 Lipid Peroxidation

wide range of environments.

**4. Fe overload in mammals** 

roots [43], Fe accumulation was not followed by Ft accumulation in soybean embryonic axes upon growth. Without any significant change in the content of Ft in the embryonic axes incubated for 24 h upon Fe supplementation, a 53% decrease in the Fe content per molecule of Ft was observed in the presence of 500 M Fe (Table 3). These data differed from previous observations showing Fe induction of Ft synthesis and accumulation in soybean [44], however, the nature of the model employed by Lescure et al. [44], cells in suspension grown heterotropically, could alter the kinetic of the response. In this regard, it should not be discarded that a transient increase in Ft content could occur under these experimental conditions before 24 h of imbibition. The observed rapid decrease in Fe content per molecule of Ft, as compared to non-added Fe conditions, could reflect an early loosing of Ft molecules altered by free radicals, or a reduction of its capacity of binding Fe, or both. The increase in the protein sensitivity to proteases would lead to an early degradation, as compared to axes grown in a non-added Fe medium. The increased rate of ROS generation could be due to the significant increase in the LIP under conditions of Fe supplementation. However, it is important to point out that the substantial increase in the total Fe content in axes grown in the presence of 500 M Fe for 24 h, as compared to seeds grown in non-added Fe medium, could not be allocated as the measured increase in the LIP that would represent only the 10% of the increase in the total Fe content. Besides the LIP critical importance as initiator of free radical reactions and the decisive requirement of keeping Fe concentration as low as possible to minimize cellular deterioration, the role of other soluble and insoluble Fe-storage proteins, the formation and contribution of Fenitrosyl complexes, glutathione, nitric oxide, etc. should be considered among other nonprotein agents, as possible candidates to handle Fe transport and storage under stress conditions since TBARS content was not significantly affected in Fe overloaded soybean embryonic axes (Table 3). Beside the apoplastic space [45], Lanquar et al. [46] identified the vacuole as a major compartment for Fe storage in plant seeds and showed that retrieval of the Fe stored in vacuoles is an essential step for successful germination in a

On the other hand, recently Simontacchi et al. [47] summarized assays performed to characterize lipid radical-dependent oxidation in photosynthetic organisms where EPR was successfully employed to evaluate not only lipidperoxidation but also to analyze the relative scavenging capacity of plant extracts, the effects of both, natural environmental challenges and oxidative stress situations, in several model and biological systems. Further studies should be oriented in this direction to explore the critical effect of Fe overload on radical-

Fe overload in mammals has been often associated with injury, fibrosis, and cirrhosis in the liver followed by cardiac disease, endocrine abnormalities, arthropathy, osteoporosis and skin pigmentation [48]. Several mechanisms has been proposed whereby excess hepatic Fe causes cellular injury, but Fe-induced peroxidative injury to phospholipids of organelle membranes is a potential unifying mechanisms underlying the major theories of cellular

dependent pathways that play a major role in plant metabolism.

Several experimental models of Fe overload have been developed. In the dietary model used by Dabbagh et al. [58] rats were fed for 10 weeks a chow diet enriched with 3% (w/w) reduced pentacarbonyl Fe (a 99%, w/w, pure form of elemental Fe). Dietary Fe overload resulted in significant increases in hepatic Fe levels; with no difference in Fe content in serum (Table 4). Lipid peroxidation was assessed by measuring TBARS and F2-isoprostanes. The latter are a series of prostaglandin-F2-like compounds derived from the free-radicalcatalyzed, non-enzymic peroxidation of arachidonic acid [59] and the *in vivo* levels of F2 isoprostanes have been shown to increase dramatically in acute hepatotoxicity [60]. Direct evidence for moderately increased lipid peroxidation products in liver was reported after dietary Fe overload. In addition to hepatic oxidative damage, Fe overload also caused changes in the plasma lipid profile. These data suggest that in this rat model of Fe overload, oxidative stress is associated with depletion of endogenous antioxidants in plasma and liver, and although no conclusive evidence for lipid peroxidation in plasma was found, hepatic F2-isoprostane levels were significantly increased in treated rats.

Experimental Fe overload in rats using dietary supplementation with carbonyl Fe is a well established model, where Fe deposition results mainly in the hepatocytes in a periportal distribution, as observed in idiopathic hemochromatosis [48]. Galleano and Puntarulo [61] used the dietary carbonyl-Fe model carried out on male Wistar rats that were fed during 6 weeks with either a) control chow diet, or b) control chow diet supplemented with 2.5% (w/w) carbonyl-Fe. Both, Fe and TBARS content, were increased in liver (Table 4). However, mild dietary Fe overload increased Fe content in plasma but did not lead to a significant increase in TBARS probably because Fe content after dietary Fe supplementation was increased less dramatically in plasma than in liver (88% and 15-fold, respectively), suggesting that plasma mechanisms for sequestering catalytically active Fe were fully operative (Table 4). Under these conditions, TBARS content in plasma does not seem to be a good indicator of oxidative stress conditions in the liver, and more sensitive techniques should be used in plasma to assess Fe-dependent oxidative stress.

Cockell et al. [62] used sucrose-based modified AIN-93G diets formulated to differ in Fe (35 mg/kg and 1500 mg/kg for control and Fe overloaded diets). Weanling male Long-Evans rats were fed these diets for 4 weeks and killed. Fe content was measured in plasma and liver. No differences in plasma between control and treated groups were found, meanwhile a significantly increase in liver between control and treated groups was observed. Since TBARS content in livers was significantly increased in Fe overloaded animals, hepatic Fe concentrations in this study were correlated positively with increases in TBARS. However, Fischer et al. [63] showed that Fe overloaded diets did not significantly alter other oxidative stress indices, such as DNA double-strand breaks or NF-κB activation despite observed increases in hepatic lipid peroxidation.


Letters indicate the units for each parameter as follows: (a) g Fe/g FW; (b) nmol/mg prot; (c) g/dl; (d) nmol/ml; (e) g Fe/g DW; (f) pmol/min/mg prot; (g) g Fe/dl; (h) nmol/l; (i) mg/l.

\*significantly different from control values p < 0.01,

\*\*p < 0.001, ANOVA.

nd stands for not-detectable.

**Table 4.** Fe effects in different organs and plasma employing several models of Fe overload

Fe-dextran treatment seems as a good model for the study of Fe toxicity resembling the pathological and clinical consequences of acute Fe overload in humans [48]. Fe supplied as Fe-dextran, is initially taken up by Kupffer cells, and when their storage capacity is exceeded the metal is accumulated by parenchymal cells producing a mild Fe overload. The increased Fe content alters the Kupffer cell functional status by inducing a progressive increase in macrophage-dependent respiration at earlier times after treatment. The effect is sensitive to macrophage inactivation by GdCl3 pretreatment, decreases the respiratory response of the Kupffer cell to particle stimulation, plays a role in the development of liver injury, and seems to condition the impairment of hepatic respiration observed at later times after Fe overload [64]. Other pathological situations that increase oxidative conditions in the cell, could enhance Fe-dependent damage. As an example, hyperthyrodism increases the susceptibility of the liver to the toxic effects of Fe, which seems to be related to the development of a severe oxidative stress status in the tissue, thus contributing to the concomitant liver injury and impairment of Kupffer cell phagocytosis and particle-induced respiratory burst activity [65]. It was also shown that acute Fe overload was responsible for oxidative stress in rat testes with a concurrent decrease of antioxidant content [66,67]. The oxidative stress has been developed using Fe-dextran intra peritoneal (ip) administration as 500 mg/kg body weight and killed after 20 h.

Spontaneous organ chemiluminescence (CL) reflects the rate of lipid peroxidation reactions through the detection of the steady-state level of excited species and is considered to be an useful technique to evaluate oxidative stress *in vivo*. Galleano and Puntarulo [68] reported an association between Fe content and light emission in rats exposed to Fe-dextran after 2-6 h. Presumably, with progressively increasing Fe deposition, the capacity of maintaining Fe in storage forms is exceeded resulting in a transient increase in the hepatic LIP. However, at longer times (20 h) the significant increase in cytosolic Fe is limited, and CL goes back to control values. Moreover, cytochrome P450 inactivation is an early event and precedes other enzyme inactivation [68]. Data included in Table 4 show that liver Fe content was increased by 7-fold after 8 h of Fe-dextran administration, and TBARS generation rate was enhanced by 3-fold (6 h after ip) suggesting that liver is deeply affected by acute Fe-overload.

100 Lipid Peroxidation

**Pentacarbonyl Fe, diet 3% (w/w)**

**Carbonyl Fe, diet 2.5% (w/w)**

**Fe-dextran, ip 500 mg/kg**

\*\*p < 0.001, ANOVA. nd stands for not-detectable.

**Sucrose-basemodified AIN-93G, diet 1500 mg/kg**

Fe/g DW; (f) pmol/min/mg prot; (g) g Fe/dl; (h) nmol/l; (i) mg/l.

\*significantly different from control values p < 0.01,

500 mg/kg body weight and killed after 20 h.

Fe content TBARS

control Fe-overload control Fe-overload

Liver [61] 69 16(a) 1091 178\*(a) 0.45 0.05(b) 0.58 0.01\*(b) Plasma [61] 179 43(c) 336 57\*(c) 0.6 0.1(d) 0.6 0.2(d)

Liver[62] 218 46(e) 895 376\*\*(e) 0.54 0.07(b) 0.78 0.19\*\*(b)

Liver [68] 257 11(e) 1837 205\*(e) 40 1(f) 110 30\*(f) Plasma [70] 126 20(g) 1538 158\*(g) 0.7 0.1(h) 2.7 0.1\*(h) Kidney [49] 14 3(e) 113 15\*(e) 29 2(f) 37 3\*(f) Letters indicate the units for each parameter as follows: (a) g Fe/g FW; (b) nmol/mg prot; (c) g/dl; (d) nmol/ml; (e) g

Plasma [62] 2.72 1.74(i) 3.82 1.21(i) - -

**Table 4.** Fe effects in different organs and plasma employing several models of Fe overload

Fe-dextran treatment seems as a good model for the study of Fe toxicity resembling the pathological and clinical consequences of acute Fe overload in humans [48]. Fe supplied as Fe-dextran, is initially taken up by Kupffer cells, and when their storage capacity is exceeded the metal is accumulated by parenchymal cells producing a mild Fe overload. The increased Fe content alters the Kupffer cell functional status by inducing a progressive increase in macrophage-dependent respiration at earlier times after treatment. The effect is sensitive to macrophage inactivation by GdCl3 pretreatment, decreases the respiratory response of the Kupffer cell to particle stimulation, plays a role in the development of liver injury, and seems to condition the impairment of hepatic respiration observed at later times after Fe overload [64]. Other pathological situations that increase oxidative conditions in the cell, could enhance Fe-dependent damage. As an example, hyperthyrodism increases the susceptibility of the liver to the toxic effects of Fe, which seems to be related to the development of a severe oxidative stress status in the tissue, thus contributing to the concomitant liver injury and impairment of Kupffer cell phagocytosis and particle-induced respiratory burst activity [65]. It was also shown that acute Fe overload was responsible for oxidative stress in rat testes with a concurrent decrease of antioxidant content [66,67]. The oxidative stress has been developed using Fe-dextran intra peritoneal (ip) administration as

Spontaneous organ chemiluminescence (CL) reflects the rate of lipid peroxidation reactions through the detection of the steady-state level of excited species and is considered to be an

Liver [58] 104 15(a) 1391 242\*(a) - - Plasma [58] 134 55(c) 124 46(c) nd nd

> Mammalian red blood cells are particularly susceptible to oxidative damage because (i) being an O2 carrier, they are exposed uninterruptedly to high O2 tension, (ii) they have no capacity to repair their damaged components, and (iii) the haemoglobin is susceptible to autoxidation and their membrane components to lipid peroxidation. Red blood cells, however, are protected by a variety of antioxidant systems which are capable of preventing most of the adverse effects of oxidative stress, under normal conditions [69]. Galleano and Puntarulo [70] reported, employing the ip Fe-dextran model of Fe overload, that 20 h after Fe-dextran injection Fe concentration in plasma of treated rats showed approximately 12 fold increase, and TBARS content in plasma showed a 285% increase as compared to control values (Table 4). On the other hand, *in vitro* studies showed that Fe can stimulate the peroxidation of erythrocytes membrane lipids. Since red blood cells from Fe overloaded rats are continuously being exposed to an increase Fe content, no differences in TBARS content were detected in red blood cells from control rats as compared to erythrocytes from Fe overloaded rats, suggesting high resistance to oxidative stress of these cells.

> Galleano et al. [71] also employed this model to comparatively studying Fe overload in kidney. Fe content in whole kidney was 8-fold increased (Table 4), and 5-fold increased in kidney mitochondria (16 ± 5 to 78 ± 1 nmol/mg prot for control and treated animals, respectively). Even thought TBARS content showed no significant differences after Fe administration, in Fe-treated rats TBARS production rate by kidney homogenates was higher in treated animals than in kidneys from control rats (Table 4). The authors suggested that Fe-dextran treatment does not affect kidney integrity, even though increases in lipid peroxidation rate occurs. α-tocopherol, one of the most efficient antioxidant in the hydrophobic phase, appeared to be effective in controlling Fe-dextran dependent damage in kidney.

> Brain tissue is thought to be very sensitive to oxidative stress. Neurons are enriched in mitochondria and possess a very high aerobic metabolism, which makes these tissues susceptible to ROS-dependent damage than other organs. Moreover, low levels of some antioxidant enzymes, high contents of polyunsaturated fatty acids in brain membranes, and high Fe content may combine their effects to make the brain a preferential target for oxidative stress-related degeneration [72]. Maaroufi et al. [73] developed a chronic Fe overload model consisting in a daily 3 mg Fe/kg administrated in adult rats during 5 days. These treatments resulted, 16 days after treatment, in a significant Fe accumulation in the

hippocampus, cerebellum, and basal ganglia. Lately, Maaroufi et al. [74] studied rats which received daily one ip injection of 3 mg FeSO4/kg dissolved in sodium chloride 0.9% (or vehicle) during 21 consecutive days, and this accumulation was correlated to behavioral deficits. No increase levels of the TBARS content in different brain structures were observed in any brain region investigated. This observation suggested that chronic Fe administration had induced adaptive responses involving stimulation of the antioxidant defenses since, both SOD and CAT activities, were increased after treatment.

Thus, different forms and quantities of Fe administrated to rats, supplemented either as diets or ip, lead to an increase in Fe content in several tissues and plasma. This Fe increase seems to be associated with an increase in lipid peroxidation. The underlying mechanisms of tissue damage are unclear, but they probably depend on the Fe administration protocol. Even though lipid damage was observed in many cases after Fe overload, antioxidant capacity seems to play a crucial role in controlling the impairment mechanisms.

## **5. Concluding remarks**

Fe metabolism is very complex since Fe is both, an essential element and a toxic compound that has to be carefully kept under a regulated concentration in a living cell. Toxic Fe activity is due to its ability of catalyzing free radical reactions. The most efficient Fe fraction to act as a free radical promoter is that forming the LIP. LIP content is the resultant of multiple dynamic equilibrium between the Fe incorporated to the cell, utilized and intracellularly stored. We have briefly reviewed the role of Fe on the oxidative damage to lipid membranes employing both *in vitro* and *in vivo* models of Fe overload in several biological systems. Much progress has still to be made in order to understand the nature and function of the LIP, the mechanisms of the Fe-catalyzed reactions *in vivo*, the contribution of Fe to oxidative stress and disease, and the development of appropriate chemotherapeutic strategies. Thus, alterations in Fe metabolism should be carefully analyzed before evaluating cellular responses to either damaging agents or xenobiotics of biomedical or ecological impact since Fe is a double-faced element that can be either good or bad to the cell, depending on whether it serves as a micronutrient or as a catalyst of free radical reactions.

Since a tight metabolic organization is required to successfully face oxidative external conditions in invertebrates, anthropogenic contamination with Fe could be toxic for animals that are adapted to their natural environment. As it could be understood from the data presented here, it is strongly suggested that natural habitats should be strictly preserved even though absolute Fe content did not seem to reach critical values to avoid cellular deterioration.

Mobilization of Fe stored in plant seeds is an essential step for germination in a wide range of environments. The analysis of these aspects would provide information that could be the key to understand Fe nutrition in plants, and will allow the designing and engineering of crop plants requiring minimal fertilizer input, contributing to a more ecological agricultural practice under optimal and sub-optimal environmental conditions avoiding reaching Fe overload conditions that would jeopardize successful plant development.

Moreover, therapeutic strategies should be designed to chelate either Fe from the LIP or Fe loosely bound to Ft to avoid Fe-related oxidative damage. Focus in chemical-related aspects of the Fe-chelator complexes should help to fulfill the new drugs designing expectances to control Fe toxicity in humans that through promoting lipid peroxidation could severely affect human health.

## **Author details**

102 Lipid Peroxidation

**5. Concluding remarks** 

deterioration.

hippocampus, cerebellum, and basal ganglia. Lately, Maaroufi et al. [74] studied rats which received daily one ip injection of 3 mg FeSO4/kg dissolved in sodium chloride 0.9% (or vehicle) during 21 consecutive days, and this accumulation was correlated to behavioral deficits. No increase levels of the TBARS content in different brain structures were observed in any brain region investigated. This observation suggested that chronic Fe administration had induced adaptive responses involving stimulation of the antioxidant defenses since,

Thus, different forms and quantities of Fe administrated to rats, supplemented either as diets or ip, lead to an increase in Fe content in several tissues and plasma. This Fe increase seems to be associated with an increase in lipid peroxidation. The underlying mechanisms of tissue damage are unclear, but they probably depend on the Fe administration protocol. Even though lipid damage was observed in many cases after Fe overload, antioxidant

Fe metabolism is very complex since Fe is both, an essential element and a toxic compound that has to be carefully kept under a regulated concentration in a living cell. Toxic Fe activity is due to its ability of catalyzing free radical reactions. The most efficient Fe fraction to act as a free radical promoter is that forming the LIP. LIP content is the resultant of multiple dynamic equilibrium between the Fe incorporated to the cell, utilized and intracellularly stored. We have briefly reviewed the role of Fe on the oxidative damage to lipid membranes employing both *in vitro* and *in vivo* models of Fe overload in several biological systems. Much progress has still to be made in order to understand the nature and function of the LIP, the mechanisms of the Fe-catalyzed reactions *in vivo*, the contribution of Fe to oxidative stress and disease, and the development of appropriate chemotherapeutic strategies. Thus, alterations in Fe metabolism should be carefully analyzed before evaluating cellular responses to either damaging agents or xenobiotics of biomedical or ecological impact since Fe is a double-faced element that can be either good or bad to the cell, depending on

Since a tight metabolic organization is required to successfully face oxidative external conditions in invertebrates, anthropogenic contamination with Fe could be toxic for animals that are adapted to their natural environment. As it could be understood from the data presented here, it is strongly suggested that natural habitats should be strictly preserved even though absolute Fe content did not seem to reach critical values to avoid cellular

Mobilization of Fe stored in plant seeds is an essential step for germination in a wide range of environments. The analysis of these aspects would provide information that could be the key to understand Fe nutrition in plants, and will allow the designing and engineering of crop plants requiring minimal fertilizer input, contributing to a more ecological agricultural practice under optimal and sub-optimal environmental conditions avoiding reaching Fe

capacity seems to play a crucial role in controlling the impairment mechanisms.

whether it serves as a micronutrient or as a catalyst of free radical reactions.

overload conditions that would jeopardize successful plant development.

both SOD and CAT activities, were increased after treatment.

Paula M. González, Natacha E. Piloni and Susana Puntarulo\* *Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires, Junín, Buenos Aires, Argentina* 

## **Acknowledgement**

This study was supported by grants from the University of Buenos Aires and CONICET. S.P. is career investigator from CONICET, and P.M.G. and N.P. are fellows from CONICET.

## **6. References**


<sup>\*</sup> Corresponding Author


[27] Boveris A (1998) Biochemistry of free radicals: from electrons to tissues. Medicina 54: 350-356.

104 Lipid Peroxidation

[11] Rue EL, Bruland KW (1995) Complexation of iron(III) by natural organic ligands in the Central North Pacific as determined by a new competitive ligand equilibration/adsorptive

[12] Watson AJ (2001) Iron limitation in the oceans. In: Turner DR, Hunter KA, editors. The

[13] Bruland, KW, Donat JR, Hutchins DA (1991) Interactive influences of bioactive metals

[14] Martin JH, Fitzwater SE, Gordon RM (1990) Iron deficiency limits phytoplankton

[15] Martin JH, Fitzwater SE, Gordon RM, Hunter CN, Tanner SJ (1993) Iron, primary productivity and carbon nitrogen flux studies during the JGOFS North Atlantic Bloom

[16] Brand LE, Sunda WG, Guillard RRL (1983) Limitation of phytoplankton reproductive

[17] Estévez MS, Malanga G, Puntarulo S (2001) Iron-dependent oxidative stress in *Chlorella* 

[18] Kolber ZS, Barber R, Coale KH, Fitzwater SE, Greene RM, Johnson KS, Lindley S, Falkowski PG (1994) Iron limitation of phytoplankton photosynthesis in the equatorial

[19] North JA, Specto AA, Buettner GR (1992) Detection of lipid radicals by electron paramagnetic resonance spin trapping using intact cells enriched with polyunsaturared

[20] Jurkiewicz BA, Buettner GR (1994) Ultraviolet light-induced free radical formation in skin: an electron paramagnetic resonance study. Photochem. Photobiol. 59: 1-4. [21] Malanga G, Puntarulo S (1995) Oxidative stress and antioxidant content in *Chlorella* 

[22] Kozak RG, Malanga G, Caro A, Puntarulo S (1997) Ascorbate free radical content in photosynthetic organisms after exposure to ultraviolet-B. Recent Res. Devel. Plant

[23] Goldberg ED (1975) The mussel watch: a first step in global marine monitoring. Mar.

[24] Viarengo A, Burlando B, Cavaletto M, Marchi B, Ponzano E, Blasco J (1999) Role of metallothionein agaist oxidative stress in the mussel *Mytilus galloprovincialis*. Am. J.

[25] Alves de Almeida E, Dias Bainy AC, de Melo Loureiro AP, Martinez GR, Miyamoto S, Onuki J, Barbosa LF, Machado Garcia CC, Manso Prado F, Ronsein GE, Sigolo CA, Barbosa Brochini C, Gracioso Martins AM, Gennari de Medeirosa MH, Di Mascio P (2007) Oxidative stress in *Perna perna* and other bivalves as indicators of environmental stress in the Brazilian marine environment: Antioxidants, lipid peroxidation and DNA

[26] González PM, Abele D, Puntarulo S (2010) Exposure to excess of iron in vivo affects oxidative status in the bivalve *Mya arenaria*. Comp. Biochem. Physiol. C 152: 167-174.

*vulgaris* after exposure to ultraviolet-B radiation. Plant Physiol. 94: 672-679.

biogeochemistry of iron in seawater. New York: Wiley and Sons. pp 85-121.

on biological production in oceanic waters. Limnol. Oceanogr. 36: 1555-1577.

cathodic stripping voltammetric method. Mar Cehm. 50: 117-138.

growth in Antarctic waters. Global Biogeochem. Cycles 4: 5-12.

rates by zinc, manganese and iron. Limnol. Oceanogr. 28: 1182-1198.

Experiment. Deep-Sea Res. 40: 115-134.

*vulgaris*. Plant Sci. 161: 9-17.

Physiol. 1: 233-239.

Poll. Bull. 6: 111-132.

Pacific Ocean. Nature 371: 145-149.

fatty acids. J. Biol. Chem. 267: 5743-5746.

Physiol. Regul. Integr. Comp. Physiol. 277: 1612-1619.

damage. Comp. Biochem. Physiol. 146(4)A: 588-600.


[58] Dabbagh AJ, Mannion T, Lynch SM, Frei B (1994) The effect of iron overload on rat plasma and liver oxidant status in vivo. Biochem. J. 300: 799-803.

106 Lipid Peroxidation

193.

312.

93: 125-134.

1890-1894.

Radic. Biol. Med. 1: 1-17.

Natl. Acad. Sci. U.S.A. 88: 8440-8444.

mediated process? Hepatology 11: 127-137.

eastern Finnish men. Circulation 86: 803-811.

59(5): 1011-1012.

EMBO J. 24: 4041-4051.

Network. pp 141-160.

[41] Schaedle M, Basshamb JA (1977) Chloroplast Glutathione Reductase. Plant Physiol.

[42] Robello E, Galatro A, Puntarulo S (2007) Iron role in oxidative metabolism of soybean

[43] Lobréaux S, Briat JF (1991) Ferritin accumulation and degradation in different organs of

[44] Lescure AM, Massenet O, Briat JF (1990) Purification and characterization of an iron induced ferritin from soybean (*Glycine max*) cell suspensions. Biochem. J. 272: 147-150. [45] Briat JF, Lobréaux S (1997) Iron transport and storage in plants. Trends Plant Sci. 2: 187-

[46] Lanquar V, Lelièvre F, Bolte S, Hamès C, Alcon C, Neumann D, Vansuyt G, Curie C, Schröder A, Krämer U, Barbie-Brygoo H, Thomine S (2005) Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron.

[47] Simontacchi M, Buet A, Puntarulo S (2011) The use of electron paramagnetic resonance (EPR) in the study of oxidative damage to lipids in plants. In: Catalá A editor. Lipid Peroxidation: Biological Implications. Kerala, India: Res. Signpost Transworld Res.

[48] Puntarulo S (2005) Iron, oxidative stress and human health. Molec. Asp. Med. 26: 299-

[49] Galleano M, Puntarulo S (1994) Mild iron overload effect on rat liver nuclei. Toxicology

[50] Galleano M, Simontacchi M, Puntarulo S (2004) Nitric oxide and iron. Effect of iron overload on nitric oxide production in endotoxemia. Molec. Asp. Med. 25: 141-154. [51] Heinecke JW, Rosen H, Chait A (1984) Iron and copper promote modification of low density lipoprotein by human arterial smooth muscle cells in culture. J. Clin. Invest. 74:

[52] Salonen JT, Nyyssonen K, Korpela H, Tuomilehto J, Seppanen R and Salonen R (1992) High stored iron levels are associated with excess risk of myocardial infarction in

[53] Loeb LA, James EA, Waltersdorph AM, Klebanoff SJ (1988) Mutagenesis by the autoxidation of iron with isolated DNA. Proc. Natl. Acad. Sci. U.S.A. 85: 3918-3922. [54] Aust SD, White BC (1985) Iron chelation prevents tissue injury following ischemia. Free

[55] Katoh S, Toyama J, Kodama I, Akita T, Abe T (1992) Deferoxamine, an iron chelator, reduces myocardial injury and free radical generation in isolated neonatal rabbit hearts

[57] Bacon BR, Britton RS (1990) The pathology of hepatic iron overload: a free radical

subjected to global ischaemia-reperfusion. J Mol Cell Cardiol. 24(11): 1267-1275. [56] Burkitt M J and Mason R P (1991) Direct evidence for in vivo hydroxyl-radical generation in experimental iron overload: an ESR spin-trapping investigation. Proc.

axes upon growth. Effect of iron overload. Plant Sci. 172: 939-947.

pea (*Pisum sativum*) during development. Biochem. J. 274: 601-606.


**Evaluation of Lipid Peroxidation Processes** 

108 Lipid Peroxidation

Neurochem. 97(6): 1634-1658.

Behav. 96(2): 343-349.

186: 39-47.

[72] Halliwell B (2006) Oxidative stress and neurodegeneration: where are we now? J.

[73] Maaroufi K, Ammari M, Jeljeli M, Roy V, Sakly M, Abdelmelek H (2009) Impairment of emotional behavior and spatial learning in adult Wistar rats by ferrous sulfate. Physiol.

[74] Maaroufi K, Save E, Poucet Sakly B, Abdelmelek H, Had-Aissouni L (2011) Oxidativestress and prevention of the adaptive response to chronic iron overload in the brain of young adult rats exposed to a 150 kilohertz electromagnetic field. Neuroscience

[75] Caro A, Puntarulo S (1995) Effect of iron-stress on antioxidant content of soybean

embryonic axes. Plant Physiol. (Life Sci. Adv.) 14: 131-136.

### **Chapter 5**

## **Trends in the Evaluation of Lipid Peroxidation Processes**

Mihaela Ilie and Denisa Margină

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46075

## **1. Introduction**

Oxidative stress occurs as a result of imbalance between the antioxidant and prooxidant systems acting at certain points in metabolic processes, in favor of the last. The oxidative stress, defined by H Sies following extensive research performed between 1981 and 1993, is the outcome of intense generation of reactive oxygen species (ROS), which are not counteracted by endogenous antioxidant molecules [Sies, 1985]. Current knowledge link**s** many types of pathologies to oxidative damage; among them, most cited are atherosclerosis, diabetes mellitus, neurodegenerative disorders, cancers, rheumatic diseases, autoimmune disorders, etc. Figure 1, sometimes referred to as "oxidative stress wheel", presents the most important diseases in which oxidative stress is involved resulting in biochemical lessions

Free radicals are chemical species containing unpaired electrons, which can increase the reactivity of atoms or molecules. Free radicals are highly reactive and unstable, due to their impaired electrons; they can react locally, accepting or donating electrons, in order to become more stable. The reaction between a radical and a non-radical compound generally leads to the propagation of the radical chain reaction, and to an increasing generation of new free radicals. During biochemical processes that normally take place in living cells, many types of free radicals are generated: oxygen-, sulfur-, bromide- and chloride- centered species [Halliwell & Gutteridge, 2007]. The most common reported cellular free radicals are singlet oxygen (1Σg+O2), hydroxyl (OH·), superoxide (O2–·) and nitric monoxide (NO·). Also, some other molecules like hydrogen peroxide (H2O2) and peroxynitrite (ONOO–) (which are not free radicals from the chemical point of view, having all-paired electrons) are reported to generate free radicals in living organisms through various chemical reactions [Halliwell, 2006].

In this context, it is extremely important to evaluate the extent and rate of the lipid peroxidation process using different methods and experimental models, ranging from

© 2012 Ilie and Margină, 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. © 2012 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.

quantitative assay of lipoperoxides end products to the evaluation of changes in certain metabolic processes under the influence of pro-oxidative or antioxidative known substances. The present article aims at reviewing different techniques, methods and experimental models for the evaluation of lipid peroxidation that can be used in clinical research and in basic biochemical research as well. Simple, rapid, cost effective, and more elaborated, expensive methods are critically evaluated, presenting the advantages and limitations of each one. A special emphasis is given to fluorescent methods, which our team is frequently using to evaluate the lipid peroxidation processes.

**Figure 1.** Implication of oxidative stress in pathology ("oxidative stress wheel")

## **2. Oxidative stress, ROS and implication in metabolic procesess**

#### **2.1. Oxygen centered reactive species (ROS)**

Reactive oxigen species is a generic term that includes both oxygen radicals and certain nonradicals that are oxidizing agents and/or are easily converted into radicals, such as H2O2, ozone (O3), singlet oxygen (Δg1O2), peroxynitrite, hypochlorous acid (HOCl), etc. [Halliwell, 2006].

ROS are generated as a result of oxygen action on nutrients or on physiological components in living organisms.

The sources for oxidative stress are either endogenous (abnormal mithocondria and peroxisomes function, lipoxygenase, NADPH-oxidase, cytochrome P450 activity), endogenous antioxidant systems dysfunction (low amount of non-enzymatic antioxidants such as gluthatione, vitamins A, C and E, reduced enzimatic activity) or exogenous agents (ultraviolet or ionizing radiation, toxins, chemotherapy, bacteria, etc.) (Figure 2).

**Figure 2.** Sources, effects and main markers of oxidative stress

112 Lipid Peroxidation

quantitative assay of lipoperoxides end products to the evaluation of changes in certain metabolic processes under the influence of pro-oxidative or antioxidative known substances. The present article aims at reviewing different techniques, methods and experimental models for the evaluation of lipid peroxidation that can be used in clinical research and in basic biochemical research as well. Simple, rapid, cost effective, and more elaborated, expensive methods are critically evaluated, presenting the advantages and limitations of each one. A special emphasis is given to fluorescent methods, which our team is frequently

using to evaluate the lipid peroxidation processes.

**Figure 1.** Implication of oxidative stress in pathology ("oxidative stress wheel")

**2.1. Oxygen centered reactive species (ROS)** 

2006].

in living organisms.

**2. Oxidative stress, ROS and implication in metabolic procesess** 

Reactive oxigen species is a generic term that includes both oxygen radicals and certain nonradicals that are oxidizing agents and/or are easily converted into radicals, such as H2O2, ozone (O3), singlet oxygen (Δg1O2), peroxynitrite, hypochlorous acid (HOCl), etc. [Halliwell,

ROS are generated as a result of oxygen action on nutrients or on physiological components

ROS can induce many damaging cellular processes, such as DNA oxidative lesions, loss of membrane integrity due to lipid peroxidation, protein and functional carbohydrate structural changes, etc. All these structural and functional changes have direct clinical consequences, leading to the acceleration of the general aging process, but also to some pathological phenomena, associated with the increase of the capillary permeability, impairment of the blood cell function, etc. ROS lesions are frequently associated with aging [Dröge & Schipper, 2007; Griffiths et al., 2011], atheroclerosis [Hulsmans & Holvoet, 2010], cardio-vascular disease [Dikalov & Nazarewicz, 2012; Puddu et al., 2009], type I or type II diabetes mellitus [Cai et al., 2004], autoimmune disorders, neurodegenerative disorders such as Parkinson [Yoritaka et al., 1996] or Alzheimer's disease [Sayre et al., 1997; Takeda et al., 2000], inflammatory diseases such as reumatoid arthritis [Griffiths et al., 2011] or different types of cancers [Lenaz, 2012; Li et al., 2009; Manda et al., 2009].

In order to counteract the damaging action of the physiologically generated ROS, the living organisms developed efficient antioxidant systems [Christofidou-Solomidou & Muzykantov 2006; Halliwell, 2006; Sies, 1997; Veskoukis et al., 2012]. Endogenous antioxidants in the human body act through different types of mechanisms:


All these antioxidant systems act differently, depending on their structure and properties, their hydrophilic or lipophilic character, and also depending on their localization (intracellular or extracellular, in cell or organelles membrane, in the cytoplasm, etc.). All the aforementioned systems act sinergically and form a network which protects living cells from the destructive action of ROS (Figure 3).

**Figure 3.** ROS neutralization by several biomolecules

#### **2.2. Nitrogen centered radical species (RNS)**

After the discovery of the physiological role of endogenously produced nitric oxide (NO), the capacity of this bio-molecule to react with other cellular components (such as proteins and lipids), specific nitrosative chemical changes have emerged as a key signaling mechanism in cell physiology. Several studies reported the involvement of excess generation of NO and its adducts in the etiology of multiple disease states, including insulin resistance and diabetes, atherosclerosis or Alzheimer's disease [Duplain et al., 2008; Parastatidis et al., 2007; Uehara, 2007; Yasukawa T et al., 2005; White et al., 2010].

The nitrosative modifications of proteins take two main forms: either S-nitrosylation of cysteine thiols or nitration of tyrosine residues. Both chemical processes may arise from protein interactions with NO or with secondary intermediates of NO, otherwise termed reactive nitrogen species (RNS) [White et al., 2010]. One of the very important members of the RNS group is represented by peroxynitrite (ONOO- ), produced from the reaction of NO with the superoxide anion (O2-), which is considered as one of the major cellular nitrating agents [Hogg, 2002; White et al., 2010]. Other nitrating agents are the nitrosonium cation (derived from the action of myeloperoxidase), produced from the reaction of nitrite with hydrogen peroxide and nitroso-peroxocarbonate, which results from the reaction of carbon dioxide with peroxynitrite. Lipid peroxyl radicals have been recently shown to promote tyrosine nitration by inducing tyrosine oxidation and also by reacting with NO2 to produce · NO2 [Bartesaghi et al., 2010; Denicola et al., 1996; Lang et al., 2000].

#### **2.3. Oxidative stress and lipid peroxidation**

114 Lipid Peroxidation

[Gordon, 2012; Halliwell, 2006]

peroxidases, lipases, etc. [Sies, 1997].

the destructive action of ROS (Figure 3).

**Figure 3.** ROS neutralization by several biomolecules

**2.2. Nitrogen centered radical species (RNS)** 

2006; Sies, 1997]





All these antioxidant systems act differently, depending on their structure and properties, their hydrophilic or lipophilic character, and also depending on their localization (intracellular or extracellular, in cell or organelles membrane, in the cytoplasm, etc.). All the aforementioned systems act sinergically and form a network which protects living cells from

After the discovery of the physiological role of endogenously produced nitric oxide (NO), the capacity of this bio-molecule to react with other cellular components (such as proteins and lipids), specific nitrosative chemical changes have emerged as a key signaling mechanism in cell physiology. Several studies reported the involvement of excess generation of NO and its adducts in the etiology of multiple disease states, including insulin resistance and diabetes, atherosclerosis or Alzheimer's disease [Duplain et al., 2008;

Parastatidis et al., 2007; Uehara, 2007; Yasukawa T et al., 2005; White et al., 2010].

Freinbichler, 2011; Halliwell & Gutteridge, 1984; Velayutham, 2011]

Among the targets of ROS and RNS, lipids are basically the most vulnerable, as their peroxidation products can result in further propagation of free radical reactions [Halliwell & Chirico, 1992]. The brain is a high oxygen-consuming organ and the nervous cell has also the greatest lipid-to-protein ratio; besides, the brain has a relatively week protection systems against ROS generation, therefore it is particularly vulnerable to oxidative stress. The agerelated increase in oxidative brain damage results in intense generation of lipid peroxidation products, protein oxidation, oxidative modifications in nuclear and mitochondrial DNA [Grimsrud et al., 2008].

Polyunsaturated fatty acids (PUFAs) and their metabolites have many physiological roles such as energy generation, direct involvement in cellular and sub-cellular membrane structure and function, implication in cell signaling processes and in the regulation of gene expression as well. They constitute the main target of ROS action in the lipid peroxidation reactions.

The general process of lipid peroxidation consists of three stages: initiation, propagation, and termination [Auroma et al., 1989; Leopold & Loscalzo, 2009]. Many species can be responsible for the initiation of the chain reaction the radicals: hydroxyl, alkoxyl, peroxyl, superoxide or peroxynitrite. As a consequence, a free radical atracts a proton from a carbon of a fatty acyl side chain leaving the remaining carbon radical accessible to molecular oxygen to form a lipid peroxyl radical. This is also highly reactive and the chain reaction is propagated further. As a result, PUFA molecules are transformed into conjugated dienes, peroxy radicals and hydroperoxides, which will undergo a cleavage mainly to aldehydes. More than 20 lipoperoxidation end-products were identified [Niki, 2009]; among the components of PUFAs oxidative degradation products, the most frequently mentioned were acrolein, malondialdehyde (MDA), 4-hydroxyalkenals and isoprostanes [Esterbauer et al., 1991; Leopold & Loscalzo, 2009].

RNS also play a major role in lipid biology, by two major pathways: targeting some enzymes (COX-2 and cytochrome P-450) and thus influencing bioactive lipid synthesis and interacting with unsaturated fatty acids (such as oleate, linoleate and arachidonate) and generating novel

nitro-fatty acids. Nitrated lipids, such as nitroalkenes, may undergo aqueous decay and release independent of thiols, isomerize to a nitrite ester with N-O bond cleavage, or generate an enol group and ·NO [Leopold & Loscalzo, 2009]. The nitro-fatty acids have distinct bioactivities from their precursor lipids [Baker et al., 2009; Freeman et al., 2008; Kim et al., 2005; Lee et al., 2008; White et al., 2010]. Studies identified a high number of nitro-fatty acid species and proved an elevated formation of RNS in hydrophobic environments, such as the lipid bilayer, suggesting that lipids might constitute candidates for nitrosative signal transduction [Jain et al., 2008; Moller et al., 2005; Moller et al., 2007; Thomas et al., 2001].

Nitroalkenes may also participate in reactions with cysteine and histidine residues in proteins and with the thiolate anion of glutathione (GSH) to initiate reversible modification(s) of proteins. Thiyl radicals may also initiate lipid peroxidation by extraction of a hydrogen atom from bis-allylic methylene groups of fatty acids generating pentadienyl radicals. These radicals, in turn, may react with oxygen to generate peroxyl radicals [Leopold & Loscalzo, 2009].

Lipid hydroperoxides (LOOHs) are intermediates of PUFAs lipid peroxidation and can be also found as minor constituents of cell membranes; these compounds are also final products of prostaglandin and leukotriene biosynthesis, and can be decomposed by transition metals to form alkoxyl and lipoperoxyl radicals. Furthermore, biomolecules such as proteins or amino-lipids, can be covalently modified by lipid decomposition products (i.e. by forming Schiff bases with aldehydes or/and by activating membrane-bound enzymes). In consequence, lipid peroxidation may alter the arrangement of proteins in bilayers and thereby interfere with their physiological role in the membrane function.

#### **2.4. Pathological involvement of the lipid peroxidation process**

Many lipid peroxidation products, either full chain or chain-shortened, have been reported to be harmful or to have pro-infl ammatory effects [Birukov 2006; Niki 2009; Salomon 2005].

Lipid peroxidation increases the permeability of cellular membranes, resulting in cell death.

The lipid peroxidation process located at the cell membrane level may lead to loss of integrity and viability, and also to altered cell signaling and finally to tissue dysfunction; the oxidation of plasma lipoproteins is probably a major contributor to the formation of lipid peroxidation products and is widely thought to be involved in atherosclerosis. Malondialdehyde and 4 hydroxy-2-nonenal (HNE), the well known products of lipid peroxidation, react with a variety of biomolecules, such as proteins, lipids and nucleic acids, and they are thought to contribute to the pathogenesis of human chronic diseases [Breusing et al., 2010].

A clear example of the pathological role of PUFAs peroxidation is the evolution of atherosclerotic lesions to cardio-vascular disease. Until 1970, it was considered that dyslipidemia was the main factor initiating the atherosclerotic lesions. Later on, researchers emphasized the involvement of inflammatory processes, growth factors, smooth muscle cells proliferation, as well as viruses, bacteria or tumor phenomenon in the atherosclerosis, besides lipids and lipoproteins.

RNS interaction with different types of proteins is directly involved in physio-pathological processes. Several studies proved that key enzymes involved in glycolysis, β-oxidation, the tricarboxylic acid cycle and electron transport chain are targets of tyrosine nitration or *S*nitrosylation. Modifications by nitrosylation that reduce the activity or function of important tricarboxylic acid cycle and electron transport proteins have the potential to slow substrate oxidation and probably lead to the build up of metabolic intermediates (particularly lipids) that could impair signaling pathways to reduce insulin action. Also, key insulin-signaling intermediaries are *S-*nitrosylated, and this could constitute a potential mechanism of insulin resistance [Chouchani et al., 2010].

#### **3. Evaluation of the end products of lipid peroxidation**

116 Lipid Peroxidation

2009].

nitro-fatty acids. Nitrated lipids, such as nitroalkenes, may undergo aqueous decay and release independent of thiols, isomerize to a nitrite ester with N-O bond cleavage, or generate an enol group and ·NO [Leopold & Loscalzo, 2009]. The nitro-fatty acids have distinct bioactivities from their precursor lipids [Baker et al., 2009; Freeman et al., 2008; Kim et al., 2005; Lee et al., 2008; White et al., 2010]. Studies identified a high number of nitro-fatty acid species and proved an elevated formation of RNS in hydrophobic environments, such as the lipid bilayer, suggesting that lipids might constitute candidates for nitrosative signal transduction [Jain et al., 2008; Moller et al., 2005; Moller et al., 2007; Thomas et al., 2001].

Nitroalkenes may also participate in reactions with cysteine and histidine residues in proteins and with the thiolate anion of glutathione (GSH) to initiate reversible modification(s) of proteins. Thiyl radicals may also initiate lipid peroxidation by extraction of a hydrogen atom from bis-allylic methylene groups of fatty acids generating pentadienyl radicals. These radicals, in turn, may react with oxygen to generate peroxyl radicals [Leopold & Loscalzo,

Lipid hydroperoxides (LOOHs) are intermediates of PUFAs lipid peroxidation and can be also found as minor constituents of cell membranes; these compounds are also final products of prostaglandin and leukotriene biosynthesis, and can be decomposed by transition metals to form alkoxyl and lipoperoxyl radicals. Furthermore, biomolecules such as proteins or amino-lipids, can be covalently modified by lipid decomposition products (i.e. by forming Schiff bases with aldehydes or/and by activating membrane-bound enzymes). In consequence, lipid peroxidation may alter the arrangement of proteins in bilayers and

Many lipid peroxidation products, either full chain or chain-shortened, have been reported to be harmful or to have pro-infl ammatory effects [Birukov 2006; Niki 2009; Salomon 2005]. Lipid peroxidation increases the permeability of cellular membranes, resulting in cell death. The lipid peroxidation process located at the cell membrane level may lead to loss of integrity and viability, and also to altered cell signaling and finally to tissue dysfunction; the oxidation of plasma lipoproteins is probably a major contributor to the formation of lipid peroxidation products and is widely thought to be involved in atherosclerosis. Malondialdehyde and 4 hydroxy-2-nonenal (HNE), the well known products of lipid peroxidation, react with a variety of biomolecules, such as proteins, lipids and nucleic acids, and they are thought to

A clear example of the pathological role of PUFAs peroxidation is the evolution of atherosclerotic lesions to cardio-vascular disease. Until 1970, it was considered that dyslipidemia was the main factor initiating the atherosclerotic lesions. Later on, researchers emphasized the involvement of inflammatory processes, growth factors, smooth muscle cells proliferation, as well as viruses, bacteria or tumor phenomenon in the atherosclerosis,

thereby interfere with their physiological role in the membrane function.

**2.4. Pathological involvement of the lipid peroxidation process** 

contribute to the pathogenesis of human chronic diseases [Breusing et al., 2010].

besides lipids and lipoproteins.

Malonidialdehyde (MDA) is one of the most cited lipoperoxidation product originating from PUFAs. Several generation mechanisms have been proposed for MDA. Pryor & Stanley (1975) considered them as bicyclic endoperoxydes coming up from nonvolatiles MDA precursors, similar to prostaglandins. This mechanism was confirmed in 1983 by Frankel & Nef. Two other mechanisms were postulated by Esterbauer (1991) and consist in the successive generation of peroxydes and -cleavage of the lipid chain or as a reaction of acroleine radical with a hydroxyl moiety (exemplification for arachidonic acid in Figure 4, adapted from Esterbauer, 1991). Hecker & Ulrich (1989) consider that MDA can be generated *in* vivo by means of enzymatic processes linked to prostaglandins.

**Figure 4.** Generation of MDA as proposed by Esterbauer (1991)

Because the evaluation of the end products of lipid peroxidation at the tissue level is considered of maximum importance in both clinical and toxicological research, several methods to assay MDA have been proposed [Del Rio, 2005]. Since 1948, the most used method to assay lipoperoxidation end products is based on the studies of Bernheim et al., consisting in MDA condensation with the thiobarbituric acid (TBA) leading to a red complex which can be quantified by visible absorption spectrophotometry (in the range 500- 600 nm, depending on the procedure used) or fluorescence spectroscopy (Figure 5). As TBA reacts also with several other aldehydes commonly present in the biological sample, the agents reacting with TBA are frequently denoted as thiobarbituric acid reacting species (TBARS). It must be said that TBARs and MDA are a rather imprecise measure of the lipid peroxidation process, since many substances that are present in human biological fluids can also react with TBA.

Moreover the reaction conditions (heating) can lead to the degradation of other molecules in the sample, increasing the amount of MDA that is available for the reaction with TBA. Therefore this assay usually gives an overestimation of free radical damage [Cherubini et al., 2005].

**Figure 5.** Mechanism of reaction for TBARS quantification

The authors generally avoid interferences by using different methods: Kwiecieñ et al., 2002, used BHT (butylated hydroxytoluene) to prevent further oxidation of the sample; deproteinisation was performed with trichloracetic acid [Cassini et al., 1986], forced the lipoperoxidation and stopped the reaction with SDS and acetic acid [Gautam et al., 2010; Ohkawa et al., 1979], added EDTA and refered the results to standards prepared from tetramethoxypropane [Houglum et al., 1990] or used the standard addition method [Sprinteroiu et al., 2010].

The specificity of the measurement is improved by HPLC to separate the MDA-TBA adduct from interfering chromogens [Agarwal & Chase, 2002; Del Rio et al., 2003; Lykkersfeldt, 2001; Templar et al., 1999].

The principles of TBARS assay is so popular, that a few companies even developed kits for clinical research to assay MDA spectrophotometrically from biological samples.

Apart from the above mentioned method, other methods were applied for the quantitative assay of lipoperoxidation end-products: direct HPLC [Karatas et al., 2002], capillary electrophoresis [Wilson et al., 1997], RP-HPLC, derivatisation with 2,4-diphenylhydrazine [Sim et al., 2003], pre-column derivatisation with diaminonaphtalene at acidic pH (for protein bound MDA) or alkaline pH (for non protein-bound MDA), followed by HPLC-UV analysis [Stegens, 2001], GC-MS analysis following derivatisation with phenylhydrazine [Cighetti et al., 2002], GC-ECD-MS after derivatisation with 2,4,6-trichlorophenylhydrazine [Stalikas & Konidari, 2001].

## **4. Evaluation of the antioxidant status**

118 Lipid Peroxidation

also react with TBA.

NH

N H

**2** +

S O

O

H H

[Sprinteroiu et al., 2010].

2001; Templar et al., 1999].

**Figure 5.** Mechanism of reaction for TBARS quantification

H H

2005].

Because the evaluation of the end products of lipid peroxidation at the tissue level is considered of maximum importance in both clinical and toxicological research, several methods to assay MDA have been proposed [Del Rio, 2005]. Since 1948, the most used method to assay lipoperoxidation end products is based on the studies of Bernheim et al., consisting in MDA condensation with the thiobarbituric acid (TBA) leading to a red complex which can be quantified by visible absorption spectrophotometry (in the range 500- 600 nm, depending on the procedure used) or fluorescence spectroscopy (Figure 5). As TBA reacts also with several other aldehydes commonly present in the biological sample, the agents reacting with TBA are frequently denoted as thiobarbituric acid reacting species (TBARS). It must be said that TBARs and MDA are a rather imprecise measure of the lipid peroxidation process, since many substances that are present in human biological fluids can

Moreover the reaction conditions (heating) can lead to the degradation of other molecules in the sample, increasing the amount of MDA that is available for the reaction with TBA. Therefore this assay usually gives an overestimation of free radical damage [Cherubini et al.,

The authors generally avoid interferences by using different methods: Kwiecieñ et al., 2002, used BHT (butylated hydroxytoluene) to prevent further oxidation of the sample; deproteinisation was performed with trichloracetic acid [Cassini et al., 1986], forced the lipoperoxidation and stopped the reaction with SDS and acetic acid [Gautam et al., 2010; Ohkawa et al., 1979], added EDTA and refered the results to standards prepared from tetramethoxypropane [Houglum et al., 1990] or used the standard addition method

<sup>O</sup> <sup>O</sup> <sup>N</sup>

2


S N

OH

OH N

OH

N

OH SH

The specificity of the measurement is improved by HPLC to separate the MDA-TBA adduct from interfering chromogens [Agarwal & Chase, 2002; Del Rio et al., 2003; Lykkersfeldt,

The principles of TBARS assay is so popular, that a few companies even developed kits for

Apart from the above mentioned method, other methods were applied for the quantitative assay of lipoperoxidation end-products: direct HPLC [Karatas et al., 2002], capillary electrophoresis [Wilson et al., 1997], RP-HPLC, derivatisation with 2,4-diphenylhydrazine [Sim et al., 2003], pre-column derivatisation with diaminonaphtalene at acidic pH (for protein bound MDA) or alkaline pH (for non protein-bound MDA), followed by HPLC-UV

clinical research to assay MDA spectrophotometrically from biological samples.

For the evaluation of the antioxidant status in biological samples several markers (either enzymatic or non-enzymatic) can be used. Generally, the results obtained from the evaluation of antioxidant status markers should be correlated with certain peroxidative parameters, in order to be able to draw conclusions from the experiments.

Among the enzymatic markers of the antioxidant defense mechanism of the biological samples, literature cites some specific enzymes, such as catalase, superoxide-dismutase, glutathione-reductase, glutathione-peroxidase, etc. Each of these enzymes can be assayed using specific kits.

Commonly used are also non-enzyimatic markers, such as reduced glutathione, some vitamins (ascorbic acid, tocopherols, carotenoides).

A series of commercial kits for measuring the antioxidant status in biological samples (blood, serum, but also food products) are also available - the so-called *total antioxidant status* kits. The assays can be colorimetric (one example is the kit using as a chromogen 2,2'-azinodi-[3-ethylbenzthiazole sulfonate], which reacts with methmyoglobin and hydrogen peroxide to give a coloured cation), chemiluminometric (using the reaction of luminol with hydrogen peroxyde), or physico-chemical (potentiometric).

These methods allow the evaluation of the total antioxidant capacity in the biological samples, thus accounting both for enzymatic and for non-enzymatic bio-molecules.

Our group developed a method enabling a distinct evaluation for the biological samples antioxidant capacity as resulting exclusively from redox hydrophilic biomolecules. The method is based on potentiometric evaluation of the status of oxidant and reducing species in samples of human serum. We used a micro Pt/AgCl combination redox electrode, with an internal reference, and a Tistand 727 Potentiometer (Metrohm AG, Switzerland). The baseline apparent redox potential of the human serum (ARP0) was measured; than a mild prooxidant chemical system (quinhydrone) was added to the biological samples. After incubating at 25°C for 1h respectively 3 hours, two final apparent redox potentials were recorded (ARPf). Quinhydrone mimics the prooxidant conditions developing *in vivo* and consumes the reducing species leading to an increase of the apparent redox potential in time. This dynamic recording of the data allowed the calculation of a difference between the final value of the ARP (ARPf) and the initial one (ARP0), thus defining the redox stability index (RSI). This parameter illustrates the serum sample capacity to counteract the prooxidant agent. The lower the RSI, the higher the activity of the hydrosoluble antioxidant protective systems in the serum [Margina et al., 2009].

## **5. Monitoring the induced-peroxidation process**

J. Goldstein, M. Brown, and D, Steinberg emphasized more than a decade ago, that low density lipoproteins can be chemically modified, loosing their ability to be recognized by the classical LDL receptors [Brown & Jessup, 1999; Steinberg, 2009]. The oxidation of LDL does not take place in the blood stream, but inside the intimae, after lipoprotein complexes crossing through the endothelium. Therefore, in the oxidation process intracellular as well as extra cellular components are involved. This change of the LDL is realized mainly by oxidation with the free radicals that appear in large amounts in hypertension, diabetes mellitus, as a consequence of smoking, or in viral and bacterial infections, but LDL may be also modified by glycation (in type II diabetes mellitus), by association with proteoglycans or by incorporation in immune complexes [Ross, 1999].

Oxidised LDL particles (LDLox) have been proved to have different proatherogenic effects that can be predominantly attributed to their lipid components. Mainly, the uptake of LDLox by macrophages is enhanced through the scavenger receptor. Products generated from the decomposition of peroxidized lipids, such as aldehydes, modify the apolipoprotein B-100 (apoB-100) structure to a more electronegative form able to interact with the macrophage scavenger receptor. The modified LDL particles are taken up by scavenger receptors on the macrophages instead of the classical LDL receptors. This process is not regulated by feed back inhibition and allows the excessive build-up of cholesterol inside the cells, leading to their transformation into foam cells that are involved in the initiation and progression of atherosclerotic lesion [Parthasarathy et al., 1999; Shamir, 1996].

Alpha-tocopherol is an antioxidant from the LDL structure, and is the first one degraded during the radical attack on these lipoproteins. When this antioxidant protection is exhausted, PUFA are changed into lipid hydroperoxides. There is a great variability between subjects regarding the amount of PUFA and of antioxidants from LDL, which explains the variability concerning the susceptibility to oxidation of LDL particles. There are also a lot of other factors that influence *in vitro* evaluation of LDLox: some endogenous compounds, diet, some medicines, and probably genetic factors. Therefore, the assay of the *in vitro* LDL susceptibility to lipid peroxidation constitutes an important marker in the evaluation of atherogenic models/patients [Parthasarathy et al., 1999; Shamir, 1996].

The susceptibility of lipoprotein particles to lipid peroxidation can be assessed, after the isolation of LDL, either by treatment with copper salts, with mixtures of ferric compounds and ascorbic acid, or other prooxidant systems.

In order to evaluate this susceptibility to oxidation Esterbauer et al. proposed in 1989 a method based on the variation of absorbance of conjugated dienes in time at 234 nm. These dienes are relatively stable products resulted by a rearrangement of the double bonds from the PUFA molecules after the radical hydrogen abstraction. The increase in absorption at 234 nm is due to the formation of conjugated dienes during the peroxidation of polyunsaturated fatty acids. Absorbance at 234 nm shows an initial slower increase (lag phase) as antioxidants are destroyed and then increases more rapidly (propagation phase) reaching a plateau phase at which the absorbance is maximal as the rate of formation of dienes approaches their rate of decomposition [Esterbauer et al., 1990].

120 Lipid Peroxidation

**5. Monitoring the induced-peroxidation process** 

or by incorporation in immune complexes [Ross, 1999].

and ascorbic acid, or other prooxidant systems.

J. Goldstein, M. Brown, and D, Steinberg emphasized more than a decade ago, that low density lipoproteins can be chemically modified, loosing their ability to be recognized by the classical LDL receptors [Brown & Jessup, 1999; Steinberg, 2009]. The oxidation of LDL does not take place in the blood stream, but inside the intimae, after lipoprotein complexes crossing through the endothelium. Therefore, in the oxidation process intracellular as well as extra cellular components are involved. This change of the LDL is realized mainly by oxidation with the free radicals that appear in large amounts in hypertension, diabetes mellitus, as a consequence of smoking, or in viral and bacterial infections, but LDL may be also modified by glycation (in type II diabetes mellitus), by association with proteoglycans

Oxidised LDL particles (LDLox) have been proved to have different proatherogenic effects that can be predominantly attributed to their lipid components. Mainly, the uptake of LDLox by macrophages is enhanced through the scavenger receptor. Products generated from the decomposition of peroxidized lipids, such as aldehydes, modify the apolipoprotein B-100 (apoB-100) structure to a more electronegative form able to interact with the macrophage scavenger receptor. The modified LDL particles are taken up by scavenger receptors on the macrophages instead of the classical LDL receptors. This process is not regulated by feed back inhibition and allows the excessive build-up of cholesterol inside the cells, leading to their transformation into foam cells that are involved in the initiation and

Alpha-tocopherol is an antioxidant from the LDL structure, and is the first one degraded during the radical attack on these lipoproteins. When this antioxidant protection is exhausted, PUFA are changed into lipid hydroperoxides. There is a great variability between subjects regarding the amount of PUFA and of antioxidants from LDL, which explains the variability concerning the susceptibility to oxidation of LDL particles. There are also a lot of other factors that influence *in vitro* evaluation of LDLox: some endogenous compounds, diet, some medicines, and probably genetic factors. Therefore, the assay of the *in vitro* LDL susceptibility to lipid peroxidation constitutes an important marker in the

progression of atherosclerotic lesion [Parthasarathy et al., 1999; Shamir, 1996].

evaluation of atherogenic models/patients [Parthasarathy et al., 1999; Shamir, 1996].

The susceptibility of lipoprotein particles to lipid peroxidation can be assessed, after the isolation of LDL, either by treatment with copper salts, with mixtures of ferric compounds

In order to evaluate this susceptibility to oxidation Esterbauer et al. proposed in 1989 a method based on the variation of absorbance of conjugated dienes in time at 234 nm. These dienes are relatively stable products resulted by a rearrangement of the double bonds from the PUFA molecules after the radical hydrogen abstraction. The increase in absorption at 234 nm is due to the formation of conjugated dienes during the peroxidation of polyunsaturated fatty acids. Absorbance at 234 nm shows an initial slower increase (lag phase) as antioxidants are destroyed and then increases more rapidly (propagation phase) reaching a Another method for the assay of lipoprotein susceptibility to lipid peroxidation is based on the ability of lipid hydroperoxides to convert iodide (I- ) to iodine (I2), which will than react with the iodide excess and form I3 that absorbs at 365 nm. There is a direct stoechiometric relationship between the amount of organic peroxides that resulted from the reaction and the concentration of I3- [Steinberg D., 1990, Esterbauer H. 1993]

One of the symplest methods for the assay of the susceptibility of LDL particles to lipid peroxidation is based on the selective precipitation of serum LDL with heparin at isoelectrical point (pH=5.4), method which is cheaper and quicker than the one based on ultracentrifugation. Personal results proved that the susceptibility of LDL to lipid peroxidation is correlated with the fasting plasma glucose level as well as with the lipid level [Margina et al., 2004].

The same type of assay can be used in order to evaluate the susceptibility to induced peroxidation for other kinds of biological samples (red blood cells, sub-cellular fractions such as mitochondria, or even tissue homogenates).

Previously published results [Margina D et al., 2011] proved that, for patients diagnosed with central obesity (BMI>30Kg/m2), adipose tissue susceptibility to lipid peroxidation correlated significantly with the total cholesterol (TC) level and with the LDL level. The susceptibility of adipose tissue to lipid peroxidation was assessed on white adipose tissue harvested from the abdominal area, homogenated in NaOH 0.015M, followed by TBARS evaluation. This parameter reflects the tendency to accumulation of free radicals in the adipose tissue of obese patients. In the same study, we pointed out that patients with impaired lipid profile (TC>220 mg/dl, LDL>150 mg/dl) had a significantly higher susceptibility of the adipose tissue to lipid peroxidation (p=0.036), associated with the decrease of the adiponectin level (Figure 6), compared to obese patients with physiologic lipid profile (TC<220mg/dl, LDL<150mg/dl).

Literature data also mention the assay of circulating LDLox, using different ELISA methods; one of these methods uses antibodies against a conformational epitope in the apolipoprotein B-100 (apoB-100) moiety of LDL that is generated as a consequence of substitution of at least 60 lysine residues of apoB-100 with aldehydes. This number of substituted lysines corresponds to the minimal number required for scavenger-mediated uptake of ox-LDL. The substituting aldehydes can be produced by peroxidation of lipids of LDL, leading to the generation of ox-LDL [Holvoet et al., 2006]. Another method might be the electrophoretic separation of LDL and LDLox particles from serum samples; studies proved that the electrophoretic mobility of oxidized LDL particles is increased compared to that of standard LDL [Lougheed et al., 1996].

Besides biochemical determination, noninvasive, real-time monitoring of lipid peroxidation using fluorescent probes has also been developed. The assays can be performed either on living cells (for example using cis-parinaric acid, fluoresceinated phosphoethanolamine, undecylamine-fluorescein, diphenyl-1-pyrenylphosphine – DPPP or other fluorescent

**Figure 6.** Obese patients with impaired plasma lipid profile (TC>220mg/dl, LDL>150mg/dl) are characterized by significantly different levels for the susceptibility of adipose tissue to induced lipid peroxidation as well as adiponectin level, compared to obese patients with normal plasma lipid profile (TC<220mg/dl, LDL<150mg/dl); \* p<0.05 for cardio-vascular group compared to normal lipid profile group)

markers) or non-living samples (liposomes, tissue homogenates, plasma, serum, etc). In the cases of experiments that are performed on living cells, common limitation of use of fluorescent probes is that the probes often are cytotoxic or affect physiological activities of the cell [Drummen et al., 2004; Margina et al., 2012, Takahashi M. et al., 2001]. DPPP stoichiometrically reduces biologically generated hydroperoxides (such as fatty acid hydroperoxides, and triacylglycerol hydroperoxides) to their corresponding alcohols, and is transformed consequently into its oxide. DPPP is essentially non-fluorescent until oxidized to a phosphine-oxide by peroxides. Due to its solubility in lipids, DPPP intercalates into the membrane leaflets and reacts with lipid hydroperoxides, thus allowing the evaluation of peroxide formation in the membranes of live cells [Kawai et al.,, 2007]. Due to these chemical properties, the probe can be used in order to evaluate the extent of lipid peroxidation of biological materials such as cell membranes [Akasaka et al., 1993; Ohshima et al., 1996; Takahashi et al., 2001]. Because DPPP molecules are incorporated into the cell membranes, hydroperoxides located in the membrane are supposed to preferably react with DPPP.

Diphenyl-1-pyrenylphosphine (DPPP) is a synthetic compound with high reactivity against hydroperoxides, which has been used as a sensitive fluorescent probe for hydroperoxide analysis for HPLC methods. H2O2, which is least lipid-soluble, does not induce the peroxidation reaction of DPPP located in cell membranes. Although H2O2 is highly permeable to the membrane, it may not stay within the membrane long enough to react with DPPP effectively. Experimental lipid peroxidation can be induced by 10μM cumene hydroperoxide (CuOOH) which generates an effective reaction with DPPP in the membrane [Gomes et al., 2005; Takahashi et al., 2001].

We proved using DPPP in cell models (U937 human macrophage cell line as well as Jurkat lymphocytic cells) that the increase of certain polyphenol concentration (quercetin or epigallocatechin gallate) induces a decrease of the CuOOH induced lipid peroxidation of cell membranes. Fluorescence signals for the cells labeled with 5 μM DPPP are presented in Figure 7 [Margina et al, 2012].

## **6. Conclusions**

122 Lipid Peroxidation

group)

**Figure 6.** Obese patients with impaired plasma lipid profile (TC>220mg/dl, LDL>150mg/dl) are characterized by significantly different levels for the susceptibility of adipose tissue to induced lipid peroxidation as well as adiponectin level, compared to obese patients with normal plasma lipid profile (TC<220mg/dl, LDL<150mg/dl); \* p<0.05 for cardio-vascular group compared to normal lipid profile

hydroperoxides located in the membrane are supposed to preferably react with DPPP.

[Gomes et al., 2005; Takahashi et al., 2001].

Diphenyl-1-pyrenylphosphine (DPPP) is a synthetic compound with high reactivity against hydroperoxides, which has been used as a sensitive fluorescent probe for hydroperoxide analysis for HPLC methods. H2O2, which is least lipid-soluble, does not induce the peroxidation reaction of DPPP located in cell membranes. Although H2O2 is highly permeable to the membrane, it may not stay within the membrane long enough to react with DPPP effectively. Experimental lipid peroxidation can be induced by 10μM cumene hydroperoxide (CuOOH) which generates an effective reaction with DPPP in the membrane

markers) or non-living samples (liposomes, tissue homogenates, plasma, serum, etc). In the cases of experiments that are performed on living cells, common limitation of use of fluorescent probes is that the probes often are cytotoxic or affect physiological activities of the cell [Drummen et al., 2004; Margina et al., 2012, Takahashi M. et al., 2001]. DPPP stoichiometrically reduces biologically generated hydroperoxides (such as fatty acid hydroperoxides, and triacylglycerol hydroperoxides) to their corresponding alcohols, and is transformed consequently into its oxide. DPPP is essentially non-fluorescent until oxidized to a phosphine-oxide by peroxides. Due to its solubility in lipids, DPPP intercalates into the membrane leaflets and reacts with lipid hydroperoxides, thus allowing the evaluation of peroxide formation in the membranes of live cells [Kawai et al.,, 2007]. Due to these chemical properties, the probe can be used in order to evaluate the extent of lipid peroxidation of biological materials such as cell membranes [Akasaka et al., 1993; Ohshima et al., 1996; Takahashi et al., 2001]. Because DPPP molecules are incorporated into the cell membranes,

Oxidative stress is among the most claimed causes of disease, as by its very definition indicates an abnormal biochemical function of the body. Among the targets of the oxidative stress, lipids are favorites, susceptible to structural changes that can decisively influence their normal function, and also generating hydroxyperoxides that further propagate the lipoperoxidation process.

Lipid peroxidation has been intensively studied in connection with normal and pathological metabolic processes; one of the main purposes was the understanding of toxicity triggered by lipoperoxidation end-products. That is why direct or indirect quantification of these products (TBARS, MDA, hydroxynonenals, prostaglandins, DNA and protein-adducts of the former, etc.) remains of interest for traditional and nowadays methods. The process of lipoperoxidation is often monitored in dynamics, even on living cells, using various techniques.

It is also of a great interest to seek for efficient antioxidants to prevent the excess lipoperoxidation, therefore one component of such kind of studies consist in finding out

efficient markers for the oxidative stress and the antioxidant status. This can be fulfilled if a right and comprehensive understanding of the lipoperoxidation process is achieved. But this is still a faraway target.

## **Author details**

Mihaela Ilie and Denisa Margină *Carol Davila University of Medicine and Pharmacy, Faculty of Pharmacy, Bucharest, Romania* 

## **Acknowledgments**

The work was performed under the CNCSIS grant PD 132/30.07.2010 (PD 29/2010).

### **7. References**


Cai W., He J.C., Zhu L., Peppa M., Lu C., Uribarri J. & Vlassara H. (2004). High Levels of Dietary Advanced Glycation End Products Transform Low-Density Lipoprotein Into a Potent Redox-Sensitive Mitogen-Activated Protein Kinase Stimulant in Diabetic Patients *Circulation*, Vol. 110, No. 3, 285-291, ISSN: 0009-7322.

124 Lipid Peroxidation

this is still a faraway target.

Mihaela Ilie and Denisa Margină

205–211, ISSN 1570- 0232

989–1003, ISSN 0891-5849.

*Res Toxicol* Vol 23, No 4, 821–835, ISSN 1520-5010.

2, Doc03.DOI: 10.3205/dgkh000146 ISSN 1863-5245

*Rep* Vol 8, No 3, 223 – 231, ISSN 1534-6242.

**Author details** 

**Acknowledgments** 

**7. References** 

1570-0232.

0021-9258

ISSN 0021-9150.

efficient markers for the oxidative stress and the antioxidant status. This can be fulfilled if a right and comprehensive understanding of the lipoperoxidation process is achieved. But

*Carol Davila University of Medicine and Pharmacy, Faculty of Pharmacy, Bucharest, Romania* 

The work was performed under the CNCSIS grant PD 132/30.07.2010 (PD 29/2010).

Agarwal R. & Chase S.D. (2002) Rapid, fluorimetric-liquid chromatographic determination of malondialdehyde in biological samples. *J Chromatogr B*, Vol. 775, No. 1, 121-126, ISSN

Akasaka K., Ohrui H., Meguro H. & Tamura M. (1993). Determination of triacylglycerol and cholesterol ester hydroperoxides in human plasma by high-performance liquid chromatography with fluorometric postcolumn detection. *J. Chromatogr. B* Vol 617, No2,

Aruoma O.I., Halliwell B., Laughton M.J., Quinlan G.J. & Gutteridge J.M.C. (1989). The mechanism of initiation of lipid peroxidation. Evidence against a requirement for an

Baker P.R., Schopfer F.J., O'Donnell V.B. & Freeman B.A. (2009). Convergence of nitric oxide and lipid signaling: anti-inflammatory nitro-fatty acids. *Free Radic Biol Med* Vol 46, No 8,

Bartesaghi S., Wenzel J., Trujillo M., Lopez M., Joseph J., Kalyanaraman B. & Radi R. (2010) Lipid peroxyl radicals mediate tyrosine dimerization and nitration in membranes. *Chem* 

Benkhai H., Lemanski S., Below H., Heiden J.U., Below E., Lademann J.,Bornewasser M., Balz T., Chudaske C. & Kramer A. (2010). Can physical stress be measured in urine using the parameter antioxidative potential? *GMS Krankenhaushyg Interdiszip.* Vol.5 No

Bernheim F., Bernheim M.L.C. & Wilbur K.M. (1948). The reaction between thiobarbituric acid and the oxidation products of certain lipids. *J Biol Chem*, Vol. 174, 257-264, ISSN

Birukov KG. (2006) Oxidized lipids: the two faces of vascular inflammation. *Curr Atheroscler* 

Brown A.J. & Jessup W. (1999) Oxysterols and atherosclerosis*. Atherosclerosis* Vol. 142, 1–28,

iron(II)-iron(III) complex. *Biochem. J.* Vol. 258, No. 2, 617-620, ISSN 0264-6021


Hecker M. & Ullrich V. (1989) On the mechanism of prostacyclin and thromboxane A2 biosynthesis. *J Biol Chem* Vol. 264, No. 1, 141-150, ISSN 1083-351X.

126 Lipid Peroxidation

Elroy-Stein O., Bernstein Y. & Groner Y. (1986). Overproduction of human Cu/Zn-superoxide dismutase in transfected cells: extenuation of paraquat-mediated cytotoxicity and

Esterbauer H., Dieber-Rothender M., Waeg G., Striegl G. & Jurgens, G. (1990). Biochemical, structural, and functional properties of oxidized low-density lipoproteins. *Chem. Res.* 

Esterbauer H., Schaur R.J, & Zollner H. (1991). Chemistry and biochemistry of 4 hydroxynonenal, malonaldehyde and related aldehydes. *Free Radic Biol Med,* Vol. 11,

Frankel E.N. & Neff W.E. (1983) Formation of malonaldehyde from lipid oxidation products,

Freeman B.A., Baker P.R., Schopfer F.J., Woodcock S.R., Napolitano A. & d'Ischia M. (2008) Nitro-fatty acid formation and signaling. *J Biol Chem* Vol 28, No 23, 15515–15519, ISSN

Freinbichler W., Colivicchi M.A., Stefanini C., Bianchi L., Ballini C., Misini B., Weinberger P., Linert W., Varešlija D., Tipton K.F. & Della Corte L. 2011. Highly reactive oxygen species: detection, formation, and possible functions. *Cell Mol Life Sci*. Vol. 68 No.12,

Gautam N., Das S., Mahapatra S.K., Chakraborty S.P., Kundu P.K. & Roy S. (2010). Age associated oxidative damage in lymphocytes. *Oxidative Medicine and Cellular Longevity*

Gomes A., Fernandes E. & Lima J.L.F.C. (2005). Fluorescence probes used for detection of reactive oxygen species. *J. Biochem. Biophys. Methods* Vol 65, No 2-3, 45–80, ISSN 0165-022X Gordon M.H. (2012). Significance of Dietary Antioxidants for Health. *Int. J. Mol. Sci.*, Vol. 13,

Griffiths H.R., Dunston C.R., Bennett S.J., Grant M.M., Phillips D.C. & Kitas G.D. 2011. Free radicals and redox signalling in T-cells during chronic inflammation and ageing.

Grimsrud P.A., Xie H., Griffin T.J & Bernlohr D.A. (2008). Oxidative Stress and Covalent Modification of Protein with Bioactive Aldehydes. *J Biol Chem* Vol. 283, No. 32, 21837–

Halliwell B. & Chirico S. (l993). Lipid peroxidation: its mechanism, measurement, and

Halliwell B. & Gutteridge J.M.C. (1984). Oxygen toxicity, oxygen radicals, transition metals

Halliwell B. (1992) Reactive oxygen species and the central nervous system. *J. Neurochem.* 

Halliwell B.& Gutteridge J.M.C. (2007). *Free radicals in Biology and Medicine*, 4-th edition

Halliwell, B. (2006). Reactive Species and Antioxidants. Redox Biology Is a Fundamental Theme of Aerobic Life. *Plant Physiology*, Vol. 141, No. 2, 312–322, ISSN 0981-9428.

significance. *J Clin Nutr* Vol 57(suppl), No. 7, 155-255, ISSN 1938-3207.

enhancement of lipid peroxidation. *EMBO J* Vol.5 No.3, 615-622, ISSN 0261-4189 Esterbauer H. (1993). Cytotoxicity and genotoxicity of lipid-oxydation products, *Am. J. Clin.* 

*Nutr.*, Vol 57, No 5, 779-785. ISSN 0002-9165.

*Toxicol.*, Vol. 3, No. 2, 77-92, ISSN 0893-228X .

*Biochim Biophys Acta* Vol. 754, No. 3, 264-270, ISSN 0006-3002

*Biochem Soc Trans.* Vol. 39, No. 5, 1273-1278, ISSN 0300-5127.

and disease. *Biochem. J.* Vol. 219, No. 1, 1-14, ISSN 0264-6021

Oxford University Press, ISBN 978-0-19-856869-8, New York.

Vol. 59, No. 5, 1609-1623, ISSN 0022-3042.

No. 1, 81-128, ISSN 0891-5849.

2067-79, ISSN 1420-682X.

Vol. 3, No.4, 275-282, ISSN 1942-0900.

No. , 173-179, ISSN 1422-0067.

21841, ISSN 0021-9258.

1083-351X.


Ultraviolet-Visible Spectrophotometry. *Clin Chem*, Vol. 47, No. 9, 1725-1727, ISSN 1530- 8561.


Ross R. (1999) Atherosclerosis–an Inflammatory Disease, *NEJM,* Vol 340, No 2, 115-126, ISSN 1533-4406.

128 Lipid Peroxidation

8561.

48, ISSN 0035-3930

1521-6551.

1539-7262.

No. 24, 3615-3617, ISSN 1099-0690.

*Sci* Vol. 16, 112, ISSN 1021-7770.

Ultraviolet-Visible Spectrophotometry. *Clin Chem*, Vol. 47, No. 9, 1725-1727, ISSN 1530-

Manda G., Nechifor M.T. & Neagu T.M. (2009). Reactive Oxygen Species, Cancer and Anti-Cancer Therapies. *Current Chemical Biology* Vol. 3, No. 1, 342-366, ISSN 1872-3136 Margina D, Gradinaru D., Panaite C., Cimponeriu D., Vladica M., Danciulescu R., Mitrea N. (2011) The association of adipose tissue markers for redox imbalance and the cardiovascular risk in obese patients, *HealthMED* Vol. 5, No 1, 194-199, ISSN 1840-2291. Margina D., Gradinaru D. & Mitrea N. (2004). Clinical study regarding the atherogenic properties of the oxidatively modified low-density lipoproteins. *Archives of the Balkan* 

Margina D., Gradinaru D., Mitrea N. (2009) Development of a potentiometric method for the evaluation of redox status in human serum, *Revue Roumaine de Chimie,* Vol 54, No 1, 45–

Moller M., Botti H., Batthyany C., Rubbo H., Radi R. & Denicola A. (2005) Direct measurement of nitric oxide and oxygen partitioning into liposomes and low density

Moller M.N., Li Q., Lancaster J.R.Jr & Denicola A. (2007) Acceleration of nitric oxide autoxidation and nitrosation by membranes. *IUBMB Life*, Vol 59, No 4-5, 243–248, ISSN

Niki E. (2009). Lipid peroxidation: physiological levels and dual biological effects. *Free Radic* 

Ohkawa H., Ohishi N. & Yagi K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. *Anal Biochem* Vol. 95, No. 2, 351-358, ISSN1096-0309. Ohshima T., Hopia A., German J.B. & Frankel E.N. (1996). Determination of hydroperoxides and structures by high-performance liquid chromatography with post-column detection with diphenyl-1-pyrenylphosphine. *Lipids* Vol 31, No 10, 1091–1096, ISSN: 1558-9307 Parastatidis I., Thomson L., Fries D.M., Moore R.E., Tohyama J., Fu X., Hazen S.L., Heijnen H.F., Dennehy M.K., Liebler D.C., Rader D.J. & Ischiropoulos H. (2007) Increased protein nitration burden in the atherosclerotic lesions and plasma of apolipoprotein A-I

Parthasarathy S., Santanam N., Ramachandran S. & Meilhac O (1999) Oxidants and antioxidants in atherogenesis: an appraisal, *J. Lipid Res.* Vol 40, No 12, 2143–2157. ISSN

Pryor W.A. & Stanley J.P. (1975). A suggested mechanism for the production of malonaldehyde during the autoxidation of polyunsaturated fatty acids. Nonenzymatic production of prostaglandin endoperoxides during autoxidation. *J Org Chem* Vol. 40,

Puddu P., Puddu G.M., Cravero E., De Pascalis S & Muscari A. (2009). The emerging role of cardiovascular risk factor-induced mitochondrial dysfunction in atherogenesis *J. Biomed* 

Rizzo M.A. & Piston D.W. (2003). Regulation of beta cell glucokinase by S-nitrosylation and association with nitric oxide synthase. *J Cell Biol* Vol 161, No 2, 243–248, ISSN 1540-8140.

lipoprotein. *J Biol Chem* Vol 280, No 10, 8850–8854, ISSN 1083-351X.

deficient mice. *Circ Res* Vol 101, No 4, 368–376, ISSN 0009-7330.

*Medical Union*, Vol 39, No. 2, 78-82, ISSN 0041-6940

*Biol Med*; Vol 47, No 5, 469 – 484. ISSN 0891-5849.


**Chapter 6** 

## **Automation of Methods for Determination of Lipid Peroxidation**

Jiri Sochor, Branislav Ruttkay-Nedecky, Petr Babula, Vojtech Adam, Jaromir Hubalek and Rene Kizek

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/45945

#### **1. Introduction**

130 Lipid Peroxidation

7716.

Vol 98, No 1, 355–360, ISSN 1091-6490.

*Chaperones* Vol. 17, No. 1, 11–21, ISSN 1355-8145

*Chem* Vol 280, No 9, 7511–7518, ISSN 1083-351X

disease. *PNAS* Vol. 93, No. 7, 2696–2701, ISSN 1091-6490.

*Endocrinol Metab,* Vol 299, No 6, E868-E878, ISSN: 0193-1849.

samples. *Clin Chem.* Vol. 43, No. 10, 1982-1984, ISSN 1530-8561

160-170, ISSN 0891-5849.

Thomas D.D., Liu X., Kantrow S.P. & Lancaster J.R. Jr. (2001) The biological lifetime of nitric oxide: implications for the perivascular dynamics of NO and O2. *Proc Natl Acad Sci USA*

Uehara T. (2007) Accumulation of misfolded protein through nitrosative stress linked to neurodegenerative disorders. *Antioxid Redox Signal* Vol 9, No 5, 597–601, ISSN: 1557-

Velayutham M., Hemann C. & Zweier J.L. (2011). Removal of H₂O₂ and generation of superoxide radical: role of cytochrome c and NADH. *Free Radic Biol Med*. Vol. 51, No. 1,

Veskoukis A.S., Tsatsakis A.M. & Kouretas D. (2012). Dietary oxidative stress and antioxidant defense with an emphasis on plant extract administration. *Cell Stress and* 

White P.J., Charbonneau A., Cooney G.J. & Marette A. (2010). Nitrosative modifications of protein and lipid signaling molecules by reactive nitrogen species, *Am J Physiol* 

Wilson D.W., Metz H.N., Graver L.M. & Rao P.S. (1997). Direct method for quantification of free malondialdehyde with high-performance capillary electrophoresis in biological

Yasukawa T., Tokunaga E., Ota H., Sugita H., Martyn J.A. & Kaneki M. (2005) Snitrosylation-dependent inactivation of Akt/protein kinase B in insulin resistance. *J Biol* 

Yoritaka A., Hattori N., Uchida K., Tanaka M., Stadtman E.R & Mizuno Y. (1996). Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson Free radicals are atoms or molecules having one (or rarely more) free electron(s). These compounds may attack most of the (bio)molecules in organisms, which leads to the oxidative stress, which belongs to the causes of pathological processes in organisms [1-6]. Oxidative stress occurs in a situation, when the imbalance between the production of free radicals and effectiveness of antioxidant defence system occurs in a healthy organism. Determination of antioxidant activity or eventually markers directly connected with this variable is one way how to monitor the damage of organisms by these compounds [7-14]. The negative effect of free oxygen radicals consists in the lipid peroxidation. This type of peroxidation is a chemical process, in which unsaturated fatty acids of lipids are damaged by free radicals and oxygen under lipoperoxides formation. Lipoperoxides are unstable and decompose to form a wide range of compounds including reactive carbonyl compounds, especially certain aldehydes (malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE)) [15- 22] that damage cells by the binding the free amino groups of amino acids of proteins. Consequently, the proteins' aggregates become less susceptible to proteolytic degradation [23-25]. In tissues, the accumulation of age pigment spots appears. In addition, free radicals effects are connected with a formation of atherosclerotic lesions. In body fluids (blood, urine) the increased levels of peroxidation end-products (MDA, 4-HNE, isoprostanes) are present [26,27]. The lipid peroxidation by free radicals occurs in three stages: initiation, propagation and termination [2,26]. Reaction (1) represents initiation, in which a fatty acid molecule of lipid is attacked by free radicals leading to a detachment of the hydrogen atom under fatty acid radical formation. In its structure, a rearrangement of the double bond to form conjugated diene occurs. This diene structure subsequently reacts with oxygen molecule to form a lipoperoxyl radical, which leads to the initiation of the second phase called propagation (2). In another part of the promotion, lipoperoxyl radical further reacts

© 2012 Sochor 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. © 2012 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.

with another molecule of fatty acid, from which a hydrogen atom is detached under formation of lipid hydroperoxide from original molecule (3). After pairing of all radicals, the last stage of the reaction called termination occurs. In addition to the above-mentioned chemical non-enzymatic peroxidation, enzymatic lipid peroxidation that is catalysed by the enzymes cyclooxygenase and lipoxygenase takes place. [26,28]. Both enzymes are involved in the formation of eicosanoids, which represent a group of biologically active lipid compounds derived from unsaturated fatty acids containing 20 carbon atoms. Cyclooxygenase is involved in the genesis of prostaglandins [29].

$$\text{(1)}\ \text{LH} + \text{R}^\* \rightarrow \text{L}^\* + \text{RH} \\ \text{(2)}\ \text{L}^\* + \text{O}\_2 \rightarrow \text{LCO}^\* \\ \text{(3)}\ \text{LCO}^\* + \text{LH} \rightarrow \text{L}^\* + \text{LCOOH}$$

**Scheme 1.** The scheme of lipid peroxidation. Initiation (1), the first part of the propagation (2), the second part of propagation (3)**.**

For the monitoring of lipid peroxidation, spectrophotometric [30,31], chromatographic [32] and immunochemical [33] methods can be used. The analysis itself may be based on the analysis of the primary products of lipid peroxidation as conjugated dienes [34] and lipid hydroperoxides [35], or secondary products, such as malondialdehyde [36], alkanes [37] or isoprostanes [32,38-40]. Chromatographic methods represent the special group of methods, which are mostly based on the decrease of unsaturated fatty acids' concentration [41]. The scope of this review was to summarize the photometric analyses of lipid peroxidation. Less common method - FOX (ferrous oxidation in xylenol orange) was suggested to be automated.

#### **1.1. Spectrophotometric methods in lipid peroxidation analysis**

Spectrophotometric methods for the analysis of lipid peroxidation (see Table 1) are well reproducible and low cost. They usually consist of several steps that can be automated without much difficulty. Determination of conjugated dienes and TBARS belong to the one of the oldest and mostlz used methods for their rapidity and simplicity. On the other hand, they are criticized for their non-specificity [42,43]. Lipid hydroperoxides may be determined by the iodometric method and FOX test [44].


**Table 1.** Summary of spectrophotometric methods used in lipid peroxidation determination. FOX –ferrous oxidation in xylenol orange, MDA – malondialdehyde, TBARS – thiobarbituric acid reactive substances

#### **1.2. Conjugated dienes**

132 Lipid Peroxidation

second part of propagation (3)**.**

automated.

**Determined analyte** 

Conjugated dienes

hydroperoxides

reactive substances

Lipid

with another molecule of fatty acid, from which a hydrogen atom is detached under formation of lipid hydroperoxide from original molecule (3). After pairing of all radicals, the last stage of the reaction called termination occurs. In addition to the above-mentioned chemical non-enzymatic peroxidation, enzymatic lipid peroxidation that is catalysed by the enzymes cyclooxygenase and lipoxygenase takes place. [26,28]. Both enzymes are involved in the formation of eicosanoids, which represent a group of biologically active lipid compounds derived from unsaturated fatty acids containing 20 carbon atoms.

Cyclooxygenase is involved in the genesis of prostaglandins [29].

**1.1. Spectrophotometric methods in lipid peroxidation analysis** 

The structures of conjugated dienes absorb in the UV spectrum of 230-235 nm

Measurement at 532 nm

by the iodometric method and FOX test [44].

TBARS/MDA TBA complex with MDA,

•• • • • •

<sup>2</sup> 1 LH R L RH 2 L O LOO 3 LOO L´H L´ LOOH **Scheme 1.** The scheme of lipid peroxidation. Initiation (1), the first part of the propagation (2), the

For the monitoring of lipid peroxidation, spectrophotometric [30,31], chromatographic [32] and immunochemical [33] methods can be used. The analysis itself may be based on the analysis of the primary products of lipid peroxidation as conjugated dienes [34] and lipid hydroperoxides [35], or secondary products, such as malondialdehyde [36], alkanes [37] or isoprostanes [32,38-40]. Chromatographic methods represent the special group of methods, which are mostly based on the decrease of unsaturated fatty acids' concentration [41]. The scope of this review was to summarize the photometric analyses of lipid peroxidation. Less common method - FOX (ferrous oxidation in xylenol orange) was suggested to be

Spectrophotometric methods for the analysis of lipid peroxidation (see Table 1) are well reproducible and low cost. They usually consist of several steps that can be automated without much difficulty. Determination of conjugated dienes and TBARS belong to the one of the oldest and mostlz used methods for their rapidity and simplicity. On the other hand, they are criticized for their non-specificity [42,43]. Lipid hydroperoxides may be determined

**Method Type of analysed sample Reference** 

Cell lysates

Iodometric method plasma, plant tissues [44,54]

FOX test plasma, serum lipoproteins,

**Table 1.** Summary of spectrophotometric methods used in lipid peroxidation determination. FOX –ferrous oxidation in xylenol orange, MDA – malondialdehyde, TBARS – thiobarbituric acid

lipids

Serum lipoproteins, tissue

plasma, urine, tissues (liver),

both animal and plant tissues

[34,45]

[36,46-53]

[35,44,55,56]

The structures of conjugated dienes (Fig. 1) with alternating double and single bonds between carbon atoms (-C=C-C=C-) absorb wavelengths of 230-235 nm in the UV region. Therefore, it is possible to use UV absorption spectrometry for their determination [41,42]. The method is used for determination of a non-specific lipid peroxidation caused by free radicals in biological samples, and is successfully used in the study of peroxidation in isolated lipoprotein fractions (LDL lipoproteins) [45]. However, its use in the direct analysis of plasma is controversial because of the presence of interfering substances, such as heme proteins, purines or pyrimidines in the UV region measurement [42,57].

**Figure 1.** Structural formula of conjugated diene arising from the fatty acids by the free radicals effects during lipid peroxidation.

Increased sensitivity of the method can be achieved by an extraction of lipids into organic solvents in combination HPLC with UV detection [34,58]. However, the result of application the method to lipid extracts from human body fluids after HPLC separation was surprising, because the majority of pre-treated lipid fraction absorbs at wavelengths typical for conjugated dienes consisting of conjugated linoleic acid isomer (*cis*-9, *trans*-11 octadecadienoic acid) [59]. The main sources of conjugated isomer of linoleic acid (CLA) are dairy products and ruminant meat, especially beef [60]. They come into human serum and tissues probably from the diet [61], but can be also produced by bacteria [62,63]. Therefore, formation of large amounts of CLA by free radicals seems unlikely. In addition, the presence of CLA was not detected in the plasma of animals suffering from oxidative stress. *In vivo* induction of lipid peroxidation in rats treated with phenylhydrazin trichlorbrommethan did not cause an increase of CLA plasma values [64]. In the case of the use this method, it is necessary to take into account the above-mentioned shortcomings in the analysis of biological fluids or tissues.

#### **1.3. TBARS, TBA-MDA adducts**

TBARS (TBA-MDA) (**T**hio**b**arbituric **A**cid **R**eactive **S**ubstances) is the most widely used method for determination of lipid peroxidation method, especially due to its simplicity and cheapness. As the name of this method implies, it is based on the ability of malondialdehyde, which is one of the secondary products of lipid peroxidation, to react with thiobarbituric acid (TBA) [65]. The principle of this method consists in the reaction of MDA with thiobarbituric acid in acidic conditions and at a higher temperature to form a pink MDA-(TBA)2 complex (Fig. 2), which can be quantified spectrophotometrically at 532 nm [17,66-70]. TBARS method measures the amount of MDA generated during lipid peroxidation, however, other aldehydes generated during lipid peroxidation, which also absorb at 532 nm, may react with TBA [71]. The results of the assay are expressed in µmol of MDA equivalents. TBARS method can be also used in the case of defined membrane systems, such as microsomes and liposomes, but its application in biological fluids and tissue extracts appears to be problematic [72-74]. The first problem is based on the fact that MDA can be formed by the decomposition of lipid peroxides under heating of the sample with TBA. This decomposition is accelerated by traces of iron in the reagents and is inhibited by the use of chelating agents [42]. At the decomposition of lipid peroxides in the analysis, the originating radicals can amplify the entire process and the amount of MDA could be overestimated [74]. To prevent the decomposition of lipid peroxides during the analysis, inhibitor of the lipid peroxidation called butylated hydroxytoluene is added to the sample [42]. One of the other problems of the TBARS method application has been found in the analysis of biological fluids. In this case, some substances, such as bile pigments and glycoproteins provide a false positive reaction with TBA [71,75]. Unspecificity TBARS test problems can be partially overcome by the using of HPLC techniques for the separation of "authentic", original MDA-(TBA)2 adduct from other chromogens absorbing at 532 nm [76]. Nevertheless, this approach cannot solve all problems. In addition, next molecules, such as aldehydes originated from lipid peroxidation, can form with TBA a original MDA-TBA2 adduct, which has been demonstrated in the deoxyribose [77]. Using of different techniques in the determination of lipid peroxides in plasma or serum of healthy people (spectrophotometric versus HPLC method) leads to significantly different results. When using spectrophotometric techniques, the content of TBARS in plasma (serum) reached values from 0.9 to 42.7 µmol·L-1 of MDA equivalents, when HPLC technique was used, the content of TBARS in human plasma (serum) reached values of 0.6 – 1.4 µmol·L-1 of MDA equivalents [78-84]. This was probably caused by the using different methods for modifying the preparation of plasma (serum) sample. Method for the non-specific index of lipid peroxidation determination in isolated purified lipid fractions seems to be most useful [42].

**Figure 2.** Chromophore produced by a condensation of MDA with TBA

#### **1.4. Lipid hydroperoxides**

#### *1.4.1. Iodometric method*

134 Lipid Peroxidation

absorb at 532 nm, may react with TBA [71]. The results of the assay are expressed in µmol of MDA equivalents. TBARS method can be also used in the case of defined membrane systems, such as microsomes and liposomes, but its application in biological fluids and tissue extracts appears to be problematic [72-74]. The first problem is based on the fact that MDA can be formed by the decomposition of lipid peroxides under heating of the sample with TBA. This decomposition is accelerated by traces of iron in the reagents and is inhibited by the use of chelating agents [42]. At the decomposition of lipid peroxides in the analysis, the originating radicals can amplify the entire process and the amount of MDA could be overestimated [74]. To prevent the decomposition of lipid peroxides during the analysis, inhibitor of the lipid peroxidation called butylated hydroxytoluene is added to the sample [42]. One of the other problems of the TBARS method application has been found in the analysis of biological fluids. In this case, some substances, such as bile pigments and glycoproteins provide a false positive reaction with TBA [71,75]. Unspecificity TBARS test problems can be partially overcome by the using of HPLC techniques for the separation of "authentic", original MDA-(TBA)2 adduct from other chromogens absorbing at 532 nm [76]. Nevertheless, this approach cannot solve all problems. In addition, next molecules, such as aldehydes originated from lipid peroxidation, can form with TBA a original MDA-TBA2 adduct, which has been demonstrated in the deoxyribose [77]. Using of different techniques in the determination of lipid peroxides in plasma or serum of healthy people (spectrophotometric versus HPLC method) leads to significantly different results. When using spectrophotometric techniques, the content of TBARS in plasma (serum) reached values from 0.9 to 42.7 µmol·L-1 of MDA equivalents, when HPLC technique was used, the content of TBARS in human plasma (serum) reached values of 0.6 – 1.4 µmol·L-1 of MDA equivalents [78-84]. This was probably caused by the using different methods for modifying the preparation of plasma (serum) sample. Method for the non-specific index of lipid peroxidation

determination in isolated purified lipid fractions seems to be most useful [42].

**Figure 2.** Chromophore produced by a condensation of MDA with TBA

Iodometric method for lipid hydroperoxides determination is one of the oldest methods and is still used to determine lipid peroxide number [42,85]. Principle of this method is based on the ability of lipid hydroperoxides to oxidize iodide (I- ) to iodine (I2), which further reacts with unreacted iodide (I- ) to triiodide anion (I3- ) [86] and can be determined spectrophotometrically at 290 or 360 nm [87]. Modification of the iodometric method using commercially available reagent used for the determination of cholesterol can also be used to determine lipid (hydro)peroxides spectrophotometrically at 365 nm [54]. The method can be applied to extracts of biological samples without present the oxidizing agents. The possible interfering factors are especially the presence of oxygen, hydrogen peroxide and protein peroxides, which are able to oxidize iodide. Oxygen interference can be avoided by the using the anaerobic cuvettes and cadmium ions, which form a complex with unreacted iodide [86]. Values of lipid hydroperoxides in human plasma determined by iodometry are about 4 µmol.L-1 [88,89].

#### *1.4.2. Ferrous oxidation in xylenol orange*

Total hydroperoxides can be determined using the oxidation of ferrous ions in the test with xylenol orange (FOX). The principle of the FOX method is based on the oxidation of ferrous ions to ferric by the hydroperoxide activity in the acidic environment [90-94]. The exact mechanism of the sequence of radical reactions is not known, but the mechanism has been designed by Gupta et al. [95] and is shown in reactions 1-4 (equation 2) [96]. The increase in the concentration of ferric ion is then detected using xylenol orange (Fig. 3), which forms a blue-violet complex with ferric ion (equation 2, reaction 5) with an absorption maximum at 560 nm [35]. However, the experimentally determined stoichiometry of 3 moles of Fe3+ xylenol orange produced from 1 mol of peroxide [96,97] cannot be explained by the mechanism proposed by Gupta [95].

$$\begin{aligned} \text{(1) } & \text{Fe}^{2+} + \text{LOOH} + \text{H}^+ \rightarrow \text{Fe}^{3+} + \text{H}\_2\text{O} + \text{LO}^\*\\ \text{(2) } & \text{LO}^\* + \text{xylenol orange} + \text{H}^+ \rightarrow \text{LOH} + \text{xylenol orange}^\*\\ \text{(3) } & \text{Xylenol orange}^\* + \text{Fe}^{2+} \rightarrow \text{xylenol orange} + \text{Fe}^{3+} \\ \text{(4) } & \text{LO}^\* + \text{Fe}^{2+} + \text{H}^+ \rightarrow \text{Fe}^{3+} + \text{LOH} \end{aligned}$$

$$\begin{aligned} \text{(5) } & \text{Fe}^{3+} + \text{xylenol orange} \rightarrow \text{blue} - \text{violet complex (560 nm)} \end{aligned}$$

Gay et al. [90] have found during comparison of the reactions of different peroxides with FOX reagents that the stoichiometry of the reaction ranged from 2.2 (H2O2) to 5.3 moles (Cu-OOH, *t*-BuOOH) Fe3+-xylenol orange (Fe-XO) generated from 1 mol of peroxide, which was observed due to determination of molar absorption coefficients of Fe-XO complexes. Therefore, it is possible to compare only the results of FOX method analyses, in which the same type of peroxide was used in calibration. Hydrogen peroxide (H2O2) and Cumene hydroperoxide (Cu-OOH) are the most often peroxides used to calibrate the FOX method.

**Figure 3.** Structural formula of xylenol orange

The literature describes two versions of the FOX method called FOX1 and FOX2.. - FOX1 method can be used for the hydroperoxides determination in water phase and FOX2 method is suitable for the hydroperoxides of the lipid phase [30,35,98]. In the FOX1 method, chemicals used for a preparation of reagents (ferrous salt and sulphuric acid) are dissolved in water, whereas in FOX2 method methanol (90 % *v*/*v*) is the solvent [35]. FOX methods are not specific to hydroperoxides, the presence of oxidizing agent(s) in sample leads to the oxidization of ferrous ions to ferric ions. In the case of FOX2, the specificity of the method is achieved by the first FOX2 test performance in the presence of triphenylphosphine (TPP), which selectively reduces hydroperoxides to alcohols. The result of this test is used as a blank. After it, the FOX test without triphenylphosphine is performed and after deduction of blank values, we get the real value of lipid hydroperoxides. Improved specificity of the method using triphenylphosphine was later achieved also in FOX1 test [99]. Peroxidation chain reactions, which might occur during the analysis, are prevented by the addition of butylated hydroxytoluene prevented into the FOX1 agent. Plasma samples collected using ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid pentasodium salt abbreviated as DETAPAC (anticoagulants or iron chelating agents) cannot be used due to interference with FOX reagents [30]. FOX1 method has been automated [100].

#### *Measurement of lipid peroxidation in (blood) plasma*

Banerjee et al. [99] enhanced sensitivity of FOX1 method by the addition of sorbitol into the FOX1 reagent in accordance with Wolff [98], and concurrently by the stabilization of pH of reagents at the values of 1.7 - 1.8. Improved specificity of method was obtained using triphenylphosphine and butylated hydroxytoluene. A comparison of both FOX1 and FOX2 methods on plasma samples of healthy individuals and diabetic patients was performed, where modified FOX1 method was more sensitive compared to the FOX2 method. Another advantage of the FOX1 method was based on the skip the centrifugation step that is necessary in FOX2 method. Nourooz-zadeh et al. [55] determined total lipid hydroperoxides in plasma by the use the FOX2 method and subsequently monitored content of lipid hydroperoxides in individual lipoprotein fractions (VLDL, LDL and HDL fractions). Content of total lipid hydroperoxides in plasma was 3.50±2.05 µmol/L. The highest rate of hydroperoxides (67 %) was detected in LDL lipoprotein fractions. Södergren et al. [101] studied the impact of the storage of samples at low temperatures on the total lipid hydroperoxide content by the use the FOX2 method. They were focused on possible reduction of total lipid hydroperoxides content during the storage of samples under these conditions. Researchers found that storage of samples for 6 weeks at -70 °C leads to the 23 % average reduction of hydroperoxides content. The finding that the content of lipid hydroperoxides in short-term stored plasma samples (6 weeks) did not differ from the content of lipid hydroperoxides in the long-term stored samples (60 weeks) was interesting too.

#### *Measurement of lipid peroxidation in animal tissues*

136 Lipid Peroxidation

26 **Figure 3.** Structural formula of xylenol orange

*Measurement of lipid peroxidation in (blood) plasma* 

same type of peroxide was used in calibration. Hydrogen peroxide (H2O2) and Cumene hydroperoxide (Cu-OOH) are the most often peroxides used to calibrate the FOX method.

The literature describes two versions of the FOX method called FOX1 and FOX2.. - FOX1 method can be used for the hydroperoxides determination in water phase and FOX2 method is suitable for the hydroperoxides of the lipid phase [30,35,98]. In the FOX1 method, chemicals used for a preparation of reagents (ferrous salt and sulphuric acid) are dissolved in water, whereas in FOX2 method methanol (90 % *v*/*v*) is the solvent [35]. FOX methods are not specific to hydroperoxides, the presence of oxidizing agent(s) in sample leads to the oxidization of ferrous ions to ferric ions. In the case of FOX2, the specificity of the method is achieved by the first FOX2 test performance in the presence of triphenylphosphine (TPP), which selectively reduces hydroperoxides to alcohols. The result of this test is used as a blank. After it, the FOX test without triphenylphosphine is performed and after deduction of blank values, we get the real value of lipid hydroperoxides. Improved specificity of the method using triphenylphosphine was later achieved also in FOX1 test [99]. Peroxidation chain reactions, which might occur during the analysis, are prevented by the addition of butylated hydroxytoluene prevented into the FOX1 agent. Plasma samples collected using ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid pentasodium salt abbreviated as DETAPAC (anticoagulants or iron chelating agents) cannot be used due

to interference with FOX reagents [30]. FOX1 method has been automated [100].

Banerjee et al. [99] enhanced sensitivity of FOX1 method by the addition of sorbitol into the FOX1 reagent in accordance with Wolff [98], and concurrently by the stabilization of pH of reagents at the values of 1.7 - 1.8. Improved specificity of method was obtained using triphenylphosphine and butylated hydroxytoluene. A comparison of both FOX1 and FOX2 methods on plasma samples of healthy individuals and diabetic patients was performed, where modified FOX1 method was more sensitive compared to the FOX2 method. Another advantage of the FOX1 method was based on the skip the centrifugation step that is Hermes-Lima et al. [96] proposed and elaborated methodology for application of FOX1 test in determination of lipid hydroperoxides in animal tissue extracts. They used methanol extracts of kidney, liver and heart from adult mice (*Mus musculus* Linnaeus), brain and lungs from adult Wistar rats (*Rattus norvegicus* Berkenhout var. *alba*), liver and adipose tissues from adult golden-mantled ground squirrels (*Spermophilus lateralis* Say), and liver and muscle tissues from adult red-eared slider turtles (*Trachemis scripta elegans*  Wied-Neuwied). The highest values of lipid hydroperoxide content were detected in mice organs. The contents of lipid peroxides in animal tissues measured by the FOX1 method well correlated with results obtained by the TBARS. Grau et al. [102] adapted the FOX2 method for the determination of lipid hydroperoxides in raw and cooked dark chicken meat. Chickens were fed by a diet with different contents of α-tocopherol and fats from different sources. They determined the absolute values of lipid hydroperoxides in different experimental groups of chickens. Eymard et al. [56] modified the FOX1 method used by Hermes-Lima et al. [96] for the determination of lipid hydroperoxides in small pelagic fish. They used methanol extracts of ground tissues of the Atlantic horse mackerel (*Trachurus trachurus* Linnaeus). The original FOX1 reagent was replaced by the FOX2 reagent used by Wolff et al. [98] with the increased content of methanol to increase a solubility of extracts.

#### *Measurement of lipid peroxidation in plant tissues*

De Long et al. [44] applied the FOX2 method in the determination of hydroperoxides in plant tissues. They used ethanol extracts of pericarp of avocado (*Persea americana* P. Mill.), periderm of potatoes (*Solanum tuberosum* L.), leaves of red cabbage (*Brassica oleracea* convar. *capitata* var. *rubra* DC. Ranost)*,* leaves of spinach (*Spinacia oleracea* L.), pericarp of the European Pear (*Pyrus communis* L.) and fruits of red pepper (*Capsicum annuum* L.) for analyses. The effect of UV radiation on lipid peroxidation was monitored. Parts of plants were exposed to UV radiation for 10-12 days prior the extraction due to induction of lipid peroxidation in plants. Lipid hydroperoxides were determined by the FOX2, the TBARS and the iodometric methods. UV radiation induced an increase in lipid peroxidation values in all samples of different plant tissues determined by the FOX method. The good correlation was found between the FOX and iodometric methods. However, the iodometric method had limitations in the determination of the low concentrations of lipid hydroperoxides. Similar results were obtained by the use the TBARS method. Griffiths et al. [103] applied the FOX2 method in determination of lipid peroxides in different types of plant tissues. They analysed plant tissues, such as extracts of bean hypocotyls (*Phaseolus* sp.) and microsomes, potato leaves (*Solanum tuberosum* L.), flowers of alstromeria (*Alstroemeria* spp.), broccoli (*Brassica oleracea* var. *italica* Plenck) and cells of green algae (*Chlamydomonas* sp.). Lipid hydroperoxide levels ranged from 26 to 602 nmol.g-1 of FW. The highest content of lipid hydroperoxides was detected in broccoli and green alga cells in their study.

## **2. Experimental section**

#### **2.1. Instruments**

For dilution of stock solutions of standards an epMotion 5075 (Eppendorf, Germany) automated pipetting system was used (Fig. 4). The pipetting provides a robotic arm with adapters (TS 50, TS 300 and TS 1000) and Gripper (TG-T). The empty microtubes are placed in the position B3 (Fig. 4) in adapter Ep0.5/1.5/2 ml. Module Reservoir is located in the position B1, where stock solutions are available. The device is controlled by the epMotion control panel. The tips are located in the A4 (ePtips 50), A3 (ePtips 300) and A2 (ePtips 1000) positions. For preparation of the standards tips of sizes 300 µl and 1000 µl (Eppendorf – Germany) were used. For determination of antioxidant activity, a BS-400 automated spectrophotometer (Mindray, China) was used. It is composed of cuvette space tempered to 37±1 °C, reagent space with a carousel for reagents (tempered to 4±1 °C), sample space with a carousel for preparation of samples and an optical detector. Transfer of samples and reagents is provided by robotic arm equipped with a dosing needle (error of dosage up to 3 % of volume). Cuvette content is mixed by an automatic mixer including a stirrer immediately after addition of reagents or samples. Contamination is reduced due to its rinsing system, including rinsing of the dosing needle as well as the stirrer by MilliQ water. For detection itself, the following range of wave lengths can be used as 340, 380, 412, 450, 505, 546, 570, 605, 660, 700, 740 and 800 nm. In addition, a SPECOL 210 two beam UV-VIS spectrophotometer (Analytik Jena AG, Germany) with cooled semiconductor detector for measurement within range from 190 to 1,100 nm with control by an external PC with the programme WinASPECT was used as the manual instrument in this study. Laboratory scales (Sartorius, Germany) and pipettes (Eppendorf Research, Germany) were used.

#### **2.2. Chemicals**

Xylenol orange disodium salt, iron D-gluconate dihydrate, glycerol, *tert*-butylhydroperoxide (t-BHP) 70% in water, sodium chloride, sulphuric acid, formic acid and water ACS reagent were purchased from Sigma Aldrich (USA).

#### **2.3. Preparation of reagents and standards**

138 Lipid Peroxidation

the iodometric methods. UV radiation induced an increase in lipid peroxidation values in all samples of different plant tissues determined by the FOX method. The good correlation was found between the FOX and iodometric methods. However, the iodometric method had limitations in the determination of the low concentrations of lipid hydroperoxides. Similar results were obtained by the use the TBARS method. Griffiths et al. [103] applied the FOX2 method in determination of lipid peroxides in different types of plant tissues. They analysed plant tissues, such as extracts of bean hypocotyls (*Phaseolus* sp.) and microsomes, potato leaves (*Solanum tuberosum* L.), flowers of alstromeria (*Alstroemeria* spp.), broccoli (*Brassica oleracea* var. *italica* Plenck) and cells of green algae (*Chlamydomonas* sp.). Lipid hydroperoxide levels ranged from 26 to 602 nmol.g-1 of FW. The highest content of lipid hydroperoxides

For dilution of stock solutions of standards an epMotion 5075 (Eppendorf, Germany) automated pipetting system was used (Fig. 4). The pipetting provides a robotic arm with adapters (TS 50, TS 300 and TS 1000) and Gripper (TG-T). The empty microtubes are placed in the position B3 (Fig. 4) in adapter Ep0.5/1.5/2 ml. Module Reservoir is located in the position B1, where stock solutions are available. The device is controlled by the epMotion control panel. The tips are located in the A4 (ePtips 50), A3 (ePtips 300) and A2 (ePtips 1000) positions. For preparation of the standards tips of sizes 300 µl and 1000 µl (Eppendorf – Germany) were used. For determination of antioxidant activity, a BS-400 automated spectrophotometer (Mindray, China) was used. It is composed of cuvette space tempered to 37±1 °C, reagent space with a carousel for reagents (tempered to 4±1 °C), sample space with a carousel for preparation of samples and an optical detector. Transfer of samples and reagents is provided by robotic arm equipped with a dosing needle (error of dosage up to 3 % of volume). Cuvette content is mixed by an automatic mixer including a stirrer immediately after addition of reagents or samples. Contamination is reduced due to its rinsing system, including rinsing of the dosing needle as well as the stirrer by MilliQ water. For detection itself, the following range of wave lengths can be used as 340, 380, 412, 450, 505, 546, 570, 605, 660, 700, 740 and 800 nm. In addition, a SPECOL 210 two beam UV-VIS spectrophotometer (Analytik Jena AG, Germany) with cooled semiconductor detector for measurement within range from 190 to 1,100 nm with control by an external PC with the programme WinASPECT was used as the manual instrument in this study. Laboratory

scales (Sartorius, Germany) and pipettes (Eppendorf Research, Germany) were used.

Xylenol orange disodium salt, iron D-gluconate dihydrate, glycerol, *tert*-butylhydroperoxide (t-BHP) 70% in water, sodium chloride, sulphuric acid, formic acid and water ACS reagent

was detected in broccoli and green alga cells in their study.

**2. Experimental section** 

**2.1. Instruments** 

**2.2. Chemicals** 

were purchased from Sigma Aldrich (USA).

FOX1 reagents were prepared according Arab et al. [100]. The general acidic reagent (acidic reagent A) final concentrations were 0.9 % NaCl, 40 mM H2SO4, 20 mM formic acid and 1.37 M glycerol in ACS water. The pH of the reagent was adjusted to the value of 1.35. The reagent R1 contained 167 µM xylenol orange disodium salt, which was dissolved in acidic reagent A. The reagent R2 contained 833 µM iron D-gluconate dehydrate, which was also dissolved in acidic reagent A. Standards were prepared from the 70% water solution of *tert*butylhydroperoxide, which was diluted by ACS water to the 20 mM pre-stock solution. From the pre-stock solution, five stock solutions: and 0.2, 3.9, 62.5, 375 and 1,000 µM were prepared daily by dilutions of pre-stock solution with 0.9 % NaCl. For further preparation of 20 standards from five stock solutions, an automated pipetting system epMotion 5075 was used to minimalize possible pipetting errors. The standards had following concentrations: 0.06, 0.12, 0.24, 0.48, 0.97, 1.9, 3.9, 7.8, 15.6, 31.2, 46.8, 62.5, 93.7, 125, 187, 250, 375, 500, 750 and 1000 µM. These standards were used for the preparation of calibration curves in both manual and automatic measurements.

#### **2.4. Working procedure for manual spectrophotometric determination**

A volume of 720 µl of the reagent R1 (167 µM xylenol orange in acidic reagent) was pipetted into plastic cuvettes. Subsequently, a volume of 100 µl of the sample was added. Absorbance was measured at λ = 591 nm. After it, a volume of 180 µl of the reagent R2 (833 µM iron D-gluconate in acidic reagent A) was pipetted to a reaction mixture and after 6 minutes of the incubation, absorbance was measured. Final value is calculated from the absorbance value of the mixture of the reagent R1 with sample and from the absorbance value after 6 minutes of incubation of the mixture with the reagent R2. The final concentrations in the cuvette of xylenol orange (R1) and iron D-gluconate (R2) were 120 and 150 µM, respectively.

#### **2.5. Working procedure for automated spectrophotometric determination**

A volume of 180 µL of the solution R1 (167 µM xylenol orange in acidic reagent) was pipetted into a plastic cuvette with subsequent addition of a 25 µL of sample. This mixture was incubated for 4.5 minutes. Subsequently, 45 µL of solution R2 (833 µM iron D-gluconate in acidic reagent) was added and the solution was incubated for next 6 minutes. Absorbance was measured at λ = 570 nm. Final value is calculated from the absorbance value of the mixture of reagent R1 with sample before the addition of the reagent 2 and from the absorbance value after 6 minutes of incubation of the mixture with the reagent 2. The final concentrations in the cuvette of xylenol orange (R1) and iron D-gluconate (R2) were 120 and 150 µM, respectively.

#### **3. Results and discussion**

Spectrophotometric methods for determination of lipid peroxidation have a relatively simple procedure of a measurement. In addition, they are relatively low-cost with easy applicability and they do not require specialized equipment or personnel. To maintain the sustainability of these methods, it is necessary to introduce these methods to automated operation, which has not been yet satisfactorily solved. Analyses of samples performed due to intensive work of personnel, which is expensive, slow, and, in addition, the human factor is responsible for a high percentage of errors. Requirement for laboratories, in which a large number of samples is analysed per day, consists in relatively simple and easy to apply method. Our aim was to automate the pre-analytical and analytical phase of the FOX1 method. For specification and comparison of this method, the method based on the use the manual spectrophotometer was also carried out.

## **3.1. Pre-analytical phase**

Pre-analytical processing of biological samples in the laboratory is a necessary and important part of laboratory work. It represents a wide range of manual, often stereotyped operations that do not require special knowledge and skills, but require maintenance of the standard procedure(s) and prevent the possibility of errors connected with this analytical phase. Pre-analytical laboratory process is destined to automation and robotics. Automation and robotics of the pre-analytical phase brings many benefits and advantages to laboratory. It reduces the number of errors, the time necessary for sample manipulation, and the response time. It significantly increases the productivity, cost savings connected with productivity, and minimizes the exposure of personnel with biological material [104].

For automation of pre-analytical phase, the epMotion 5075 automated pipetting system was used. Stock solutions of *tert*-butylhydroperoxide (*t*-BHP) at the concentrations of 1000, 375, 62.5, 3.9 and 0.2 µM prepared in 0.9 % NaCl solution were applied into five vials. Sixth vial contained diluting solution (0.9% NaCl). Twenty empty Eppendorf tubes (1.5 ml) were placed into the metal holder. Scheme of the preparation of standards is shown in Table 2. Pipetting robot first pipetted different volumes of diluting solution (0.9% NaCl) into vials and after it, different volumes of stock solutions of various concentrations of *t*-BHP were pipetted. When pipetting the stock solution into the dilution buffer in micro test tube, robot three times mixed the solution by a pipetting.

**Figure 4.** epMotion 5075 automated pipetting system from frontal part.



**Table 2.** Volume of the solution in the preparation of standards using epMotion 5075 automated pipetting system.

Using the epMotion 5075 automated pipetting system, work time of 20 minutes was saved (time, when laboratory staff was not needed). The only time-demanding operation consisted in replenishment of vials and initiation of the program. Potential errors that arise due to human activity were avoided. Accuracy of a pipetting was verified by weighing, the average error was approximately 1.8 %.

#### **3.2. Analytical phase**

140 Lipid Peroxidation

manual spectrophotometer was also carried out.

three times mixed the solution by a pipetting.

**Figure 4.** epMotion 5075 automated pipetting system from frontal part.

**3.1. Pre-analytical phase** 

biological material [104].

applicability and they do not require specialized equipment or personnel. To maintain the sustainability of these methods, it is necessary to introduce these methods to automated operation, which has not been yet satisfactorily solved. Analyses of samples performed due to intensive work of personnel, which is expensive, slow, and, in addition, the human factor is responsible for a high percentage of errors. Requirement for laboratories, in which a large number of samples is analysed per day, consists in relatively simple and easy to apply method. Our aim was to automate the pre-analytical and analytical phase of the FOX1 method. For specification and comparison of this method, the method based on the use the

Pre-analytical processing of biological samples in the laboratory is a necessary and important part of laboratory work. It represents a wide range of manual, often stereotyped operations that do not require special knowledge and skills, but require maintenance of the standard procedure(s) and prevent the possibility of errors connected with this analytical phase. Pre-analytical laboratory process is destined to automation and robotics. Automation and robotics of the pre-analytical phase brings many benefits and advantages to laboratory. It reduces the number of errors, the time necessary for sample manipulation, and the response time. It significantly increases the productivity, cost savings connected with productivity, and minimizes the exposure of personnel with

For automation of pre-analytical phase, the epMotion 5075 automated pipetting system was used. Stock solutions of *tert*-butylhydroperoxide (*t*-BHP) at the concentrations of 1000, 375, 62.5, 3.9 and 0.2 µM prepared in 0.9 % NaCl solution were applied into five vials. Sixth vial contained diluting solution (0.9% NaCl). Twenty empty Eppendorf tubes (1.5 ml) were placed into the metal holder. Scheme of the preparation of standards is shown in Table 2. Pipetting robot first pipetted different volumes of diluting solution (0.9% NaCl) into vials and after it, different volumes of stock solutions of various concentrations of *t*-BHP were pipetted. When pipetting the stock solution into the dilution buffer in micro test tube, robot

> Our goal was to introduce the FOX1 method to an automated operation and improve both analysis itself and conditions of analysis. The experiment was carried out using *tert*butylhydroperoxide standard prepared at the concentrations from 0.06 to 1000 µM. Furthermore, the spectral curves of generated chromatic complexes were observed and the concentration dependence on temperature and time were determined. In addition, reaction kinetics during the reaction was established.

#### *3.2.1. Monitoring the spectral courses at different concentrations and times*

Spectral changes in the *t*-BHP concentration range from 0.06 to 1000 µM (Figures 5A and 4B) were observed. Two peaks at the wavelengths of 444 and 591 nm were detected in the formed complex at the recommended temperature of interaction of 37 °C.

**Figure 5.** Courses of spectra of *t*-BHP in the concentrations from 0.06 to 1000 µM - **a)** 1000, **b)** 500, **c)** 250, **d)** 125, **e)** 62.50, **f)** 31., **g)** 7.8, **h)** 1.9, **i)** 0.4, **j)** 0.06 in the time of 6 **(A)** and 60 **(B)** minutes. **(C)** Comparison of values of absorption maximum at the wavelength of 591 nm and a time period of 6 and 60 minutes. The courses were measured in the interval form 350 to 700 nm using the SPECORD 210 apparatus. All analyses were carried out in triplicates.

Absorption maximum at low concentrations (up to the concentration of 0.122 µM) was at 444 nm, and with the increasing concentrations (higher than 0.122 µM) the absorption maximum was sifted and observed at 591 nm. Interaction of sample and reagents proceeded in six minutes, after this time, absorbance could be measured and the final value of lipid peroxidation calculated. We wanted to determine the changes in the absorbance during one hour. Comparison of absorbance values at the time of 6 and 60 min at λ = 591 nm is shown in Figure 5C. Absorbance values during the monitoring decreased for about 13 % on an average. When interlaying the trends points in the linear concentration part from 0.12 to 125 µM, the determination factor decreased from 0.996 (for the 6-minute reaction time) to 0.987 (for the 60-minute reaction time). This fact can be explained by unequal reaction kinetics during the analysis (see the reaction kinetics, Chapter 3.2.3) and oxidation of the sample during the analysis.

#### *3.2.2. Monitoring the reaction under different temperature conditions*

142 Lipid Peroxidation

*3.2.1. Monitoring the spectral courses at different concentrations and times* 

formed complex at the recommended temperature of interaction of 37 °C.

Spectral changes in the *t*-BHP concentration range from 0.06 to 1000 µM (Figures 5A and 4B) were observed. Two peaks at the wavelengths of 444 and 591 nm were detected in the

**Figure 5.** Courses of spectra of *t*-BHP in the concentrations from 0.06 to 1000 µM - **a)** 1000, **b)** 500, **c)** 250, **d)** 125, **e)** 62.50, **f)** 31., **g)** 7.8, **h)** 1.9, **i)** 0.4, **j)** 0.06 in the time of 6 **(A)** and 60 **(B)** minutes. **(C)** Comparison of values of absorption maximum at the wavelength of 591 nm and a time period of 6 and 60 minutes. The courses were measured in the interval form 350 to 700 nm using the SPECORD

Absorption maximum at low concentrations (up to the concentration of 0.122 µM) was at 444 nm, and with the increasing concentrations (higher than 0.122 µM) the absorption maximum was sifted and observed at 591 nm. Interaction of sample and reagents proceeded in six minutes, after this time, absorbance could be measured and the final value of lipid peroxidation calculated. We wanted to determine the changes in the absorbance during one hour. Comparison of absorbance values at the time of 6 and 60 min at λ = 591 nm is shown in Figure 5C. Absorbance values during the monitoring decreased for about 13 % on an average. When interlaying the trends points in the linear concentration part from 0.12 to 125 µM, the determination factor decreased from 0.996 (for the 6-minute reaction time) to 0.987 (for the 60-minute reaction time). This fact can be explained by unequal reaction kinetics during the analysis (see the reaction kinetics, Chapter 3.2.3) and oxidation of the sample

210 apparatus. All analyses were carried out in triplicates.

during the analysis.

Dependences of representative concentration (62.5 µM) on the temperature conditions (17, 27, 37 and 47 °C) and the time from 0 to 30 minutes and absorption maximum of 591 nm is shown in Figure 6. The absorbance increased with the increasing temperature; after 6 minutes of reaction, the difference of absorbance value between the lowest (17 °C) and the highest (47 °C) temperature was about 0.64 AU. In other words, the value of absorbance at 47 °C was higher for 71 % compared to the absorbance determined at 17 °C. The highest values of absorbance and concurrently the most prominent difference was detected at 47 °C, therefore, this temperature was the most suitable for our purposes. On the other hand, this temperature may lead to degradation of biological samples. Due to this fact, the temperature of interaction of 37 °C was selected for further analyses.

**Figure 6.** Dependences of representative concentration (62.5 µM) of applied *t*-BHP on temperature conditions (17, 27, 37 and 47 °C) and the time of interaction. Detected at 591 nm, interval of record is 1 minute, interval period 0 - 30 minutes. All analyses were carried out in triplicates.

#### *3.2.3. Determination of reaction kinetics*

Reaction kinetics at the temperature of 37 °C in the shortest time intervals in all concentrations (0.06 – 1000 µM) was monitored. Automated analyser BS-400 was used for this purpose. All samples could be studied at all once. This is not possible using the manual spectrophotometer, thus, use the automated analyser represents one of the most important steps in the analysis automation.

The curves were used for the calculating the reaction rate constants indicating the course and conception of the impact of the effect of *t*-BHP concentration on the reaction rate. The constant was calculated as the change in the absorbance per time unit (second, minute) according to the equation x = A/t, where x is the rate constant, A the value of absorbance after 6 minutes and t time for which the rate constant was related (second, minute). The effect of each of concentrations on the change in absorbance value was determined.

**Figure 7.** Monitoring of reaction curves of *t*-TBH in the concentrations from 0.06 to 1000 µM - **a)** 1000, **b)** 750, **c)** 500, **d)** 375, **e)** 250, **f)** 187, **g)** 125 **h)** 94, **i)** 63, **j)** 47., **k)** 31, **l)** 15.6, **m)** 7.8, **n)** 3.9, **o)** 1.9, **p)** 0.9, **q)** 0.4, **r)** 0.2, **s)** 0.1, and **t)** 0.06 µM in the time interval from 0 to 6 minutes. All analyses were carried out in triplicates.


**Table 3.** Mathematical formularization of the course of reaction curves for *t*-TBH in the concentration range from 0.06 to 1000 µM by the use the logarithmic equation. Reaction rate constant is expressed as a change in absorbance per second, and per minute. In addition, change in absorbance per minute recalculated to 1 µM t-BHP is introduced.

#### *3.2.4. Dependence on concentration*

144 Lipid Peroxidation

in triplicates.

**Concentration Logarithmic equation** 

**Figure 7.** Monitoring of reaction curves of *t*-TBH in the concentrations from 0.06 to 1000 µM - **a)** 1000, **b)** 750, **c)** 500, **d)** 375, **e)** 250, **f)** 187, **g)** 125 **h)** 94, **i)** 63, **j)** 47., **k)** 31, **l)** 15.6, **m)** 7.8, **n)** 3.9, **o)** 1.9, **p)** 0.9, **q)** 0.4, **r)** 0.2, **s)** 0.1, and **t)** 0.06 µM in the time interval from 0 to 6 minutes. All analyses were carried out

> **Change in absorbance per second**

1000 y = 3.7532ln(x) - 5.899 0.02304 1.383 0.0013 750.0 y = 3.6495ln(x) - 5.544 0.02211 1.345 0.0017 500.0 y = 3.4895ln(x) - 5.241 0.02168 1.301 0.0028 375.0 y = 3.1895ln(x) - 4.872 0.01987 1.258 0.0036 250.0 y = 3.2076ln(x) - 4.677 0.01853 1.112 0.0044 187.5 y = 2.7574ln(x) - 4.375 0.01534 0.924 0.0052 125.0 y = 2.2477ln(x) - 3.945 0.01298 0.779 0.0062 93.75 y = 1.7316ln(x) - 2.968 0.01000 0.600 0.0060 62.50 y = 1.2213ln(x) - 1.998 0.00705 0.423 0.0068 46.87 y = 1.0049ln(x) - 1.596 0.00580 0.348 0.0070 31.25 y = 0.7102ln(x) - 1.054 0.00410 0.246 0.0079 15.62 y = 0.3846ln(x) - 0.445 0.00222 0.133 0.0085 7.812 y = 0.2525ln(x) - 0.183 0.00146 0.088 0.0112 3.906 y = 0.1765ln(x) - 0.037 0.00102 0.061 0.0157 1.953 y = 0.1303ln(x) + 0.033 0.00075 0.045 0.0231 0.976 y = 0.1177ln(x) + 0.073 0.00068 0.041 0.0418 0.488 y = 0.1031ln(x) + 0.089 0.00060 0.036 0.0732 0.244 y = 0.0965ln(x) + 0.101 0.00057 0.034 0.1370 0.122 y = 0.0926ln(x) + 0.105 0.00055 0.033 0.2629 0.061 y = 0.0957ln(x) + 0.131 0.00053 0.032 0.5434 **Table 3.** Mathematical formularization of the course of reaction curves for *t*-TBH in the concentration range from 0.06 to 1000 µM by the use the logarithmic equation. Reaction rate constant is expressed as a

**Change in absorbance per minute** 

**Change in abs. per minute recalculated to 1 µM t-BHP** 

By the using manual spectrophotometer and automated analyser, the dependence of *t*-TBH concentration (0.06 – 1000 µM) on the changes of coloured complex was determined. The calibration curves were calculated from final values.

**Figure 8.** Dependence of absorbance on applied *t*-BHP concentration measured by manual spectrophotometer SPECOL 210 and automated analyser BS-400. All analyses were carried out in triplicates. For other experimental detail, see Fig. 7.

The analysis of 60 samples (20 samples in a standard three repetitions) took using the BS-400 automated analyser only 24 minutes. The analysis of 60 samples including delays for the pipetting, mixing and displacement of samples using the manual spectrophotometer took about 7 hours (6 minutes per sample + one minute of delay, 60 × 6 minutes of sample analysis). By using the fully automated analyser, results were obtained in more than 17 times less time compared to manual spectrophotometer. Shortening of the time of analysis contributes especially to higher quality of results due to reduction of possibility of chemical modification including degradation of the measured samples. This fact resulted in the preparation of calibration curves, where the determination factor for the calibration curve obtained using the automatic analyser was R2 = 0.9996, while the determination factor for the results from manual spectrophotometer was R2 = 0.9966. In addition, a limit of detection (LOD) and limit of quantification (LOQ) were determined. In the case of both automated and manual analyses, the LOD was determined as LOD = 0.06 µM of *t*-BHP, limit of quantification (LOQ) was also determined as LOQ = 0.2 µM of *t*-BHP (see Table 3). All measurements of all concentrations of *t*-BHP (concentration range from 0.06 to 1000 µM) were carried out in 3 repetitions and repeatability (RSD) was determined. In the case of automated method, the repeatability was RSD = 2.6 % compared to manual spectrophotometer, where RSD = 3.8 %.

Technical development is responsible for a tendency to increase the speed of analysis and analytical process itself. Automatic analysers allow analysing more samples at the same time, reducing the time required to analyse one sample and errors caused by incorrect

pipetting and manipulation with sample, and generally provide higher data quality compared to manual analysis. Due to automation, the risk of sample confusion is significantly reduced. In addition, the whole process is much faster, the consumption of reagents and demands of personnel staff are reduced. The aim of automation is to eliminate stereotypical incompetent operation, eliminate the possibility of error, and to accelerate operations under significant increase of capacity while maintaining the precise performance of all necessary operations. The disadvantage, however, consists in still high acquisition costs and the need for compete service [105,106].


**Table 4.** Analytic parameters for the FOX1 method for *t*-BHP analysis using manual SPECOL and automated BS-400 analysers.

#### **4. Conclusion**

This chapter brought a comprehensive overview of photometric methods used in the study of lipid peroxidation. Main attention was devoted to the detection of lipid peroxidation by using the less common FOX1 method. The proposal to automation the pre-analytical and analytical phases of the sample was introduced. In addition, conditions and parameters influencing the photometric reaction were studied and described. The comparison of results obtained using the manual and automated apparatuses (manual/automated operation) is introduced and discussed.

## **Author details**

Jiri Sochor, Branislav Ruttkay-Nedecky, Vojtech Adam, Jaromir Hubalek and Rene Kizek\* *Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Brno, Czech Republic, European Union Department of Microelectronics, Faculty of Electrical Engineering and Communication,* 

*Brno University of Technology, Brno, Czech Republic, European Union* 

#### Petr Babula

*Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic, European Union* 

<sup>\*</sup> Corresponding Author

#### **Acknowledgement**

This project was supported by SIX research centre CZ.1.05/2.1.00/03.0072. The authors wish to express their thanks to Lukáš Nejdl for excellent technical assistance.

#### **5. References**

146 Lipid Peroxidation

costs and the need for compete service [105,106].

SPECOL 591 0.06 0.2 0.012

BS-400 570 0.06 0.2 0.012

(nm) LOD LOQ Measuring

Apparatus Wavelength

automated BS-400 analysers.

introduced and discussed.

*Brno, Czech Republic, European Union* 

*Department of Natural Drugs, Faculty of Pharmacy,* 

**Author details** 

Petr Babula

Corresponding Author

 \*

**4. Conclusion** 

pipetting and manipulation with sample, and generally provide higher data quality compared to manual analysis. Due to automation, the risk of sample confusion is significantly reduced. In addition, the whole process is much faster, the consumption of reagents and demands of personnel staff are reduced. The aim of automation is to eliminate stereotypical incompetent operation, eliminate the possibility of error, and to accelerate operations under significant increase of capacity while maintaining the precise performance of all necessary operations. The disadvantage, however, consists in still high acquisition

range (µM)



This chapter brought a comprehensive overview of photometric methods used in the study of lipid peroxidation. Main attention was devoted to the detection of lipid peroxidation by using the less common FOX1 method. The proposal to automation the pre-analytical and analytical phases of the sample was introduced. In addition, conditions and parameters influencing the photometric reaction were studied and described. The comparison of results obtained using the manual and automated apparatuses (manual/automated operation) is

Jiri Sochor, Branislav Ruttkay-Nedecky, Vojtech Adam, Jaromir Hubalek and Rene Kizek\* *Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno,* 

*University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic, European Union* 

*Department of Microelectronics, Faculty of Electrical Engineering and Communication,* 

*Brno University of Technology, Brno, Czech Republic, European Union* 

**Table 4.** Analytic parameters for the FOX1 method for *t*-BHP analysis using manual SPECOL and

Calibration equation

y=0.0105x

y=0.0107x

Confidence coefficient (R2)

+0.006 0.9969 3.8 420

+0.0128 0.9996 2.6 24

RSD

Time analysis of 60 samples (min)


stages of ripening on chemical compounds in Medlar (Mespilus germanica L.). Molecules. 16: 74-91.


[25] Esterbauer H, Comporti M, Benedetti A (1980) Biochemical effects of 4 hydroxyalkenals, in particular 4-hydroxynonenal produced by microsomal lipidperoxidation. J. Am. Oil Chem. Soc. 57: A144-A145.

148 Lipid Peroxidation

Molecules. 16: 74-91.

Care. 15: 99-106.

145: 217-221.

S104.

128.

from Central Europe. J. Food Qual. 34: 187-194.

breast cancer. J. Cell. Physiol. 227: 1577-1582.

m(5)dC rates. Ecotox. Environ. Safe. 76: 63-70.

Environ. Mutagen. 726: 98-103.

Biochim. Biophys. Acta. 620: 281-296.

Mutat. Res.-Fundam. Mol. Mech. Mutagen. 729: 41-51.

product of lipid peroxidation. J. Proteomics. 74: 2360-2369.

stages of ripening on chemical compounds in Medlar (Mespilus germanica L.).

[14] Rop O, Jurikova T, Sochor J, Mlcek J, Kramarova D (2011) Antioxidant capacity, scavenging radical activity and selected chemical composition of native apple cultivars

[15] Balestrieri ML, Dicitore A, Benevento R, Di Maio M, Santoriello A, Canonico S, Giordano A, Stiuso P (2012) Interplay between membrane lipid peroxidation, transglutaminase activity, and Cyclooxygenase 2 expression in the tissue adjoining to

[16] Cai F, Dupertuis YM, Pichard C (2012) Role of polyunsaturated fatty acids and lipid peroxidation on colorectal cancer risk and treatments. Curr. Opin. Clin. Nutr. Metab.

[17] Zalejska-Fiolka J, Wielkoszynski T, Kasperczyk S, Kasperczyk A, Birkner E (2012) Effects of Oxidized Cooking Oil and alpha-Lipoic Acid on Blood Antioxidants: Enzyme Activities and Lipid Peroxidation in Rats Fed a High-Fat Diet. Biol. Trace Elem. Res.

[18] Flohr L, Fuzinatto CF, Melegari SP, Matias WG (2012) Effects of exposure to soluble fraction of industrial solid waste on lipid peroxidation and DNA methylation in erythrocytes of Oreochromis niloticus, as assessed by quantification of MDA and

[19] Janowska B, Kurpios-Piec D, Prorok P, Szparecki G, Komisarski M, Kowalczyk P, Janion C, Tudek B (2012) Role of damage-specific DNA polymerases in M13 phage mutagenesis induced by a major lipid peroxidation product trans-4-hydroxy-2-nonenal.

[20] Demir E, Kaya B, Soriano C, Creus A, Marcos R (2011) Genotoxic analysis of four lipidperoxidation products in the mouse lymphoma assay. Mutat. Res. Genet. Toxicol.

[21] Guo LL, Chen ZY, Cox BE, Gragg S, Zhang YQ, Amarnath V, van Lenten B, Epand R, Davies SS (2011) Lipid Peroxidation Generates Aldehyde-Modified Phosphatidylethanolamines That Induce Inflammation. Free Radic. Biol. Med. 51: S103-

[22] Guo J, Prokai L (2011) To tag or not to tag: A comparative evaluation of immunoaffinity-labeling and tandem mass spectrometry for the identification and localization of posttranslational protein carbonylation by 4-hydroxy-2-nonenal, an end-

[23] Esterbauer H, Schaur RJ, Zollner H (1991) Chemistry and biochemistry of 4 hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11: 81-

[24] Benedetti A, Comporti M, Esterbauer H (1980) Identification of 4-hydroxynoneal as a cyto-toxic product originating from the peroxidation of liver microsomal lipids.


Peroxidation and Antioxidant Enzymes Activity of Wistar Rats Experimentally Infected with Leptospira interrogans. Acta Sci. Vet. 39: 966-976.

[54] Elsaadani M, Esterbauer H, Elsayed M, Goher M, Nassar AY, Jurgens G (1989) A spectrophotometric assay for lipid peroxides in serum-lipoproteins using a commercially available reagent. J. Lipid Res. 30: 627-630.

150 Lipid Peroxidation

11: 4631-4659.

22: 200-209.

308.

78: 1776-1784.

40-46.

[39] Janicka M, Kot-Wasik A, Kot J, Namiesnik J (2010) Isoprostanes-Biomarkers of Lipid Peroxidation: Their Utility in Evaluating Oxidative Stress and Analysis. Int. J. Mol. Sci.

[40] Soffler C, Campbell VL, Hassel DM (2010) Measurement of urinary F(2)-isoprostanes as markers of in vivo lipid peroxidation: a comparison of enzyme immunoassays with gas chromatography mass spectrometry in domestic animal species. J. Vet. Diagn. Invest.

[41] Gutteridge JMC, Halliwell B (1990) The measurement and mechanism of lipid-

[42] Halliwell B, Chirico S (1993) Lipid-peroxidation - its mechanism, measurement, and

[43] Devasagayam TPA, Boloor KK, Ramasarma T (2003) Methods for estimating lipid peroxidation: An analysis of merits and demerits. Indian J. Biochem. Biophys. 40: 300-

[44] DeLong JM, Prange RK, Hodges DM, Forney CF, Bishop MC, Quilliam M (2002) Using a modified ferrous oxidation-xylenol orange (FOX) assay for detection of lipid

[45] Esterbauer H, Striegl G, Puhl H, Rotheneder M (1989) Continuous monitoring of invitro oxidation of human low-density lipoprotein. Free Rad. Res. Commun. 6: 67-75. [46] Yagi K (1984) Assay for blood-plasma or serum Methods Enzymol. 105: 328-331.

[47] Hong R, Kang TY, Michels CA, Gadura N (2012) Membrane Lipid Peroxidation in Copper Alloy-Mediated Contact Killing of Escherichia coli. Appl. Environ. Microbiol.

[48] Martins DB, Mazzanti CM, Franca RT, Pagnoncelli M, Costa MM, de Souza EM, Goncalves J, Spanevello R, Schmatz R, da Costa P, Mazzanti A, Beckmann DV, Cecim MD, Schetinger MR, Lopes STD (2012) 17-beta estradiol in the acetylcholinesterase activity and lipid peroxidation in the brain and blood of ovariectomized adult and

[49] Kwok CT, van de Merwe JP, Chiu JMY, Wu RSS (2012) Antioxidant responses and lipid peroxidation in gills and hepatopancreas of the mussel Perna viridis upon exposure to the red-tide organism Chattonella marina and hydrogen peroxide. Harmful Algae. 13:

[50] Ahmad MK, Syma S, Mahmood R (2011) Cr(VI) Induces Lipid Peroxidation, Protein Oxidation and Alters the Activities of Antioxidant Enzymes in Human Erythrocytes.

[51] Naziroglu M, Akkus S, Celik H (2011) Levels of lipid peroxidation and antioxidant vitamins in plasma and erythrocytes of patients with ankylosing spondylitis. Clin.

[52] Pimentel VC, Pinheiro FV, Kaefer M, Moresco RN, Moretto MB (2011) Assessment of uric acid and lipid peroxidation in serum and urine after hypoxia-ischemia neonatal in

[53] Tonin AA, Thome GR, Calgaroto N, Baldissarelli J, Azevedo MI, Escobar TP, dos Santos LG, Da Silva AS, Badke MRT, Schetinger MR, Mazzanti CM, Lopes STD (2011) Lipid

peroxidation in biological-systems. Trends Biochem.Sci. 15: 129-135.

hydroperoxides in plant tissue. J. Agric. Food Chem. 50: 248-254.

significance Am. J. Clin. Nutr. 57: S715-S725.

middle-aged rats. Life Sciences. 90: 351-359.

Biol. Trace Elem. Res. 144: 426-435.

Biochem. 44: 1412-1415.

rats. Neurol. Sci. 32: 59-65.


[86] Jessup W, Dean RT, Gebicki JM (1994) Iodometric determination of hydroperoxides in lipids and proteins. Oxygen Radicals in Biological Systems, Pt C. 233: 289-303.

152 Lipid Peroxidation

[69] Mohebbi-Fani M, Mirzaei A, Nazifi S, Shabbooie Z (2012) Changes of vitamins A, E, and C and lipid peroxidation status of breeding and pregnant sheep during dry seasons on

[70] Keles H, Ince S, Kucukkurt I, Tatli, II, Akkol EK, Kahraman C, Demirel HH (2012) The effects of Feijoa sellowiana fruits on the antioxidant defense system, lipid peroxidation,

[71] Kosugi H, Kato T, Kikugawa K (1987) Formation of yellow, orange, and red pigments in the reaction of alk-2-enals with 2-thiobarbituric acid. Anal. Biochem. 165: 456-464. [72] Frankel EN (1991) Recent advances in lipid oxidation. J. Sci. Food Agric. 54: 495-511. [73] Bonnestaourel D, Guerin MC, Torreilles J (1992) Is malonaldehyde a valuable indicator

[74] Lapenna D, Cuccurullo F (1993) TBA test and free MDA assay in evaluation of lipidperoxidation and oxidative stress in tissue systems Am. J. Physiol. 265: H1030-H1031. [75] Gutteridge JMC, Tickner TR (1978) Thiobarbituric acid reactivity of bile-pigments.

[76] Largilliere C, Melancon SB (1988) Free malondialdehyde determination in humanplasma by high-performance liquid-chromatography. Anal. Biochem. 170: 123-126. [77] Halliwell B, Gutteridge JMC, Aruoma OI (1987) The deoxyribose method - a simple testtube assay for determination of rate constants for reactions of hydroxyl radicals. Anal.

[78] Wade CR, Vanrij AM (1989) Plasma malondialdehyde, lipid peroxides, and the

[79] Wade CR, Jackson PG, Vanrij AM (1985) Quantitation of malondialdehyde (MDA) in plasma, by ion-pairing reverse phase high-performance liquid-chromatography.

[80] Wade CR, Vanrij AM (1988) Plasma thiobarbituric acid reactivity - Reaction conditions and the role of iron, antioxidants and lipid peroxy-radicals on the quantitation of

[81] Wong SHY, Knight JA, Hopfer SM, Zaharia O, Leach CN, Sunderman FW (1987) Lipoperoxides in plasma as measured by liquid-chromatographic separation of

[82] Santos MT, Valles J, Aznar J, Vilches J (1980) Determination of plasma malondialdehyde-like material and its clinical-application in stroke patients. J. Clin.

[83] Mezes M, Bartosiewicz G (1983) Investigations on vitamin-E and lipid peroxide status

[84] Kedziora J, Bartosz G, Gromadzinska J, Sklodowska M, Wesowicz W, Scianowski J (1986) Lipid peroxides in blood-plasma and enzymatic antioxidative defense of

[85] Takagi T, Mitsuno Y, Masumura M (1978) Determination of peroxide value by colorimetric iodine method with protection of iodide as cadmium complex. Lipids. 13:

malondialdehyde thiobarbituric acid adduct. Clin. Chem. 33: 214-220.

erythrocytes in Downs-syndrome. Clin. Chim. Acta. 154: 191-194.

medium-to-low quality forages. Trop. Anim. Health Prod. 44: 259-265.

and tissue morphology in rats. Pharm. Biol. 50: 318-325.

of lipid-peroxidation Biochem. Pharmacol. 44: 985-988.

thiobarbituric acid reaction. Clin. Chem. 35: 336-336.

plasma-lipid peroxides. Life Sciences. 43: 1085-1093.

in rheumatic diseases. Clin. Rheumatol. 2: 259-263.

Biochem. Med. 19: 127-132.

Biochem. 165: 215-219.

Biochem. Med. 33: 291-296.

Pathol. 33: 973-976.

147-151.


## **Liposomes as a Tool to Study Lipid Peroxidation**

Biljana Kaurinovic and Mira Popovic

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46020

#### **1. Introduction**

154 Lipid Peroxidation

[102] Grau A, Codony R, Rafecas M, Barroeta AC, Guardiola F (2000) Lipid hydroperoxide determination in dark chicken meat through a ferrous oxidation-xylenol orange

[103] Griffiths G, Leverentz M, Silkowski H, Gill N, Sanchez-Serrano JJ (2000) Lipid

[104] Huska D, Adam V, Babula P, Trnkova L, Hubalek J, Zehnalek J, Havel L, Kizek R (2011) Microfluidic robotic device coupled with electrochemical sensor field for handling of paramagnetic micro-particles as a tool for determination of plant mRNA.

[105] Sochor J, Salas P, Zehnalek J, Krska B, Adam V, Havel L, Kizek R (2010) An assay for spectrometric determination of antioxidant activity of a biological extract. Lis. Cukrov.

[106] Sochor J, Ryvolova M, Krystofova O, Salas P, Hubalek J, Adam V, Trnkova L, Havel L, Beklova M, Zehnalek J, Provaznik I, Kizek R (2010) Fully automated spectrometric protocols for determination of antioxidant activity: Advantages and disadvantages.

hydroperoxide levels in plant tissues. J. Exp. Bot. 51: 1363-1370.

method. J. Agric. Food Chem. 48: 4136-4143.

Microchim. Acta. 173: 189-197.

Repar. 126: 416-417.

Molecules. 15: 8618-8640.

Lipid peroxidation is used as a marker of cellular oxidative stress and contributes to the oxidative damage that occurs as a result of xenobiotics metabolism, inflammatory processes, ischemia, reperfusion injuries and chronic diseases such as atherosclerosis and cancer [1,2].

Cell membrane lipids (phospholipids, glycolipids and cholesterol) are the most common substrates of oxidative attack. Once initiated reaction autocatalytic continues, it has progradient flow, and the ultimate consequence is the structural-functional changes of the substrate. Lipid peroxidation is one of the best studied processes of cell damage under conditions of oxidative stress [3-5]. In 1960s Hochstein et al. [6] found that the initiation of lipid peroxidation require the presence of iron ions. From that moment the mechanism of lipid peroxidation process has been studied in many *in vitro* systems. However, accurate and precise mechanism is still not fully understood. Peroxidation in liposomes is usually studied after adding iron ions (Fe2+ plus ascorbic acid). Although the mechanism is not fully understood, it is known that redox chemistry of iron plays an important role in the occurrence and the rate of lipid peroxidation. Many studies have shown that the irondependent lipid peroxidation in systems comprised initially of Fe2+ and liposomes requires Fe2+ oxidation. In their research work, Minotti and Aust [7] assumed that the complex is formed between Fe2+ and Fe3+ ions could be initiator of iron-dependet lipid peroxidation. However, the existence of this complex has never been proven. In contrast, Aruoma et al. [8] argue against the participation of a Fe2+-Fe3+-O2 complex, or a critical 1:1 ratio of Fe2+ to Fe3+, in the initiation of lipid peroxidation in liposomes. Study of Tang et al. [9] showed that whether adding 100 or 150 mM Fe2+ initially or adding 100 mM Fe2+ initially and then 50 mM Fe2+ later at various times during the latent period in the liposomal system, the concentration of the remaining Fe2+ at the end of the latent period was almost the same every time.

Since lipid peroxidation causes oxidative damage to cell membranes and all other systems that contain lipids, in investigation of total antioxidative activity of plant extracts it is necessary to investigate their effects on lipid peroxidation. However, the impact of various

© 2012 Kaurinovic and Popovic, 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. © 2012 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.

natural products (isolated compounds and extracts) on the intensity of lipid peroxidation is studied in a number of substrate (linoleic acid, liposomes, various fatty oils, liver homogenates or hepatocytes isolated from it). Some substrates (liposomes and linolenic acids) are used more frequently than others mainly because of the simpler ways of performing the method. Also, due to the complex composition, examining the process of lipid peroxidation in fatty oils and liver homogenates makes research more difficult.

#### **2. Liposomes as a model system**

Liposomes are microscopic structure consisting of the one or more lipid bilayer enclosing the same number of water compartments. First, they were produced in Great Britain in 1961 by Alex D. Bangham while he was studying blood clotting. It was discovered that when phospholipids were combined with water they immediately formed a sphere. This is due to the fact that one end of each molecule is water soluble, while the oposite end is water insoluble. Water-soluble medications added to the water were trapped inside the aggregation of the hydrophobic ends; fat-soluble medications were incorporated into phospholipids layer and then – an important delivery system was born! Generally, such a structure formed polar lipids (such as phospholipids) [10]. Liposomes could be characterized as particles, similar to the structure and composition of cell membrane (Figure 1.). They occur in nature and could be artificially prepared [11].

**Figure 1.** Example of a) empty liposome; b) liposome (2007 Encyclopadeia Britannica, Inc.)

The behaviour of liposomes in physical and biological systems is governed by the factors such as physical size, membrane permeability, percent entrapped solutes, chemical composition (estimation of phospholipids, phospholipids oxidation, and analysis of cholesterol), and quantity and purity of the starting material. Therefore, liposomes are characterized for physical attributes: shape, size, and its distribution; percentage drug capture; entrapped volume; lameliarity; percentage drug release. Based on the structure and size, we distinguish between different types of liposomes: Multilamellar Vesicles (MLV, size >0.5μm), Oligolamellar Vesicles (OLV, size 0.1-1μm), Unilamellar Vesicles (UV, all size ranges), Multivesicular Vesicle (MVV/MV, size >1μm). Unilamellar Vesicles are further divided into Small Unilamellar Vesicles (SUV, size 20-50nm), Medium Unilamellar Vesicles (MUV, size 50-100nm), Large Unilamellar Vesicles (LUV, size >100nm) and Giant Unilamellar Vesicles (GUV) (Figure 2.).

**Figure 2.** Example of a) unilamellar liposome; b) multilamellar liposome

**2. Liposomes as a model system** 

Unilamellar Vesicles (GUV) (Figure 2.).

1.). They occur in nature and could be artificially prepared [11].

**Figure 1.** Example of a) empty liposome; b) liposome (2007 Encyclopadeia Britannica, Inc.)

The behaviour of liposomes in physical and biological systems is governed by the factors such as physical size, membrane permeability, percent entrapped solutes, chemical composition (estimation of phospholipids, phospholipids oxidation, and analysis of cholesterol), and quantity and purity of the starting material. Therefore, liposomes are characterized for physical attributes: shape, size, and its distribution; percentage drug capture; entrapped volume; lameliarity; percentage drug release. Based on the structure and size, we distinguish between different types of liposomes: Multilamellar Vesicles (MLV, size >0.5μm), Oligolamellar Vesicles (OLV, size 0.1-1μm), Unilamellar Vesicles (UV, all size ranges), Multivesicular Vesicle (MVV/MV, size >1μm). Unilamellar Vesicles are further divided into Small Unilamellar Vesicles (SUV, size 20-50nm), Medium Unilamellar Vesicles (MUV, size 50-100nm), Large Unilamellar Vesicles (LUV, size >100nm) and Giant

natural products (isolated compounds and extracts) on the intensity of lipid peroxidation is studied in a number of substrate (linoleic acid, liposomes, various fatty oils, liver homogenates or hepatocytes isolated from it). Some substrates (liposomes and linolenic acids) are used more frequently than others mainly because of the simpler ways of performing the method. Also, due to the complex composition, examining the process of

Liposomes are microscopic structure consisting of the one or more lipid bilayer enclosing the same number of water compartments. First, they were produced in Great Britain in 1961 by Alex D. Bangham while he was studying blood clotting. It was discovered that when phospholipids were combined with water they immediately formed a sphere. This is due to the fact that one end of each molecule is water soluble, while the oposite end is water insoluble. Water-soluble medications added to the water were trapped inside the aggregation of the hydrophobic ends; fat-soluble medications were incorporated into phospholipids layer and then – an important delivery system was born! Generally, such a structure formed polar lipids (such as phospholipids) [10]. Liposomes could be characterized as particles, similar to the structure and composition of cell membrane (Figure

lipid peroxidation in fatty oils and liver homogenates makes research more difficult.

Based on composition and applications, liposomes are divided into conventional liposomes (CL), fusogenic liposomes, pH sensitive liposomes, cationic liposomes, long circulatory (stealth) liposomes (LCL) and immuno-liposomes [12]. It is very difficult to measure dirrectly the phospholipid concentration, since dried lipids can often contain considerable quantities of residual solvent. Because of that, the method most widely used is an indirect one in which the phosphate content of the sample is first measured. The phospholipid concentration is measured using two methods - Bartlett and Stewart. In the Bartlett method the phospholipid phosphorous in the sample is first hydrolyzed to inorganic phosphate. This is converted to phospho-molybdic acid by the addition of ammonium molybdate and phospho-molybdic acid is quantitatively reduced to a blue colored compound by aminonaphthyl-sulfonic acid. The intensity of the blue color is measured spectrophotometrically and is compared with the curve of standards to give phosphorous and hence phospholipid content. This method is very sensitive. The problem is that test is easily upset by trace contamination with inorganic phosphate. In the other test, Stewart test, the phospholipid forms a complex with ammonium ferrothiocyanate in organic solution. The advantage of this method is that the presence of inorganic phosphate does not interfere with the test.

Until recently, liposomes are used as inert particles, carries of active principles, mostly for cosmetic purposes [13]. Today liposomes are used as very useful models, reagents and tools in various scientific disciplines, including biophysics (properties of cell membranes and channels), chemistry (catalysis, energy conversion, photosynthesis), biochemistry (the function of membrane proteins) and biology (excretion, cellular functions, transports and signaling, the transfer of genes and their functions). Liposomal formulation of several active molecules are currently in pre-clinical and clinical trials in different fields, with promising results. Two of the key problems in drug therapy (biodistribution throught the body and targeting to specific receptors) can be overcome by using liposomal formulations – liposomes protect encapsulated molecules from degradation and can passively target tissues or organs that have a discontinuous endothelium, such as liver, spleen, and bone marrow [14]. Comercial use of liposome was based on their colloidal, chemical and surface and microcapsuled proporties. These products include dosage formes of drugs (anti-cancer and antifugal agents, vaccines), cosmetic formulation (skin care products, shampoos), diagnostic products, a variety of applications in the food chemistry, as well as oral nutrient transport (liposomal vitamins, minerals and plants extracts for oral use). Liposome stability is an importrant aspect that must be met to be able to apply. By selecting the optimal value and size, pH and ionic strenght and the addition of complexing agents, liquid liposomical formulations could be stable for years.

Liposomal models have helped us to better understand the structure and dynamics of natural biomembrane systems. The concepts of structure and function of biomembranes, such as membrane fluidity, phase transition, the movement of lipids and proteins, triggering prosesses that affect metal ions or pH, have been very developed in this way. Modulatind effect of internal molecules (such as cholesterol) and insight into the mechanisms of membrane permeabillity for non-electrolytes and ions, are obtained by testing the model membranes. Liposomes that contains proteins as a components of membrane (reconstructes liposomes) were used in testing lipid-protein interactions in biological membranes, in examining the activities of active components such as membrane ionophores, anesthetics and divalent cations and mechanisms of antybody-antigen interactions [10].

Liposomes are very good models because they show the selectivity of the membrane to ions, osmotic swelling and response of range to agents that speed up or slow down the loss of ions and molecules from the particles in a way that at least, qualitatively mimic their activity in the natural membrane systems. Liposomes have also been successfully applied to "exclude the role" of membranes lipids and other components in biomembranes interact with the physical or chemical agents. Nevertheless, liposomal systems are useful because they allow the manipulation of membrane lipid composition, pH, temperature, content of different compounds in a limited way and provide the ability to determine the individual effect of the investigation product [15].

An example of the advantages of liposome in investigation of lipid peroxidation is that the influence of free radicals can be explored in the absence of chemical systems that produce free radicals, which may affect the test reaction. It is also possible to control the chemical composition of the liposome. This is particularly useful for the determination of lipid peroxidation induced by different systems for the generation of free radicals, and monitoring the overall effect of the combined system, or synergistic effects of combined systems that may arise. In addition to this it is possible to determine the antioxidant activity of tested compounds and determine which system works the best, by simple monitoring of lipid peroxidation. The tests used to determine the power of antioxidants to exert suppression of lipid peroxidation based on an assessment of the strength of oxidation of lipid substrate in the presence or absence of potential antioxidant molecules of plant extracts. There are four different strategies for assessing antioxidant capacity of molecules to the lipid substrate. They include the determination of oxygen consumed, the loss of substrate and formation of primary and secondary oxidation products [16]. The first method for determining the degree of lipid peroxidation, which includes the determination of oxygen consumed, based on following of initiation phase and its extension in the absence of antioxidants. The second method is based on measuring the loss of substrate in systems such as samples of food or biological samples and is very complicated, because they are full of potential oxiable substrates that are difficult to identify and characterize. The third method is based on monitoring the formation of primary oxidation products. It is a method that is well adapted to study such complex model systems and often involves the spectrophotometric determination of hydroperoxide, the dominant primary products of lipid peroxidation. Monitoring of secondary products of oxidation is the most commonly used method for the study of lipid model systems and lipid isolated from their natural environment. Both the *in vitro* and *in vivo* conditions are very often used TBA (thiobarbituric acid) test for detection of MDA (malondialdehyde), a secondary product of oxidation. This test is based on the reaction between TBA and MDA, which produces red chromophore with maximum of absorbance at 532 nm. This reaction is widely used and is performed by means of determination of many oxiable substrates (free fatty acids, LDL, body fluids). However, this method has some drawbacks. One is that the MDA is formed from free fatty acids which contain at least three double bonds. The next disadvantage is that the TBA is not specific for MDA because it can react with other aldehydes, such as occurs brown color that comes from the reactions of decomposition of sugar, amino acids, proteins and nucleic acids. Finally, MDA is not generated during the oxidation of many lipids and is often less important secondary oxidation product, and therefore not representative enough for the individual measurements. However, the TBA test was held for examination of lipid peroxidation, due to the simplicity of the method.

158 Lipid Peroxidation

formulations could be stable for years.

effect of the investigation product [15].

(liposomal vitamins, minerals and plants extracts for oral use). Liposome stability is an importrant aspect that must be met to be able to apply. By selecting the optimal value and size, pH and ionic strenght and the addition of complexing agents, liquid liposomical

Liposomal models have helped us to better understand the structure and dynamics of natural biomembrane systems. The concepts of structure and function of biomembranes, such as membrane fluidity, phase transition, the movement of lipids and proteins, triggering prosesses that affect metal ions or pH, have been very developed in this way. Modulatind effect of internal molecules (such as cholesterol) and insight into the mechanisms of membrane permeabillity for non-electrolytes and ions, are obtained by testing the model membranes. Liposomes that contains proteins as a components of membrane (reconstructes liposomes) were used in testing lipid-protein interactions in biological membranes, in examining the activities of active components such as membrane ionophores, anesthetics

Liposomes are very good models because they show the selectivity of the membrane to ions, osmotic swelling and response of range to agents that speed up or slow down the loss of ions and molecules from the particles in a way that at least, qualitatively mimic their activity in the natural membrane systems. Liposomes have also been successfully applied to "exclude the role" of membranes lipids and other components in biomembranes interact with the physical or chemical agents. Nevertheless, liposomal systems are useful because they allow the manipulation of membrane lipid composition, pH, temperature, content of different compounds in a limited way and provide the ability to determine the individual

An example of the advantages of liposome in investigation of lipid peroxidation is that the influence of free radicals can be explored in the absence of chemical systems that produce free radicals, which may affect the test reaction. It is also possible to control the chemical composition of the liposome. This is particularly useful for the determination of lipid peroxidation induced by different systems for the generation of free radicals, and monitoring the overall effect of the combined system, or synergistic effects of combined systems that may arise. In addition to this it is possible to determine the antioxidant activity of tested compounds and determine which system works the best, by simple monitoring of lipid peroxidation. The tests used to determine the power of antioxidants to exert suppression of lipid peroxidation based on an assessment of the strength of oxidation of lipid substrate in the presence or absence of potential antioxidant molecules of plant extracts. There are four different strategies for assessing antioxidant capacity of molecules to the lipid substrate. They include the determination of oxygen consumed, the loss of substrate and formation of primary and secondary oxidation products [16]. The first method for determining the degree of lipid peroxidation, which includes the determination of oxygen consumed, based on following of initiation phase and its extension in the absence of antioxidants. The second method is based on measuring the loss of substrate in systems such as samples of food or biological samples and is very complicated, because they are full

and divalent cations and mechanisms of antybody-antigen interactions [10].

Despite all these advantage, liposomal systems remain different from the natural cellular systems. For this reason, regardless of the results obtained by testing the liposomes, they could not be reproduced on the natural membrane system, but they can provide useful information.

Therefore, the liposomes are still mostly used as a model system of biological membrane for testing the LP, especially when testing extracts and essential oils from plants on the intensity of LP. These studies are important because free radical oxidation of lipid components of food is a major strategic problem of food producers. The degree of oxidation of fatty acids and their esters in foods depends on the chemical structure of fatty acids, food processing technology, the temperature at which food is stored or prepared for eating and the presence of antioxidants. Synthetic antioxidants are widely used in many foods to retard undesirable changes as a result of oxidation. Chemicals, like tert-butyl-4-hydroxyanisole (BHA) and tertbutyl hydroxytoluene (BHT), can be used as antimicrobial and antioxidants agents. However, the use of some of these chemicals is restricted in several countries, as they may be dangerous to human health [17]. Therefore, the search for new natural antioxidant sources has been greatly intensified. For this reason, there is a growing interest in the studies of natural additives as potential antioxidants. The antioxidant properties of many herbs and spices are reported to be effective in retarding the process of lipid peroxidation in oils and fatty foods and have gained the interest of many research groups. A number of studies on the antioxidant activities of various aromatic plants have been reported over the last 20 years [18,19]. Their aroma is associated with essential oils, complex mixtures of volatile compounds, dominated by mono- and sesquiterpenes. It is known that essential oils exhibit significant biological and pharmacological activities such as anti-inflammatory, antimicrobial, spasmolytic, stimulant effect on the CNS and the like. New research shows that they possess significant antitumor activity [20], and act as inhibitors of growth of breast tumors [21]. It was confirmed that essential oils of some aromatic plants possess a high antioxidant potential [22]. Widely used in the food industry to improve the flavor of foods.

In addition to essential oils, aromatic plants and characterized by the presence of plant phenolic compounds, primarily phenylpropanoids and coumarins which are proven to have multiple pharmacological activities. Studies of these secondary biomolecules have become intensified when some commercial synthetic antioxidants found to be expressed toxic, mutagenic and carcinogenic activities [23]. In addition, it was found that excessive production of oxygen radicals in the body initiates oxidation and degradation of polyunsaturated fatty acids. It is known that free radicals attack the highly unsaturated fatty acid of membrane system and induce lipid peroxidation, which is a key process in many pathological conditions, and one of the reactions caused by oxidative stress. Particularly vulnerable are the biological membrane lipids in the spinal cord and brain because they contain high oxiable polyunsaturated fatty acids. These features facilitate the formation of oxygen radicals involved in the processes of aging, Alzheimer's and Parkinson's disease, ischemic damage, arthritis, myocardial infarction, arteriosclerosis and cancer. Phenolic antioxidants "stop" oxygen free radicals and free radicals formed from the substrate by giving hydrogen atom or an electron. Some flavonoids have strong inhibitory effect on lipid peroxidation processes. This action is based on their ability to chelate transition metal ions, thereby preventing the formation of radicals (initiators of LP), caught radicals initiators of LP (ROS), scavenge lipid-alkoxyl and lipid-peroxyl radicals and regenerate -tocopherol by reduction of -tocopheryl radicals. Flavonoids have the following characteristics: 3 ', 4' dihydroxy group in ring B, or 4-keto and 3-hydroxy group in C ring, or 4'-keto group in C ring and 5-hydroxy group in A ring have the metal chelated properties (Figure 3.).

**Figure 3.** Possible places on flavonoids for chelating the transition metal ions in the process of lipid peroxidation.

Different metals have different binding affinity of the flavonoids [24]. Thus, for example, iron has the highest binding affinity for 3-OH group of ring C, then catechol group ring B and at the end of 5-OH group of ring A, while the copper ions bind to the first ring catechol group B [25]. Solubility of flavonoids in the lipid phase and the ability to penetrate the lipid membrane is small, since flavonoids in nature are mostly in the form of polar glycosides. Numerous tests of the inhibitory effects of flavonoids on lipid peroxidation were carried out on models of cell membranes. Based on these studies, it is assumed that quercetin and other flavonoids probably located on the surface membrane could easily capture radicals from the aqueous phase and thus prevent the initiation of LP. Thus located, flavonoids faster capture radicals initiators LP than -tocopherol, which is located within phospholipid bilayer and that the switch is a typical chain reaction. Prevention of initial attacks radicals from the aqueous phase to membrane phospholipids is essential in the antioxidant protection of biomembranes because free radicals are constantly generated in the aqueous phase of cellular and sub cellular structure [25,26].

In the present chapter, lipid peroxidation in a liposomal system was initiated by Fe2+ ascorbic acid system and the effects of four different Lamiaceae species (*Melittis melissophyllum*, *Marrubium peregrinum*, *Ocimum basilicum,* and *Origanum vulgare*) extracts and essential oils were investigated. Particular attention was paid to the chemical composition of extracts and essential oils and their capability to reduce lipid peroxidation. The plant leaves were dried in air and ground in a mixer. Finely powdered material (200 g) was macerated three times in 70% methanol (MeOH) with 4 L during a 24-h period. The macerates were collected, filtered, and evaporated to dryness under vacuum. The residues were dissolved in water and successively extracted with four solvents of increasing polarity: ether (Et2O), chloroform (CHCl3), ethyl acetate (EtOAc), and *n*-butanol (*n*-BuOH). The extraction was carried out until a colorless extract was obtained. The residue was the aqueous extract. All of five extracts (Et2O, CHCl3, EtOAc, *n*-BuOH, and H2O) were evaporated to dryness and then dissolved in 50% ethanol to make 10% (w=v) solutions. Both, these and the diluted solutions, were further used for examination. Essential oil was made when air-dried plant material was submitted to hydrodistillation according to Eur. Pharm. 4 [27], using *n*-hexane as a collecting solvent. The solvent was removed under vacuum. The oils were dried over anhydrous sodium sulphate and kept at +4 ºC. The inhibition of LP was determined by measuring the formation of secondary components (malondialdehyde) of the oxidative process, using liposomes as an oxidizable substrate [28-30]. However, because the thiobarbituric acid test is not specific for MDA, other non-lipid substances present in plant extracts, or peroxidation products other then malondialdehyde, could react positevely with TBA. These interfering compounds distort the results and therefore all the final results of investigated extracts have been corrected using the absorbances of the investigated extracts after the TBA-test (without liposomes) [31]. The commercial preparation of liposomes 'PRO-LIPO S' (Lucas-Meyer) pH = 5–7 was used as a model system of biological membranes. The liposomes, 225–250 nm in diameter, were obtained by dissolving the commercial preparation in demineralized water (1:10), in an ultrasonic bath.

#### **3. Lamiaceae (Labiatae) family**

160 Lipid Peroxidation

peroxidation.

exhibit significant biological and pharmacological activities such as anti-inflammatory, antimicrobial, spasmolytic, stimulant effect on the CNS and the like. New research shows that they possess significant antitumor activity [20], and act as inhibitors of growth of breast tumors [21]. It was confirmed that essential oils of some aromatic plants possess a high antioxidant potential [22]. Widely used in the food industry to improve the flavor of foods.

In addition to essential oils, aromatic plants and characterized by the presence of plant phenolic compounds, primarily phenylpropanoids and coumarins which are proven to have multiple pharmacological activities. Studies of these secondary biomolecules have become intensified when some commercial synthetic antioxidants found to be expressed toxic, mutagenic and carcinogenic activities [23]. In addition, it was found that excessive production of oxygen radicals in the body initiates oxidation and degradation of polyunsaturated fatty acids. It is known that free radicals attack the highly unsaturated fatty acid of membrane system and induce lipid peroxidation, which is a key process in many pathological conditions, and one of the reactions caused by oxidative stress. Particularly vulnerable are the biological membrane lipids in the spinal cord and brain because they contain high oxiable polyunsaturated fatty acids. These features facilitate the formation of oxygen radicals involved in the processes of aging, Alzheimer's and Parkinson's disease, ischemic damage, arthritis, myocardial infarction, arteriosclerosis and cancer. Phenolic antioxidants "stop" oxygen free radicals and free radicals formed from the substrate by giving hydrogen atom or an electron. Some flavonoids have strong inhibitory effect on lipid peroxidation processes. This action is based on their ability to chelate transition metal ions, thereby preventing the formation of radicals (initiators of LP), caught radicals initiators of LP (ROS), scavenge lipid-alkoxyl and lipid-peroxyl radicals and regenerate -tocopherol by reduction of -tocopheryl radicals. Flavonoids have the following characteristics: 3 ', 4' dihydroxy group in ring B, or 4-keto and 3-hydroxy group in C ring, or 4'-keto group in C

ring and 5-hydroxy group in A ring have the metal chelated properties (Figure 3.).

HO O

HO

O

**Figure 3.** Possible places on flavonoids for chelating the transition metal ions in the process of lipid

Different metals have different binding affinity of the flavonoids [24]. Thus, for example, iron has the highest binding affinity for 3-OH group of ring C, then catechol group ring B and at the end of 5-OH group of ring A, while the copper ions bind to the first ring catechol group B [25]. Solubility of flavonoids in the lipid phase and the ability to penetrate the lipid

Men+

OH

HO

Men+

Men+

OH

The Lamiaceae family is one of the largest and most distinctive families of flowering plants, with about 220 genera and almost 4000 species worldwide [32]. Lamiaceae are best known for the essential oils common to many members of the family [33]. The family was

established by De Jussieu in 1789 as the order Labiatae. This was the original family name, so given because the flowers typically have petals fused into an upper lip and a lower lip, the flower thus having an open mouth. Although this is still considered an acceptable alternative name, most botanists now use the name "Lamiaceae" in referring to this family. The main centre of diversity is the Mediterranean region to central Asia. Members are found in tropical and temperature regions [34]. All Lamiaceae are aromatic plants. The essential oil contains mainly monoterpenes, sesquiterpenes and phenylpropanoid compounds. Also, the plant species of Lamiaceae have been shown as rich sources of phenolic compounds mostly flavonoids and phenolic acids.

#### **3.1. Balm (***Melittis melissophyllum* **L.)**

The name *melittis* of the genus derives from a Greek words *Melissa* or *Melitta*, meaning "honey bee" and refers to the properties of flowers of attracting these insects. The name *melissophyllum* of the species simply means "with leaves similar to melissa". This is a tall plant which likes shady places and is ideal for a sunny woodland edge or scrubby border, where it will be attractive to bees and other insects. Bastard balm is a strongly aromatic plant that smells like fresh mowed grass and has erect hairy stems. It blooms white with a large pinkish purple blotch on the lower lip. The flowers are hermaphrodite and get pollinated by bees and moths. It has oval, bluntly-toothed, leaves in opposite pairs up the stems. Bastard balm is a herb native to the Mediterranean region.

Main flavonoids in balm are glycosides of apigenin and luteolin. However, presence of some other flavonoids as kaempherol, quercetin (Figure 4.) and ramnocitrin have been also reported [35].

**Figure 4.** Structures of two flavonoids present in *M. melissophyllum*

Balm is characterized by the presence of the other important plant phenolic substances such as phenolic acids (caffeic, rosmarinic and chlorogenic acid) (Figure 5.).

Also, balm leaf is characterized by the presence of pentacyclic triterpenes (ursolic, pomolic and oleanolic acid) (Figure 6.). The main biopharmacological effects shared by ursolic and oleanolic acid are anti-inflammatory, hepatoprotective, antitumor, and antioxidative [36-39].

Essential oil is present in all parts of the plant. The largest amount of oil obtained from aerial parts of plants, harvested in late summer. Balm leaves contain no more than 0.13% of essential oil which is of complex and variable composition. Among the more than 50 compounds identified to date, citronellal (dominantly the (*R*) enantiomer), β-caryophyllene, �-caryophyllene oxide, germacrene-D, nerol, geranial, citronellol, and geraniol amount to about 70% of the oil (Figure 7.) [40]. The composition is similar to that of lemongrass, but balm oil can be identified by its typical pattern of chiral compounds; for example, almost enantiomerically pure (*R*)-(+�)-methyl citronellate is a good indicator of true balm oil. For distinguish between two oils there is used the carbon isotopic ratio (IRMS-*isotope ratio mass spectrometry)* [41]. The essential oil exhibits spasmolitic action and acts as a muscle relaxant, sedative, narcotic, antibacterial, and antifungal [42,43].

**Figure 5.** Structures of phenolic acids in *M. Melissophyllum*

162 Lipid Peroxidation

reported [35].

flavonoids and phenolic acids.

**3.1. Balm (***Melittis melissophyllum* **L.)** 

stems. Bastard balm is a herb native to the Mediterranean region.

**Figure 4.** Structures of two flavonoids present in *M. melissophyllum*

as phenolic acids (caffeic, rosmarinic and chlorogenic acid) (Figure 5.).

established by De Jussieu in 1789 as the order Labiatae. This was the original family name, so given because the flowers typically have petals fused into an upper lip and a lower lip, the flower thus having an open mouth. Although this is still considered an acceptable alternative name, most botanists now use the name "Lamiaceae" in referring to this family. The main centre of diversity is the Mediterranean region to central Asia. Members are found in tropical and temperature regions [34]. All Lamiaceae are aromatic plants. The essential oil contains mainly monoterpenes, sesquiterpenes and phenylpropanoid compounds. Also, the plant species of Lamiaceae have been shown as rich sources of phenolic compounds mostly

The name *melittis* of the genus derives from a Greek words *Melissa* or *Melitta*, meaning "honey bee" and refers to the properties of flowers of attracting these insects. The name *melissophyllum* of the species simply means "with leaves similar to melissa". This is a tall plant which likes shady places and is ideal for a sunny woodland edge or scrubby border, where it will be attractive to bees and other insects. Bastard balm is a strongly aromatic plant that smells like fresh mowed grass and has erect hairy stems. It blooms white with a large pinkish purple blotch on the lower lip. The flowers are hermaphrodite and get pollinated by bees and moths. It has oval, bluntly-toothed, leaves in opposite pairs up the

Main flavonoids in balm are glycosides of apigenin and luteolin. However, presence of some other flavonoids as kaempherol, quercetin (Figure 4.) and ramnocitrin have been also

Balm is characterized by the presence of the other important plant phenolic substances such

Also, balm leaf is characterized by the presence of pentacyclic triterpenes (ursolic, pomolic and oleanolic acid) (Figure 6.). The main biopharmacological effects shared by ursolic and oleanolic acid are anti-inflammatory, hepatoprotective, antitumor, and antioxidative [36-39]. Essential oil is present in all parts of the plant. The largest amount of oil obtained from aerial parts of plants, harvested in late summer. Balm leaves contain no more than 0.13% of essential oil which is of complex and variable composition. Among the more than 50

**Figure 6.** Structures of triterpenoids compounds present in *M. melissophyllum* leaves.

**Figure 7.** Sesquiterpenes in *M.melissophyllum* leaves

Beneficial effects of plants introduced by ancient Greeks and Romans.There is overlap with the use of plants in folk medicine and science. In relation to its complex composition it has multiple medicinal effects. Its herb has wide applications in the folk medicine. Due to the soothing action balm leaves enters into the composition of tea for calming, which is recommended for hysteria and neuralgia. Balm leaves mixed with bitter herbs are a great tool to enhance appetite. Various preparations containing extract or essential oil balm leaves are used as an addition to baths against rheumatism. In the folk medicine of Belarus alcoholic extract is drunk for stomach ulcer and duodenum, to calm the pain in the stomach, intestines, the liver, heart, and women's diseases. Terpenes found in essential oil of balm leaves, have a relaxing and antiviral effects. Eugenol calms muscle spasms and destroy bacteria [44]. It is also used as a carminative and sedative. Recent results indicate that the balm extract acts as depressants and have sedative effect on central nervous system of mice [45]. In the folk medicine of central Italy inflorescences of this plant, called "Erba Lupa", were used under infusion as antispasmodic, against insomnia and eyes inflammations [46,47].

Our research on balm was recently extended to the comprehensive *in vitro* and *in vivo* studies of antioxidant properties of balm essential oil and extracts measuring their capability to reduce lipid peroxidation in liposomes and effect on some enzymes of antioxidant defense systems [48]. Investigation of balm essential oil showed that with increasing concentration of essential oil reduces the intensity of lipid peroxidation compared to hexane-control (Table 1). Also, only the most diluted solution of essential oil of balm (0.213 and 0.535 g/mL) has a weaker protective effect than the synthetic antioxidant BHT. The capability to reduce lipid peroxidation of essential oil was dose-dependent. This high inhibitory effect of balm essential oil was found to be in correlation with the content of monoterpene alcohols and ketones.


**Table 1.** Inhibition of LP (%) in Fe2+/ascorbate system of induction by essential oil of balm leaves and BHT (as a positive control) in the TBA assay.

The protective effects on lipid peroxidation of balm extracts have been evaluated using the Fe2+/ascorbate system of induction, by the TBA-assay (Table 2.). In general, all of the examined extracts (except *n*-BuOH extract) expressed strong antioxidant capacity and ability to reduce lipid peroxidation in liposomes. The largest inhibitory activity was exhibited by EtOAc and H2O extracts because the 5% solutions show better protective effect than BHT. All extracts of the highest concentrations (10%) exhibited a better inhibitory effect than BHT. Protective activity of these extracts and its components towards Fe2+-dependent LP of liposomes can be explained by present of phenolic acids and flavonoids and their influence on antioxidative capacity of ascorbic acid, which doesn`t show a strong antioxidative effect in lipid phase, but different phenolic compounds can result increase of its antioxidant activity [49].

164 Lipid Peroxidation

inflammations [46,47].

monoterpene alcohols and ketones.

BHT (as a positive control) in the TBA assay.

**Figure 7.** Sesquiterpenes in *M.melissophyllum* leaves

Beneficial effects of plants introduced by ancient Greeks and Romans.There is overlap with the use of plants in folk medicine and science. In relation to its complex composition it has multiple medicinal effects. Its herb has wide applications in the folk medicine. Due to the soothing action balm leaves enters into the composition of tea for calming, which is recommended for hysteria and neuralgia. Balm leaves mixed with bitter herbs are a great tool to enhance appetite. Various preparations containing extract or essential oil balm leaves are used as an addition to baths against rheumatism. In the folk medicine of Belarus alcoholic extract is drunk for stomach ulcer and duodenum, to calm the pain in the stomach, intestines, the liver, heart, and women's diseases. Terpenes found in essential oil of balm leaves, have a relaxing and antiviral effects. Eugenol calms muscle spasms and destroy bacteria [44]. It is also used as a carminative and sedative. Recent results indicate that the balm extract acts as depressants and have sedative effect on central nervous system of mice [45]. In the folk medicine of central Italy inflorescences of this plant, called "Erba Lupa", were used under infusion as antispasmodic, against insomnia and eyes

Our research on balm was recently extended to the comprehensive *in vitro* and *in vivo* studies of antioxidant properties of balm essential oil and extracts measuring their capability to reduce lipid peroxidation in liposomes and effect on some enzymes of antioxidant defense systems [48]. Investigation of balm essential oil showed that with increasing concentration of essential oil reduces the intensity of lipid peroxidation compared to hexane-control (Table 1). Also, only the most diluted solution of essential oil of balm (0.213 and 0.535 g/mL) has a weaker protective effect than the synthetic antioxidant BHT. The capability to reduce lipid peroxidation of essential oil was dose-dependent. This high inhibitory effect of balm essential oil was found to be in correlation with the content of

> **Concentration (g/mL) BHT 0.213 0.535 1.065 1.598 2.130**

**LP** 26.15 13.05 21.03 39.62 46.09 56.81 **Table 1.** Inhibition of LP (%) in Fe2+/ascorbate system of induction by essential oil of balm leaves and

The protective effects on lipid peroxidation of balm extracts have been evaluated using the Fe2+/ascorbate system of induction, by the TBA-assay (Table 2.). In general, all of the


**Table 2.** Inhibition of LP (%) in Fe2+/ascorbate system of induction by extracts of balm leaves and BHT (as a positive control) in the TBA assay.

It is known that quercetin, like many other flavonoids, prevents oxidation of LDL cholesterol, and its anti-inflammatory activity comes from inhibition of the enzyme lipooxigenase and inhibition of inflammatory mediators [50]. Kaempferol acts synergistically with quercetin to reduce the proliferation of malignant cells, and treatments are a combination of quercetin and kaempherol efficient than their single use [51]. It is, also, known that rutin has strong antioxidant effects, as well as a feature to built chelates with metal ions (e.g. iron) and reduces the Fenton reaction in which the resulting harmful oxygen radicals. It is supposed to stabilize vitamin C. If rutin is taken together with vitamin C, increases the activity of ascorbic acid [52]. In addition, HPLC-DAD analysis showed that the aqueous extract, in large quantities, present phenolic acids (rosmarinic, chlorogenic and caffeic acid), which are known antioxidants. It was determined that rosmarinic acid has stronger antioxidant activity than vitamin E. Rosmarinic acid prevents cell damage caused by free radicals and reduce the risk of cancer and atherosclerosis. In contrast to the histamines, rosmarinic acid prevents activation of the immune system cells that cause swelling and fluid collection. Also, it is known that the caffeic acid by far surpassing other antioxidants because it reduces the production of -toxin for more than 95% [35]. Furthermore, it can be supposed that the reduction process of lipid peroxidation is caused, besides flavonoids, also by triterpenoids acids (especially ursolic, oleanolic, and pomolic acid) since non-polar extracts (Et2O and CHCl3) also exhibited high antioxidant potential [39]. The *n*-BuOH extract shows a prooxidative effect that is increased by increasing concentration of added extract. It can be supposed that compounds with polar groups were extracted by *n*-BuOH, and are present in high concentration in the extract. It is notable that molecules which show antioxidant activity, when they are present in high concentration, might behave as prooxidants [53], so *n*-BuOH extract of balm leaves probably have this kind of activity. The antioxidant activities of all extracts of balm leaves were dose dependent.

The represented antioxidant activity results show that extracts of examined plant species, especially EtOAc and H2O extracts are efficient in the protection of tissues and cells from oxidative stress. Anyway, according to variations in regard to antioxidant activity of tested by different *in vitro* models, there are also requiste *in vivo* test that would confirm the capability of extracts to reduce the lipid peroxidation. *In vivo* tests are also necessary because a lot of plant phenols are biotransformed during their active metabolism. *In vivo* effects are evaluated on LP in the mice liver (Table 3.) and blood hemolysate (Table 4.) after treatment with examined balm extracts, or in combination with carbon tetrachloride (CCl4).


**Table 3.** Effect of extracts of balm leaves on intensity of lipid peroxidation (nmol malondialdehyde/mg of proteines) in liver homogenate before and after treatment with CCl4

As compared with control, intensity of LP is statistically significant reduced during the treatment with ethylacetate and water extracts of balm leaves. The result derived by treatment with ethylacetate and water extracts is in according with amounts got *in vitro* experiment. Using CHCl3 extract leads to a significant increase of LP intensity, whereas the other two extracts had no effect on this parameter. All extracts of balm leaves combine with CCl4 have showed a statistically significant decrease of LP intensity, and this behavior of the extract probably results from the presence of secondary biomolecules like flavonoids and phenolic acids. Handa *et al.* [54] determined that secondary biomolecules such as flavonoids, xanthones and tannins in combination with CCl4 have protective effects on liver. Phenolic components present in balm leaves (rutin, luteolin, kaempherol) are known as strong inhibitors of CCl4 induced LP [55]. Flavonoids could affect the initiation phase of lipid peroxidation, where they influence the metabolism of CCl4, they scavenge the free radicals, or they decrease the microsomal enzyme systems that are claimed for CCl4 metabolism [56]. In continuation of this process, flavonoids can scavenge lipoperoxides and their radicals or they can act as chelating agents for Fe2+ ion, and in this way can stop Fenton reactions [57]. Furthermore, Afanas′ev et al. [28] found that quercetin and rutin exhibited a high inhibitory effect on the Fe2+-induced liposomal LPx and NADPH-dependent CCl4-induction LPx in liver microsomes. Luteolin, one of the main active component in the balm, is responsible for the inhibitory effect on the former reaction. In addition to the above-mentioned mechanism (chelate formation with Fe2+) it is possible that these compounds (of flavonoid type) act as scavengers of OH radicals, whereby they are transformed in the corresponding radical form which is stabilized by resonance. On the basis of these results, it can be concluded that all of extracts of balm leaves showed protection effect in relation to the CCl4-induced lipid peroxidation.

Similar results were obtained during examining the effects of extracts of bastard balm on LP in blood hemolysate in mice (Table 4.). Three extracts, CHCl3, EtOAc and H2O, induced a significant decrease of LP intensity, while Et2O and *n*-BuOH ones decreased the level of this enzyme insignificantly.


**Table 4.** Effect of extracts of balm leaves on intensity of lipid peroxidation (nmol malondialdehyde/mL erythrocytes) in blood hemolysate before and after treatment with CCl4

The LP value showed a statistically insignificant increase with CCl4-treated animals compared with the untreated ones. A clear protective effect was seen in experimental animals administered H2O extract and CCl4 compared with untreated animals. Furthermore, EtOAc extract also significantly decreased the activity of LP, while Et2O, CHCl3 and *n*-BuOH extracts did not change notably the levels of lipid peroxidation. These results suggest that these two extracts (EtOAc and H2O) had a protective effect. According to the literature data [58], the reduction of the serum LP might be the result of antioxidant activity of several classes of plant phenolic constituents, such as cinnamic acids (ferulic, caffeic, and chlorogenic), flavonoids and biflavonoids, 1,3,6,7-tetrahydroxyxynthones, and acylphoroglycinols such as hyperforin and adhyperforin. Cock and Samman [59] showed that quercetin and rutin and their glycosides show strong inhibitory effect in respect of LP. The observed differences in the action of particular balm extracts are probably due to the different contents of flavonoids, but the potential protective effects of some other groups of compounds can not be ruled out.

#### **3.2. Horehound (***Marrubium peregrinum* **L.)**

166 Lipid Peroxidation

The represented antioxidant activity results show that extracts of examined plant species, especially EtOAc and H2O extracts are efficient in the protection of tissues and cells from oxidative stress. Anyway, according to variations in regard to antioxidant activity of tested by different *in vitro* models, there are also requiste *in vivo* test that would confirm the capability of extracts to reduce the lipid peroxidation. *In vivo* tests are also necessary because a lot of plant phenols are biotransformed during their active metabolism. *In vivo* effects are evaluated on LP in the mice liver (Table 3.) and blood hemolysate (Table 4.) after treatment

with examined balm extracts, or in combination with carbon tetrachloride (CCl4).

of proteines) in liver homogenate before and after treatment with CCl4

protection effect in relation to the CCl4-induced lipid peroxidation.

enzyme insignificantly.

**Parameter Control Et2O CHCl3 EtOAc** *n***-BuOH H2O LP** 7.19±0.23 7.36±0.21 7.91±0.19 6.71±0.16 7.12±0.23 6.19±0.27 **LP + CCl4** 8.91±0.29 7.12±0.21 7.06±0.24 6.92±0.17 6.98±0.24 6.81±0.24 **Table 3.** Effect of extracts of balm leaves on intensity of lipid peroxidation (nmol malondialdehyde/mg

As compared with control, intensity of LP is statistically significant reduced during the treatment with ethylacetate and water extracts of balm leaves. The result derived by treatment with ethylacetate and water extracts is in according with amounts got *in vitro* experiment. Using CHCl3 extract leads to a significant increase of LP intensity, whereas the other two extracts had no effect on this parameter. All extracts of balm leaves combine with CCl4 have showed a statistically significant decrease of LP intensity, and this behavior of the extract probably results from the presence of secondary biomolecules like flavonoids and phenolic acids. Handa *et al.* [54] determined that secondary biomolecules such as flavonoids, xanthones and tannins in combination with CCl4 have protective effects on liver. Phenolic components present in balm leaves (rutin, luteolin, kaempherol) are known as strong inhibitors of CCl4 induced LP [55]. Flavonoids could affect the initiation phase of lipid peroxidation, where they influence the metabolism of CCl4, they scavenge the free radicals, or they decrease the microsomal enzyme systems that are claimed for CCl4 metabolism [56]. In continuation of this process, flavonoids can scavenge lipoperoxides and their radicals or they can act as chelating agents for Fe2+ ion, and in this way can stop Fenton reactions [57]. Furthermore, Afanas′ev et al. [28] found that quercetin and rutin exhibited a high inhibitory effect on the Fe2+-induced liposomal LPx and NADPH-dependent CCl4-induction LPx in liver microsomes. Luteolin, one of the main active component in the balm, is responsible for the inhibitory effect on the former reaction. In addition to the above-mentioned mechanism (chelate formation with Fe2+) it is possible that these compounds (of flavonoid type) act as scavengers of OH radicals, whereby they are transformed in the corresponding radical form which is stabilized by resonance. On the basis of these results, it can be concluded that all of extracts of balm leaves showed

Similar results were obtained during examining the effects of extracts of bastard balm on LP in blood hemolysate in mice (Table 4.). Three extracts, CHCl3, EtOAc and H2O, induced a significant decrease of LP intensity, while Et2O and *n*-BuOH ones decreased the level of this Marrubium genus includes about 40 species. Species of this genus growing in dry pastures, abandoned the places along the roads in central and southern Europe, but also in North Africa, in parts of Asia and the Americas. Horehound is a perennial plant with a rectangular stem, branched in the upper part. Rhizomes of this species are ligneous, leaves oblong, flowers grouped in loose inflorescence [60]. A common plant blooms from July to September and harvested in that period. It has a bitter and pungent taste and smell. It is the drug of Herba *Marrubii albi*. This plant doesn`t require special conditions for growth.

In previous phytochemical investigations on *M. peregrinum*, different groups of chemicals were isolated: flavones (apigenin and luteolin) [61] (Figure 8.), flavonols (kaempferol) [62], glycosylated flavonoids (quercetin-3-O-β-D-rutinoside, naringenin-7-O-β-D-glucoside, kaempferol-3-O-β-D-rutinoside, quercetin-3-O-β-D-glucoside) [63], caffeic acid derivatives [64], and four diterpenoids (peregrinin, peregrinol, marrubiin and premarrubiin) [65]. T. Hennebelle et al. [66] have established the presence of acteoside, forsythoside B, arenarioside and terniflorine (apigenin-7-*O*-[6″-E-p-coumaroyl]β-D-glucopyranoside) in the MeOH extract of *M. peregrinum*.

*Marrubium peregrinum* essential oil yield between 0.02-0.07% [67]. Dominant monoterpenes are: α-pinene, sabinene, limonene, camphene and α-terpinolene. In a Greek sample, βphellandrene, epi-bicyclosesquiphellandrene and bicyclogermacrene proved to be the major compounds [68], whereas the essential oil of a sample from Central Europe was rich in βcaryophyllene and its oxide, bicyclogermacrene and germacrene D [69]. The main sesquiterpene compounds are Z- and E- β-farnesene (~12%), β-caryophyllene (~8.5%), heksahidrofarnesil acetone (~ 6.5%), spathullenol (~5%) i germacrene D (~4.5%) (Figure 9.) [68].

**Figure 8.** Figure 8. Structures of two main flavonoids in *M. peregrinum*.

**Figure 9.** Main constituents of *M. peregrinum* essential oil

Some species of *Marrubium* are used in traditional and modern medicine. Many studies have shown various activities in this genus, such as hypoglycemic effect, anti-schistosoma, antioxidant, calcium channel blocker and hypotensive activity [70]. As a medicinal plant, *M. peregrinum* have been employed against vascular diseases (antihypertensive, antispasmolitic) [61].

In our comprehensive study of chemical and biochemical investigation of *M. peregrinum*  from three different locations (Backo Gradiste-Rimski Sanac (No 1.); Novi Knezevac (No 2.) and Senta (No 3.)), we have identified more than 40 compounds in essential oil of *M. peregrinum* (44 for *M. peregrinum* from Senta locality, 42 for *M. peregrinum* from Novi Knezevac locality and 41 for *M. peregrinum* from Rimski Sanac locality, representing 96.15%, 87.60% and 83.66% of the total oil contents, respectively), in which dominant compounds were β-caryophyllene (13.20-17.99%), bicyclogermacrene (6.42-9.80%) and germacrene-D (6.79-9.05%). Besides sesquiterpene hydrocarbons, oxygenated sesquiterpenes, spathulenol (3.76-5.78%) and caryophyllene oxide (3.73-4.78%) are also present in relevant quantities. However, we must point out that the amounts of these components in essential oil from different localities are very different. Essential oil obtain from plant collected in Senta is the richest of sesquiterpene hydrocarbons (62.71%), while oxygenated sesquiterpenes are most represented (11.84%) in essential oil from plants collected in the Rimski Sanac area.


9.) [68].

caryophyllene and its oxide, bicyclogermacrene and germacrene D [69]. The main sesquiterpene compounds are Z- and E- β-farnesene (~12%), β-caryophyllene (~8.5%), heksahidrofarnesil acetone (~ 6.5%), spathullenol (~5%) i germacrene D (~4.5%) (Figure

Some species of *Marrubium* are used in traditional and modern medicine. Many studies have shown various activities in this genus, such as hypoglycemic effect, anti-schistosoma, antioxidant, calcium channel blocker and hypotensive activity [70]. As a medicinal plant, *M. peregrinum* have been employed against vascular diseases (antihypertensive,

In our comprehensive study of chemical and biochemical investigation of *M. peregrinum*  from three different locations (Backo Gradiste-Rimski Sanac (No 1.); Novi Knezevac (No 2.) and Senta (No 3.)), we have identified more than 40 compounds in essential oil of *M. peregrinum* (44 for *M. peregrinum* from Senta locality, 42 for *M. peregrinum* from Novi Knezevac locality and 41 for *M. peregrinum* from Rimski Sanac locality, representing 96.15%, 87.60% and 83.66% of the total oil contents, respectively), in which dominant compounds were β-caryophyllene (13.20-17.99%), bicyclogermacrene (6.42-9.80%) and germacrene-D (6.79-9.05%). Besides sesquiterpene hydrocarbons, oxygenated sesquiterpenes, spathulenol (3.76-5.78%) and caryophyllene oxide (3.73-4.78%) are also present in relevant quantities. However, we must point out that the amounts of these components in essential oil from different localities are very different. Essential oil obtain from plant collected in Senta is the richest of sesquiterpene hydrocarbons (62.71%), while oxygenated sesquiterpenes are most

represented (11.84%) in essential oil from plants collected in the Rimski Sanac area.

**Figure 8.** Figure 8. Structures of two main flavonoids in *M. peregrinum*.

**Figure 9.** Main constituents of *M. peregrinum* essential oil

antispasmolitic) [61].

**Table 5.** Inhibition of LP (%) in Fe2+/ascorbate system of induction by essential oil of *M. peregrinum* from three different location, and BHT (as a positive control) in the TBA assay.

Also, our study showed that all of the examined essential oils express strong antioxidant activity and capability to reduce lipid peroxidation (Table 5.). The largest inhibitory activity was exhibited by essential oil from plant collected at Senta locality (No. 3.). Solution of all concentrations, except the most diluted (0.213 μg/mL), have exhibited a stronger protective effect (from 37.02 to 71.32% of inhibition of LP) than BHT (26.15%). The other two essential oils (from Rimski sanac and Novi Knezevac), at higher concentration (from 1.065 to 2.130 μg/mL), have also exhibited more intense protective effect than BHT [71].

The effect of crude MeOH extracts of *M. peregrinum* was preliminarily determined from the three localities. There were taken three concentrations of MeOH extracts (1, 5, and 10% extracts). All of the examined extracts expressed stronger antioxidant capacity as compared to the 50% solution of MeOH. In particular, the largest inhibitory activity was established by the MeOH extracts of *M. peregrinum* collected from Senta locality. Also, the best results were obtained using solutions of the highest concentrations [72]. Because of all this there was carried out successive extractions of *M. peregrinum* from all three localities, and for further work 10% extracts are prepared. Successive extraction was performed as the extraction of antioxidant substances of different chemical structure, was achieved using solvents of different polarity. Numerous investigations of qualitative composition of plant extracts revealed the presence of high concentration of phenols in the extracts obtained using polar solvents [73]. The extracts that perform the highest antioxidant activity have the highest concentration of phenols. Phenols are very important plant constituents because of their scavenging ability on free radicals due to their antioxidant action [74]. The examination of capability to reduce intensity of lipid peroxidation of plant extracts from *M. peregrinum* showed different values (Table 6.). The first two extracts (Et2O and CHCl3) obtained from plants from all three locality are exhibited weaker protective effect than BHT, while the other three extracts (EtOAc, *n*-BuOH and H2O) showed better protective properties than synthetic antioxidant.The largest inhibitory activity, again, was exhibited by the EtOAc and H2O extracts of *M. peregrinum* collected from Senta locality.

Obtained results can be related to the experiments in which the total amount of flavonoids was determined, which show that EtOAc and H2O extracts from Senta locality contains the largest amounts of total flavonoids, namely of luteolin, either being present as free or in the form of its glucosides. The suggested mechanism of flavonoid antioxidative action is as follows: the double bond in position 2, 3 is conjugated with C4-carbonyl group, and free OH groups (C5, C3 and C7) can form chelates with ions of d-elements. Once formed, complex with Fe2+ ion prevents formation of OH• radicals in Fenton's reaction [59]. Also, luteolin is thought to play an important role in the human body as an antioxidant, a free radical scavenger, an agent in the prevention of inflammation, a promoter of carbohydrate metabolism, and an immune system modulator. These characteristics of luteolin are also believed to play an important part in the prevention of cancer. Multiple research experiments describe luteolin as a biochemical agent that can dramatically reduce inflammation and the symptoms of septic shock [75]. Furthermore, it is well known that some other flavonoids isolated from *M. peregrinum* possess certain biological and pharmacological activity. For example, apigenin, one of the flavonoids present in *M. peregrinum*, was shown to express strong antioxidant effects, increasing the activities of antioxidant enzymes and, related to that, decreasing the oxidative damage to tissues [61].


**Table 6.** Inhibition of LP (%) in Fe2+/ascorbate system of induction by extracts of *M. peregrinum* and BHT (as a positive control) in the TBA assay.

#### **3.3. Basil (***Ocimum basilicum* **L.)**

Basil is originally native to India and other tropical regions of Asia, having been cultivated there for more then 5.000 years. Ocimum genus includes about 150 species [76]. There are many varieties of *Ocimum basilicum*, as well as several related species or species hybrids also called basil. These varieties differ in morphological and general structure, and also in the content and composition of essential oil. The chemotype is determined by chemical composition of essential oil and it is basic for chemotaxonomy within the genus Ocimum and species *Ocimum basilicum* [77].

The word market has several types of essential oils that differ in chemical structure, composition and fragrance. The dominant compounds of basil essential oil occur in two different biochemical pathways: phenylpropanoids (methyl chavicole, eugenol, methyl eugenol, and methyl cinnamate) through shicimic acid, and terpenoids (linalool and geraniol) through mevalonic acid. Based on chemical content, basils can be divided into four groups: European (French) *O. basilicum* (contains lower amounts of phenols); Exotic (contains methyl chavicol (40-80%)); Reunion and Javanean. European type of essential oil is the finest quality, has the finest fragrance and the highest price in the market. Other components that can be found in higher concentrations in this type of oil are: linalool, methyl chavicol (estragole) (Figure 10.), 1,8-cineole, eugenol, geraniol, germacrene D, α-terpinolene, β-caryophyllene, ocimene, sabinene, thujone, and γ-terpinene [78].

Among phenolic constituents flavonoids and their glucosides are dominant. The major flavonoids are: quercetin, kaempferol, apigenin, luteolin and rutin. Quercetin-3-O-

diglucoside and kaempferol-3-O-β-rutinoside have been also identified. Beside, basil is rich in triterpenoid acids (ursolic and oleanolic), cinnamic acid (caffeic and rosmarinic), vitamin C and β-carotene, as well with calcium, copper, magnesium, sodium and potassium [79].

**Figure 10.** Main constituents of *O. basilicum* essential oil

170 Lipid Peroxidation

with Fe2+ ion prevents formation of OH• radicals in Fenton's reaction [59]. Also, luteolin is thought to play an important role in the human body as an antioxidant, a free radical scavenger, an agent in the prevention of inflammation, a promoter of carbohydrate metabolism, and an immune system modulator. These characteristics of luteolin are also believed to play an important part in the prevention of cancer. Multiple research experiments describe luteolin as a biochemical agent that can dramatically reduce inflammation and the symptoms of septic shock [75]. Furthermore, it is well known that some other flavonoids isolated from *M. peregrinum* possess certain biological and pharmacological activity. For example, apigenin, one of the flavonoids present in *M. peregrinum*, was shown to express strong antioxidant effects, increasing the activities of antioxidant enzymes and, related to that, decreasing the oxidative damage to tissues [61].

 **BHT Et2O CHCl3 EtOAc** *n***-BuOH H2O**  *M. peregrinum* **(No 1.)** 26.15 9.38 14.22 29.41 27.39 32.35 *M. peregrinum* **(No 2.)** 26.15 14.27 21.19 29.54 26.83 37.55 *M. peregrinum* **(No 3.)** 26.15 17.11 23.52 38.83 28.73 41.18 **Table 6.** Inhibition of LP (%) in Fe2+/ascorbate system of induction by extracts of *M. peregrinum* and

Basil is originally native to India and other tropical regions of Asia, having been cultivated there for more then 5.000 years. Ocimum genus includes about 150 species [76]. There are many varieties of *Ocimum basilicum*, as well as several related species or species hybrids also called basil. These varieties differ in morphological and general structure, and also in the content and composition of essential oil. The chemotype is determined by chemical composition of essential oil and it is basic for chemotaxonomy within the genus Ocimum

The word market has several types of essential oils that differ in chemical structure, composition and fragrance. The dominant compounds of basil essential oil occur in two different biochemical pathways: phenylpropanoids (methyl chavicole, eugenol, methyl eugenol, and methyl cinnamate) through shicimic acid, and terpenoids (linalool and geraniol) through mevalonic acid. Based on chemical content, basils can be divided into four groups: European (French) *O. basilicum* (contains lower amounts of phenols); Exotic (contains methyl chavicol (40-80%)); Reunion and Javanean. European type of essential oil is the finest quality, has the finest fragrance and the highest price in the market. Other components that can be found in higher concentrations in this type of oil are: linalool, methyl chavicol (estragole) (Figure 10.), 1,8-cineole, eugenol, geraniol, germacrene D,

α-terpinolene, β-caryophyllene, ocimene, sabinene, thujone, and γ-terpinene [78].

Among phenolic constituents flavonoids and their glucosides are dominant. The major flavonoids are: quercetin, kaempferol, apigenin, luteolin and rutin. Quercetin-3-O-

 **Extracts**

BHT (as a positive control) in the TBA assay.

**3.3. Basil (***Ocimum basilicum* **L.)** 

and species *Ocimum basilicum* [77].

Basilici herba has been used in traditional and homeopathic medicine to treat number of diseases. Essential oil (*Basilici aetheroleum*) extracted from fresh leaves and flowers can be used as aroma additives in foods, pharmaceuticals, and cosmetics [80]. Traditionally, basil has been used as a medicinal plant in the treatment of headaches, coughs, diarrhea, constipation, warts, worms, and kidney malfunction. Major aroma compounds from volatile extracts of basil present anti-oxidative activity [81]. Among the many studies to determine the antioxidant activities of basil, most have focused mainly on the antioxidant activities of crude extracts, using methanol, acetone, or water as a solvent [82,83].


**Table 7.** Inhibition of LP (%) in Fe2+/ascorbate system of induction by essential oil of basil leaves and BHT (as a positive control) in the TBA assay.

In our investigation, the examined essential oil expressed strong antioxidant activity (Table 7.). Solutions of all concentrations, except the most diluted (0.213 μg/mL), have exhibited a stronger protective effect (from 35.17 to 79.14% of inhibition of LP) than BHT (26.15%). The largest inhibitory activity was achieved by using the solution of the highest concentration. For the inhibition of LP, the most responsible compounds were the oxygenated phenolic monoterpens (methyl chavicole) and the mixture of mono- and sesquiterpene hydrocarbons. These findings are in correlation with the earlier published data on the antioxidant activities of the investigated essential oil and selected oil components [84,85].


**Table 8.** Inhibition of LP (%) in Fe2+/ascorbate system of induction by extracts of basil leaves and BHT (as a positive control) in the TBA assay.

The data presented in Table 8. show that the last three extracts of *O. basilicum* (EtOAc, *n*-BuOH and H2O) reduced the intensity of lipid peroxidation, while the first two extracts (Et2O and CHCl3) increased the intensity of LP, but statistically insignificant. The largest inhibitory activity was exhibited by ethyl acetate extract. High inhibitory effect of this three extracts can be related to the presence of the amount of total phenolic compounds and content of total flavonoids in the extracts, because a considerably content of total phenolic compounds and total flavonoids was determined in EtOAc and H2O extract of *O. basilicum*. Preliminary 2D-TLC (Two Dimensional - Thin Layer Chromatography) analysis showed that the dominant flavonoid in the EtOAc extract of *O. basilicum* is derivative of quercetin. It is known that quercetin shows high antioxidant activity because of present OH groups in position 3' ring B (includes a 3', 4'-dihydroxy group). In the same experiment we established the presence of caffeic acid and its derivatives in the H2O extract, which has two hydroxyl groups in ortho position. This was confirmed once again that the antioxidant capacity depends not only on quantity, but also depends on of the type of phenols and flavonoids present in the extracts. However, in extracts of *O. basilicum* higher content of total phenols and flavonoids from the EtOAc extract had an H2O extract. From all this it can be assumed that the polarity of flavonoid components affects their ability to inhibit the process of LP. Specifically, in this test we used liposomes as a model system of biological membranes, and the least polar flavonoids present in the EtOAc extract could help to approach the scene and engage in the process of defense from the LP, compared to more polar compounds that are found in H2O extract. A little less of total flavonoids was determined in *n*-BuOH extracts, while the smallest quantity of these compounds was found in Et2O and CHCl3 extracts. Differences in the amount of total phenolic compounds and flavonoid content between extracts can be explained by different number of secretory structures in various plant tissues [86]. Furthermore, the obtained results could be related to the protective role of phenolics, especially the flavonoid aglycones, in plants collected on the outskirts of big cities. One of the functions of these biomolecules, which are produced in response to ecological stress factors like pollution, is to serve as UV-B filters in plants [87]. It was established that flavonoids act as mighty scavengers of free radicals [88]. Different flavonoids inhibit LP *in vitro* and the most pronounced effect is exhibited by quercetin whose presence is found in extracts of *O. basilicum* using 2D-TLC [89]. More investigation is required to explain the enhanced production of phenolics in certain geographic areas [90]. Also, from the presented results we can conclude that the increase in concentration of the extracts does not significantly affect the inhibition of lipid peroxidation.

#### **3.4. Oregano (***Origanum vulgare* **L.)**

172 Lipid Peroxidation

peroxidation.

 **Extracts**

(as a positive control) in the TBA assay.

**Concentration** BHT Et2O CHCl3 EtOAc *n*-BuOH H2O **1%** 26.15 -0.87 -0.86 37.42 26.31 31.74 **5%** 26.15 -0.94 -0.89 38.91 27.06 35.29 **10%** 26.15 -1.01 -1.04 41.56 28.83 36.54 **Table 8.** Inhibition of LP (%) in Fe2+/ascorbate system of induction by extracts of basil leaves and BHT

The data presented in Table 8. show that the last three extracts of *O. basilicum* (EtOAc, *n*-BuOH and H2O) reduced the intensity of lipid peroxidation, while the first two extracts (Et2O and CHCl3) increased the intensity of LP, but statistically insignificant. The largest inhibitory activity was exhibited by ethyl acetate extract. High inhibitory effect of this three extracts can be related to the presence of the amount of total phenolic compounds and content of total flavonoids in the extracts, because a considerably content of total phenolic compounds and total flavonoids was determined in EtOAc and H2O extract of *O. basilicum*. Preliminary 2D-TLC (Two Dimensional - Thin Layer Chromatography) analysis showed that the dominant flavonoid in the EtOAc extract of *O. basilicum* is derivative of quercetin. It is known that quercetin shows high antioxidant activity because of present OH groups in position 3' ring B (includes a 3', 4'-dihydroxy group). In the same experiment we established the presence of caffeic acid and its derivatives in the H2O extract, which has two hydroxyl groups in ortho position. This was confirmed once again that the antioxidant capacity depends not only on quantity, but also depends on of the type of phenols and flavonoids present in the extracts. However, in extracts of *O. basilicum* higher content of total phenols and flavonoids from the EtOAc extract had an H2O extract. From all this it can be assumed that the polarity of flavonoid components affects their ability to inhibit the process of LP. Specifically, in this test we used liposomes as a model system of biological membranes, and the least polar flavonoids present in the EtOAc extract could help to approach the scene and engage in the process of defense from the LP, compared to more polar compounds that are found in H2O extract. A little less of total flavonoids was determined in *n*-BuOH extracts, while the smallest quantity of these compounds was found in Et2O and CHCl3 extracts. Differences in the amount of total phenolic compounds and flavonoid content between extracts can be explained by different number of secretory structures in various plant tissues [86]. Furthermore, the obtained results could be related to the protective role of phenolics, especially the flavonoid aglycones, in plants collected on the outskirts of big cities. One of the functions of these biomolecules, which are produced in response to ecological stress factors like pollution, is to serve as UV-B filters in plants [87]. It was established that flavonoids act as mighty scavengers of free radicals [88]. Different flavonoids inhibit LP *in vitro* and the most pronounced effect is exhibited by quercetin whose presence is found in extracts of *O. basilicum* using 2D-TLC [89]. More investigation is required to explain the enhanced production of phenolics in certain geographic areas [90]. Also, from the presented results we can conclude that the increase in concentration of the extracts does not significantly affect the inhibition of lipid Origanum is one of the most variable genera of Lamiaceae family. Originates from Europe, but is now cultivated throughout the world including USA, India and South America. This is an extremely variable species with several subspecies and named cultivars grown for ornamental, culinary and medicinal uses. Oregano is a bushy, semi-woody sub-shrub with upright or spreading stems and branches. Some varieties grow in mound like mats, spreading by underground stems (called rhizomes), and others with a more upright habit. The aromatic leaves are oval-shaped. Oregano will grow in a pH range between 6.0 (mildly acid) and 9.0 (strongly alkaline) with a preferred range between 6.0 and 8.0. The flowers are purple, 3–4 mm long, produced in erect spikes.

As the other three Lamiaceae species oregano is characterized by the presence of essential oil, flavonoids, phenolic acids (caffeic, chlorogenic and rosmarinic), triterpenoid acids (oleanolic and ursolic) and tannins. The oregano essential oil yield between 0.35-0.55% [91]. According to Arnold et al. [92], the content of essential oil in *Origanum ssp.* may come up even to 8.8%. Essential oils obtained from different parts of plant have a similar chemical profile. The dominant components are oxygenated phenolic monoterpenes thymol and carvacrol (Figure 11.), as well as sabinene, linalool, terpine-4-ol, α-pinene, caryophyllene, caryophyllene-oxide and 1,8-cineole.

**Figure 11.** Oxygenated phenolic monoterpens from *O. vulgare* essential oil

According to Duke [93], flavonoids are found in the leaves and whole plant, mostly as kaempferol, quercetin, apigenin, luteolin and rutin. Beside, oregano is rich in apigenin-7-Oβ-D-glucoside and luteolin-7-O-β-D-glucuronide. In oregano flavanon naringenin and flavanon glucoside (naringin), have also been identified (Figure 12.).

Most of the healing properties are attributed to the essential oil and flavonoids. It has been widely used in agricultural, pharmaceutical and cosmetic industries as a culinary herb, flavoring substances in food products, alcoholic beverages and perfumery for its spicy fragrance [94]. Regarding the nonvolatile components, the extracts of oregano have the most effective antioxidant activity among aromatic herbs [95]. Oregano family, is widely known as possessing therapeutic properties (diaphoretic, carminative, antispasmodic, antiseptic, tonic) being used in traditional medicine systems in many countries. Different groups of researchers [96,97] studied oregano alcohol extracts. The antioxidant effect of the mentioned extracts is generally due to the presence of rosmarinic and caffeic acid [98].

**Figure 12.** Structures of main flavonoids of *O. vulgare*


**Table 9.** Inhibition of LP (%) in Fe2+/ascorbate system of induction by essential oil of oregano leaves and BHT (as a positive control) in the TBA assay.

Our tests showed that only concentrated solutions of essential oil exhibit a greater ability to inhibit LP in liposomes of synthetic antioxidant BHT. The antioxidant activities were dose dependent, but it is noticeable that the values obtained using two most concentrated solution of essential oils (1.598 and 2.130 μg/mL) are very close (49.58 and 51.13% of inhibition of LP). For the inhibition of LP, the most responsible compounds were the oxygenated phenolic monoterpens (thymol and carvacrol) and the mixture of mono- and sesquiterpene hydrocarbons [98].


**Table 10.** Inhibition of LP (%) in Fe2+/ascorbate system of induction by extracts of oregano leaves and BHT (as a positive control) in the TBA assay.

The data presented in Table 10. show that the last three extracts of *O. vulgare* (EtOAc, *n*-BuOH and H2O) reduced the intensity of lipid peroxidation while the first two extracts (Et2O and CHCl3) have prooxidative effect (but not statistically significant). The largest inhibitory activity was exhibited by ethyl acetate extract. High inhibitory effect of this extract and its components towards Fe2+-dependent LP of liposomes can be related to the presence of flavonoids in the extract. It was established that flavonoids that antiradical potential of flavonoids are the most pronounced towards OH, peroxy- and alkoxy radicals, which are formed in the process of lipid peroxidation [99]. Also, these results are consistent with 2D-TLC analysis which showed that the dominant component of the EtOAc extract was kaempferol monoglycoside, while the H2O extract contains multiple kaempferol diglycosides. From the literature it is known that additional glycosylation reduces the antioxidant activity and capability to reduce lipid peroxidation [100].The antioxidant and prooxidant activities of all extracts of oregano leaves were dose dependent.

## **4. Conclusions**

174 Lipid Peroxidation

researchers [96,97] studied oregano alcohol extracts. The antioxidant effect of the mentioned

**Concentration (g/mL)** 

 **BHT 0.213 0.535 1.065 1.598 2.130 LP** 26.15 17.31 24.35 37.17 49.58 51.13 **Table 9.** Inhibition of LP (%) in Fe2+/ascorbate system of induction by essential oil of oregano leaves and

Our tests showed that only concentrated solutions of essential oil exhibit a greater ability to inhibit LP in liposomes of synthetic antioxidant BHT. The antioxidant activities were dose dependent, but it is noticeable that the values obtained using two most concentrated solution of essential oils (1.598 and 2.130 μg/mL) are very close (49.58 and 51.13% of inhibition of LP). For the inhibition of LP, the most responsible compounds were the oxygenated phenolic monoterpens (thymol and carvacrol) and the mixture of mono- and

**Concentration BHT Et2O CHCl3 EtOAc** *n***-BuOH H2O 1%** 26.15 -0.46 -0.92 24.17 11.31 13.58 **5%** 26.15 -0.77 -0.94 26.04 14.57 16.49 **10%** 26.15 -0.91 -0.97 30.28 19.78 23.24 **Table 10.** Inhibition of LP (%) in Fe2+/ascorbate system of induction by extracts of oregano leaves and

The data presented in Table 10. show that the last three extracts of *O. vulgare* (EtOAc, *n*-BuOH and H2O) reduced the intensity of lipid peroxidation while the first two extracts (Et2O and CHCl3) have prooxidative effect (but not statistically significant). The largest inhibitory

extracts is generally due to the presence of rosmarinic and caffeic acid [98].

**Figure 12.** Structures of main flavonoids of *O. vulgare*

BHT (as a positive control) in the TBA assay.

sesquiterpene hydrocarbons [98].

BHT (as a positive control) in the TBA assay.

 **Extracts**

It was found that excessive production of oxygen radicals in the body initiates oxidation and degradation of polyunsaturated fatty acids. It is known that free radicals attack the highly unsaturated fatty acid of membrane system and induce lipid peroxidation. Since lipid peroxidation causes oxidative damage to cell membranes and all other systems that contain lipids, in any investigation of total antioxidative activity of extracts and essential oils it is necessary to investigate their effects on lipid peroxidation. Some substrates (for example liposomes) are used more frequently than others, mainly because of the simplicity of the methods involved. In this way we get very useful information to direct future research. The results of our *in vitro* assays of examined four different Lamiaceae species extracts expressed significant protective effects on LP, which was found to be correlated to different compounds. It can be concluded that ethyl acetate and water proved to be the best solvent for extraction of plant material. Also, a very strong protective activity of the EtOAc and H2O extracts in lipid peroxidation processes was recorded, which means that they may have a protective role in oxidative stress. Experimental results indicate that the essential oil of *M. peregrinum* collected from the Senta locality (No.3) exhibited the strongest inhibitory effect on lipid peroxidation. Furthermore, the present chapter on the chemistry and biological activity of four well known Lamiaceae species explicitly prove that these plants may be an important sources of pharmalogically active substances, and thus can be used in the preparation of various herbal medicine.

## **Author details**

Biljana Kaurinovic and Mira Popovic *Department of Chemistry, Biochemistry and Environmental Protection, University of Novi Sad, Novi Sad, Republic of Serbia* 

## **Acknowledgement**

This work was supported by the Ministry of Science and Environmental Protection of the Republic of Serbia (Project No. 172058) and by the Provincial Secretariat for Science and Technological Development, Autonomous Province of Vojvodina, Republic of Serbia.

#### **5. References**


[20] Crowell PL, Ayoubi S, Burke YD (1996) Antitumorigenic Effects of Limopnene and Perillyl Alcohol Against Pancreatic and Breast Cancer. Adv. exp. med. biol. 401: 131-136.

176 Lipid Peroxidation

**5. References** 

pathol. 44: 169-181.

Species. Lab. invest. 62: 670-679.

biophys. res. commun. 14: 323-328.

Iron(III) Complex. Biochem. j. 258: 617-620.

Peroxidation. Free rad. biol. med. 4: 51-72.

in Delivery System Design. PSTT. 3: 417-425.

(in Serbian). Master Thesis. Novi Sad: PMF. 48 p.

phys. lipids. 44: 191-208.

Fitoterapia. 65: 397-401. [12] http://www.pharmaxchange.info

res. 46: 244-282.

58: 686-690.

Histol. histopathol. 197: 391-406.

lebensmitt.- unters. forsch. 197: 20-23.

and Antioxidant Therapy. Lancet. 23: 1396-1397.

[1] Dargel R (1992) Lipid Peroxidation – A Common Pathogenic Mechanism? Exp. toxicol.

[2] Ursini F, Maiorino M, Sevanian A (1991) Membrane Hydroperoxides. In: Sies H, editor. Oxidative Stress: Oxidants and Antioxidants. London: Academic Press. pp. 319-336. [3] Kagan VE (1988) Lipid Peroxidation in Biomembranes. Boca Raton: CRC Press. 1 p. [4] Farber JL, Kyle ME, Coleman JB (1990) Mechanisms of Cell Injury by Activated Oxygen

[5] Halliwell B, Gutteridge JMC. (1984) Lipid Peroxidation, Oxygen Radicals, Cell Damage

[6] Holchestein P, Nordenbrand K, Ernster L (1964) Evidence for the Involvement of Iron in the ADP-Activated Peroxidation of Lipids in Microsomes and Mitochondria. Biochem.

[7] Minotti G, Aust SD (1987) The Role of Iron in the Initiation of Lipid Peroxidation. Chem.

[8] Aruoma OI, Halliwell B, Laughton MJ, Quinlan J, Gutteridge JMC (1989) The Mechanism of Initiation of Lipid Peorxidation. Evidence Against a Requirement for an Iron(II)-

[9] Tang L, Zhang Y, Qian Z, Shen X (2000) The Mechanism of Fe2+-Initiated Lipid Peroxidation in Liposomes: The Dual Function of Ferrous Ions, the Roles of the Pre-

[11] Bombardelli E, Cristoni A, Morazzobi P (1994) PHYTOSOME in Functional Cosmetics.

[13] Foldvari M (2000) Non-Invasive Administration of Drugs Through the Skin. Challenges

[14] Immordino ML, Dosio F, Cattel L (2006) Stealth Liposomes: Review of the Basic Science, Rationale, and Clinical Applications, Existing and Potential. IJN 1(3): 297-315. [15] Kujundžić S (2002) Biochemical Investigaion of Plant Species from the Apiaceae Family

[16] Laguerre M, Lecomte J, Villeneuve P (2007) Evaluation of the Ability of Antioxidants to Counteract Lipid Oxidation: Existing Methods, New Trends and Challenges. Prog. lipid

[17] Safer AM, Al-Nughamish AJ (1999) Hepatotoxicity Induced by the Antioxidant Food Additive Butylated Hydroxytoluene (BHT) in Rats: An Electron Microscopical Study.

[18] Brraco U, Loliger J., Viret J (1981) Production and Use of Natural Antioxidants. JAOCS.

[19] Lagouri V, Blekas G, Tsimidou M, Kokkini S, Boskou D (1993) Composition and Antioxidant Activity of Essential Oil from Oregano Plants Grown in Greece. Z.

Existing Lipid Peroxides and the Lipid Peroxyl Radical. Biochem. j. 352: 27-36. [10] Chatterjee SN, Agarwal S (1988) Liposomes as Membrane Model for Study of Lipid

	- [41] http://www.ang.kfunigraz.ac.at/~katzer/engl.html
	- [42] Skrzypczak-Pietraszek E, Hensel A (2000) Polysaccharides from *Melittis melissophyllum*  L. Herb and Callus. Pharmazie. 55: 768-771.
	- [43] Skrzypczak, E.; Skrzypczak, L. (1993) The Tissue Culture and Chemical Analysis of *Melittis melissophyllum* L. Acta hort. 330: 263-267.
	- [44] http://www.umm.edu/altmed.html
	- [45] http://www.hort.purdue.edu/newcrop/med-aro.html
	- [46] Guarrera PM (2005) Traditional Phytotherapy in Central Italy (Marche, Abruzzo, and Latium). Fitoterapia. 76: 1–25.
	- [47] Maggi F, Bílek T, Lucarini D, Papa F, Sagratini G, Vittori S (2009) *M. melissophyllum* L. subsp. *melissophyllum* (Lamiaceae) from Central Italy: A New Source of a Mushroom-Like Flavour. Food chem. 113: 216-221.
	- [48] Kaurinovic B, Popovic M, Vlaisavljevic S, Raseta M (2011) Antioxidant Activities of *Melittis melissophyllum* L. (Lamiaceae). Molecules. 16: 3152-3167.
	- [49] Doba T, Burton GW, Ingold KU (1985) Antioxidant and Co-Oxidant Activity of Vitamin C. The Effects of Vitamin C, Either Alone or in the Presence of Vitamin E or a Water-Soluble Vitamin E Analogue, Upon the Peroxidation of Aqueous Multimalleral Phospholipid Liposomes. Biochim. biophys. acta. 835: 298-303.
	- [50] http://www.phytochemicals.info/phytochemicals/quercetin.php
	- [51] Acland ML, van de Waarsenburg S, Jones R (2005) Synergistic Antiproliferative Action of the Flavonols Quercetin and Kaempferol in Cultured Human Cancer Cell Lines. In vivo. 19(1): 69-76.
	- [52] http://www.phytochemicals.info/phytochemicals/rutin.php
	- [53] Decker EA (1997) Phenolics: Prooxidants or Antioxidants? Nutr. rev. 55: 396-407.
	- [54] Handa SS, Sharma A, Chakraborti KK (1986) Natural Products in Plants as Liver Protecting Drugs. Fitoterapia. 57: 307-310.
	- [55] Cholbi MR, Paya M, Alcaraz MJ (1991) Inhibitory Effect of Phenolic Compounds on CCl4-Induced Microsomal Lipid Peroxidation. Experimentia. 47: 195-198.
	- [56] Sousa RL, Marletta MA (1985) Inhibition of Cytochrome P-450 Activity in Rat Liver Microsomes by the Natural Occuring Flavonoid, Quercetin. Arch. biochem. biophys. 240: 345-348.
	- [57] Rekka E, Kourounakis PN (1991) Effect of Hydroxyethyl Rutoside and Related Compounds on Lipid Peroxidation and Free Radical Scavening Activity. Some Structural Aspects. J. pharmac. pharmacol. 43: 486-490.
	- [58] Larson RA (1988) The Antioxidant of Higher Plants. Phytochemistry. 27: 969-978.
	- [59] Cock C, Samman S (1996) Flavonoids Chemistry Metabolism, Cardioprotectioeffects and Dietary Sources. Nutrit. biochem. 7: 66-76.
	- [60] Stanković SM (2011) Total Phenolic Content, Flavonoid Concentration and Antioxidant Activity of *Marrubium peregrinum* L. Extracts. Kragujevac j. sci. 33: 63-72.
	- [61 Vlaisavljevic S (2007) Antioxidant Activities of *Marrubium peregrinum* L. Essential Oil and Extracts. Master Thesis. Novi Sad: PMF. 36 p.
	- [62] Nagy M, Gergel D, Grancai D, Novomesky P, Ubik K (1996) Antilipoperoxidative Activity of Some Phenolic Constituents from *Marrubium peregrinum* L. Farmaceuticky-Obzor. 65: 283-285.

[63] Sahpaz S, Hennebelle T, Bailleul F (2002) Marruboside, a New Phenylethanoid Glycoside from *Marrubium vulgare* L. Nat. prod. lett. 16: 195-199.

178 Lipid Peroxidation

[41] http://www.ang.kfunigraz.ac.at/~katzer/engl.html

L. Herb and Callus. Pharmazie. 55: 768-771.

[44] http://www.umm.edu/altmed.html

Latium). Fitoterapia. 76: 1–25.

vivo. 19(1): 69-76.

345-348.

Obzor. 65: 283-285.

Like Flavour. Food chem. 113: 216-221.

*Melittis melissophyllum* L. Acta hort. 330: 263-267.

[45] http://www.hort.purdue.edu/newcrop/med-aro.html

[42] Skrzypczak-Pietraszek E, Hensel A (2000) Polysaccharides from *Melittis melissophyllum* 

[43] Skrzypczak, E.; Skrzypczak, L. (1993) The Tissue Culture and Chemical Analysis of

[46] Guarrera PM (2005) Traditional Phytotherapy in Central Italy (Marche, Abruzzo, and

[47] Maggi F, Bílek T, Lucarini D, Papa F, Sagratini G, Vittori S (2009) *M. melissophyllum* L. subsp. *melissophyllum* (Lamiaceae) from Central Italy: A New Source of a Mushroom-

[48] Kaurinovic B, Popovic M, Vlaisavljevic S, Raseta M (2011) Antioxidant Activities of

[49] Doba T, Burton GW, Ingold KU (1985) Antioxidant and Co-Oxidant Activity of Vitamin C. The Effects of Vitamin C, Either Alone or in the Presence of Vitamin E or a Water-Soluble Vitamin E Analogue, Upon the Peroxidation of Aqueous Multimalleral

[51] Acland ML, van de Waarsenburg S, Jones R (2005) Synergistic Antiproliferative Action of the Flavonols Quercetin and Kaempferol in Cultured Human Cancer Cell Lines. In

[54] Handa SS, Sharma A, Chakraborti KK (1986) Natural Products in Plants as Liver

[55] Cholbi MR, Paya M, Alcaraz MJ (1991) Inhibitory Effect of Phenolic Compounds on

[56] Sousa RL, Marletta MA (1985) Inhibition of Cytochrome P-450 Activity in Rat Liver Microsomes by the Natural Occuring Flavonoid, Quercetin. Arch. biochem. biophys. 240:

[57] Rekka E, Kourounakis PN (1991) Effect of Hydroxyethyl Rutoside and Related Compounds on Lipid Peroxidation and Free Radical Scavening Activity. Some

[59] Cock C, Samman S (1996) Flavonoids Chemistry Metabolism, Cardioprotectioeffects

[60] Stanković SM (2011) Total Phenolic Content, Flavonoid Concentration and Antioxidant

[61 Vlaisavljevic S (2007) Antioxidant Activities of *Marrubium peregrinum* L. Essential Oil and

[62] Nagy M, Gergel D, Grancai D, Novomesky P, Ubik K (1996) Antilipoperoxidative Activity of Some Phenolic Constituents from *Marrubium peregrinum* L. Farmaceuticky-

[58] Larson RA (1988) The Antioxidant of Higher Plants. Phytochemistry. 27: 969-978.

Activity of *Marrubium peregrinum* L. Extracts. Kragujevac j. sci. 33: 63-72.

[53] Decker EA (1997) Phenolics: Prooxidants or Antioxidants? Nutr. rev. 55: 396-407.

CCl4-Induced Microsomal Lipid Peroxidation. Experimentia. 47: 195-198.

*Melittis melissophyllum* L. (Lamiaceae). Molecules. 16: 3152-3167.

Phospholipid Liposomes. Biochim. biophys. acta. 835: 298-303.

[50] http://www.phytochemicals.info/phytochemicals/quercetin.php

[52] http://www.phytochemicals.info/phytochemicals/rutin.php

Structural Aspects. J. pharmac. pharmacol. 43: 486-490.

and Dietary Sources. Nutrit. biochem. 7: 66-76.

Extracts. Master Thesis. Novi Sad: PMF. 36 p.

Protecting Drugs. Fitoterapia. 57: 307-310.


**Chapter 8** 

## **Liposomes as a Tool to Study Lipid Peroxidation in Retina**

Natalia Fagali and Angel Catalá

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46074

## **1. Introduction**

180 Lipid Peroxidation

[82] Gülçin I, Elmastaş M, Aboul-Enein YH (2007) Determination of Antioxidant and Radical Scavenging Activity of Basil (*Ocimum basilicum* L. Family Lamiaceae) Assayed by

[83] Lee SJ, Umano K, Shibamoto T, Lee KG (2005) Identification of Volatile Components in Basil (*Ocimum basilicum* L.) and Thyme Leaves (*Thymus vulgaris* L.) and Their

[84] Ruberto G, Baratta MT (2000) Antioxidant Activity of Selected Essential Oil

[85] Lewinsohn E, Ziv-Raz I, Dudai N, Tadmor Y, Lastochkin E, Larkov O, Chaimovitsh D, Ravid U, Putievsky E, Pichersky E, Shoham Y (2000) Biosynthesis of Estragole and Methyl Eugenol in Sweet Basil (*Ocimum basilicum* L.). Developmental and Chemotypic

[88] Robak J, Gryglewski J (1988) Flavonoids are Scavengers of Superoxide Anion. Biochem.

[89] Kaurinovic B, Popovic M, Vlaisavljevic S, Trivic S (2011) Antioxidant Capacity of *Ocimum basilicum* L. and *Origanum vulgare* L. Extracts. Molecules. 16: 7401-7416. [90] Lakic N, Mimica-Dukic N, Isak J, Božin B (2010) Antioxidant Properties of *Galium verum* 

[91] W'glarz Z, Osidska E, Geszprych A, Przybyb J (2006) Intraspecific Variability of Wild Marjoram (*Origanum vulgare* L.) Naturally Occurring in Poland. Rev. bras. pl. med.

[92] Arnold N, Bellomaria B, Valentini G (2000) Composition of the Essential Oil of Different

[93] Duke J (1997) The Green Pharmacy, the Ultimate Compendium of Natural Remedies from the World's Foremost Authority on Healing and Herbs. New York: St. Martin's Press. [94] Souza EL, Stamford TLM, Lima EO, Trajano VN (2007) Effectiveness of *Origanum vulgare*  L. Essential Oil to Inhibit the Growth of Food Spoiling Yeasts. Food Control. 18: 409-413. [95] Vekiari SA, Oreopoulou V, Tzia C, Thomopoulos CD (1993) Oregano Flavonoids as

[96] Chevolleau S, Mallet JF, Ucciani E, Gamisans J, Gruber M (1992) Effects of Rosemary Extracts and Major Constituents on Lipid Oxidation and Soybean Lipoxygenase

[98] Milos M, Mastelic J, Jerkovic I (2000) Chemical Composition and Antioxidant Effect of Glycosidically Bound Volatile Compounds from Oregano (*Origanum vulgare* L. ssp.

[99] Sichel G, Corsaro C, Scalia M, Dibilio J, Bonomo R (1991) *In Vitro* Scavenger Activity of

[100] Rice-Evans C, Miller NJ, Paganga G (1997) Antioxidant Properties of Phenolic

Species of *Origanum* in the Eastern Mediterrenean. JEOR. 12: 192-196.

[97] Kramer RE (1985) Antioxidants in Clove. J. am. oil chem. soc. 62: 111-113.

Some Flavonoids and Melanins Against O2. Free rad. biol. med. 11: 1-8.

Association of Allaphenol O-Methyltransferase Activities. Plant sci. 160: 27-35. [86] Tucakov J (1997) Healing With Herbs (Phytotherapy) (in Serbian). Beograd: Rad. 34 p. [87] Kootstra A (1994) Protection from UV-B-Induced DNA Damage by Flavonoids. Plant

Components in Two Lipid Model Systems. Food chem. 68: 167-174.

Different Methodologies. Phytother. res. 21: 354-361.

Antioxidant Properties. Food chem. 91: 131-137.

L. (Rubiaceae) Extracts. Cent. eur. j. biol. 5: 331-337.

Lipid Antioxidants. J. am. oil chem. soc. 70: 483-487.

Activity. J. am. oil chem. soc. 69: 1269-1271.

Compounds. Trends plant sci. 2: 152-159.

*Hirtum*). Food chem. 71: 79-83.

mol. biol. 26: 771-774.

pharmacol. 37: 837-841.

botucatu. 8: 23-26.

In living organisms, the oxidative stress is associated with several physio- pathological affections (e.g. atherosclerosis, cancer, aging, neurodegenerative diseases). The oxidative stress is generally initiated by generation of reactive oxygen (ROS) and nitrogen species (RNS) (Halliwell & Gutteridge, 1990). ROS are continuously formed during cellular metabolism and are removed by antioxidants defences. ROS from endogenous and exogenous sources results in continuous and accumulative oxidative damage to cellular components and alters many cellular functions. The most vulnerable molecules to oxidative damage are proteins, lipids and DNA (Kohen & Nyska, 2002; Catalá, 2009, 2011a, 2011b).

In mammalian retina, free radicals and lipoperoxides seem to play important roles in the evolution of different retinopathies including glaucoma, cataractogenesis, diabetic retinopathy, ocular inflammation and retinal degeneration (Ueda et al., 1996; De La Paz & Anderson, 1992). Because of free radicals production induces the lipid peroxyl radical formation, known as secondary free radicals products; this chain reaction of lipid peroxidation can damage the retina, especially the membranes that play important roles in visual function (Catalá, 2006). The retina is the neurosensorial tissue of the eye. It is very rich in membranes and therefore in polyunsaturated fatty acids (PUFAs) such as docosahexenoic acid (22:6 n-3), that are quite vulnerable to lipid peroxidation. Also, the human retina is a well oxygenated tissue. High-energy short-wavelength visible light promotes the formation of ROS which can initiate lipid peroxidation in the macula and elsewhere. The macular carotenoids are thought to combat light-induced damage mediated by ROS by absorbing the most damaging incoming wavelengths of light prior to the formation of ROS and by chemically quenching ROS once they are formed.

Although peroxidation in model membranes may be very different from peroxidation in biological membranes, the results obtained in model membranes may be used to progress

© 2012 Fagali and Catalá, 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. © 2012 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.

our understanding of subjects that cannot be studied in biological membranes. Nevertheless, in spite of the relative simplicity of peroxidation of liposomal lipids model, these reactions are still relatively complex because they depend in a complex fashion on liposome type, reaction initiator and reaction medium (Fagali & Catalá, 2009). This complexity is the most likely cause of the apparent contradictions of literature results.

Biological membranes are complex systems. In view of this complexity and in order to avoid collateral effects that may arise during lipid peroxidation process of whole retinal membranes, we have attempted to gain understanding of the mechanisms responsible for peroxidation in a simple model system, made by dispersing retinal lipids in the form of liposomes.

This chapter describes a very useful method to prepare liposomes with natural phospholipids and the necessary methodology to follow the lipid peroxidation of these liposomes.

## **2. Materials and methods**

### **2.1. Materials**

Chloroform, methanol, trizma base, butylated hydroxytoluene (BHT), NaCl, FeSO4 heptahydrate and 2-thiobarbituric acid (TBA) were purchased from Sigma Chemical Co. Suitable plastic lab ware was used throughout this study to avoid effects of adventitious metals. Other reagents were of the highest quality commercially available. All solutions were prepared using distilled water treated with a Millipore Q system.

## **2.2. Isolation of bovine retina**

Eyes were enucleated at slaughter (Frigorífico Gorina), transported in ice to laboratory where retinas were taken out within 1–2 h. Under red light and with all tubes and solutions in ice buckets, corneas were excised; lenses and vitreous were subsequently removed. Eye cups were inverted and retinas were carefully peeled from the eyes. Retinas were briefly homogenized in 0.15 M NaCl (1 ml/retina) 120 s (20on-20off) at 4 ºC in an Ultraturrax X25 homogenizer at 7000 rpm.

#### **2.3. Lipid extraction**

Total lipids were extracted from retinal homogenates with chloroform/methanol (2:1 v/v) (Folch et al, 1957) at 4 ºC (sample:Folch = 1:5). A volume of water corresponding to 20 % of total volume was added. This mixture was shaken and kept in rest in cold to allow phases separation. Chloroformic phase was kept at -22 °C.

#### **2.4. Preparation of liposomes made of retinal lipids**

Total lipids obtained from retinal homogenates dissolved in chloroform were evaporated under nitrogen until constant weight and submitted to vacuum to remove traces of chloroform. Resultant films were dispersed at room temperature in a saline solution (0.15 M NaCl). Dispersed lipids were mixed to homogeneity using a vortex-mixer to obtain nonsonicated liposomes (NSL). Sonicated liposomes (SL) were prepared by sonication of NSL under nitrogen and ice cooling (Huang, 1969), using a Sonics vibra cell, probe-sonicator Model VCX 750 (750 W, 20kHz) at 75% of maximal output. Preparation of liposomes required about 2.5 min of sonication to reach apparently minimal optical density values.

#### **2.5. Determination of liposomes size by Dynamic Light-Scattering (DLS)**

182 Lipid Peroxidation

liposomes.

**2.1. Materials** 

**2. Materials and methods** 

**2.2. Isolation of bovine retina** 

homogenizer at 7000 rpm.

separation. Chloroformic phase was kept at -22 °C.

**2.4. Preparation of liposomes made of retinal lipids** 

**2.3. Lipid extraction** 

our understanding of subjects that cannot be studied in biological membranes. Nevertheless, in spite of the relative simplicity of peroxidation of liposomal lipids model, these reactions are still relatively complex because they depend in a complex fashion on liposome type, reaction initiator and reaction medium (Fagali & Catalá, 2009). This complexity is the most

Biological membranes are complex systems. In view of this complexity and in order to avoid collateral effects that may arise during lipid peroxidation process of whole retinal membranes, we have attempted to gain understanding of the mechanisms responsible for peroxidation in a simple model system, made by dispersing retinal lipids in the form of

This chapter describes a very useful method to prepare liposomes with natural phospholipids

Chloroform, methanol, trizma base, butylated hydroxytoluene (BHT), NaCl, FeSO4 heptahydrate and 2-thiobarbituric acid (TBA) were purchased from Sigma Chemical Co. Suitable plastic lab ware was used throughout this study to avoid effects of adventitious metals. Other reagents were of the highest quality commercially available. All solutions

Eyes were enucleated at slaughter (Frigorífico Gorina), transported in ice to laboratory where retinas were taken out within 1–2 h. Under red light and with all tubes and solutions in ice buckets, corneas were excised; lenses and vitreous were subsequently removed. Eye cups were inverted and retinas were carefully peeled from the eyes. Retinas were briefly homogenized in 0.15 M NaCl (1 ml/retina) 120 s (20on-20off) at 4 ºC in an Ultraturrax X25

Total lipids were extracted from retinal homogenates with chloroform/methanol (2:1 v/v) (Folch et al, 1957) at 4 ºC (sample:Folch = 1:5). A volume of water corresponding to 20 % of total volume was added. This mixture was shaken and kept in rest in cold to allow phases

Total lipids obtained from retinal homogenates dissolved in chloroform were evaporated under nitrogen until constant weight and submitted to vacuum to remove traces of

and the necessary methodology to follow the lipid peroxidation of these liposomes.

were prepared using distilled water treated with a Millipore Q system.

likely cause of the apparent contradictions of literature results.

The time correlation *G*(*q*,*t*) of the light-scattering intensity was measured at 90° with a goniometer, ALV/CGS-5022F, with a multiple-τ digital correlator, ALV-5000/EPP, covering a 10-6-103 s time range. The light source was a helium/neon laser with a wavelength of 633 nm operating at 22 mW. Each correlation function was analyzed by the well known cumulant fit yielding the apparent mean diffusion coefficient and the distribution δD of this value (Koppel, 1972). The measurements were carried out with 80 μl of SL and NSL (lipid concentration= 2 mg/ml) in water, 0.15 M NaCl and Tris-HCl buffer 20 mM (final volume= 2 mL).

#### **2.6. Measurements of lipid peroxidation by detection of conjugated dienes and trienes production**

In order to determine conjugated dienes and trienes production, absorption spectra were recorded by means of a Shimadzu UV-1800 spectrophotometer, in the range 200 to 300 nm, at 22 °C, with 1 cm path length quartz cell. Liposomes made of retinal lipids (80 μl, 2 g/l of lipids) were diluted to 2 ml with water, 0.15 M NaCl or 20 mM Tris-HCl pH 7.4, and oxidation was initiated by the addition of FeSO4 (final concentration = 25 μM). Lipid peroxidation was assessed continuously by measuring the increase in absorbance at 234 nm (formation of conjugated dienes) and 270 nm (formation of conjugated trienes) taken at 1 min intervals. Oxidation rates were determined as the slope of a regression line drawn through linear range of absorbance versus time curve. Lag times were determined as time corresponding to intersection of oxidation rate regression line with a regression line drawn through initial phase of oxidation (Sargis & Subbaiah, 2003).

#### **2.7. Measurements of lipid peroxidation by detection of Thiobarbituric Reactive Substances (TBARS)**

During Fe2+- initiated reactions, extent of liposomal lipid peroxidation was assessed using a TBA assay. In this procedure, 850 μL of TBA (0.375% w/v TBA, 0.25 N HCl) were added to aliquots of 150 μl of reaction mixture containing BHT (0.1 % w/v in ethanol) to prevent possible peroxidation of liposomes during incubation. The aliquots were taken at different intervals of time. Samples were heated for 30 min at 75 **°**C. Absorbance was measured at 532 nm for determination of aldehydic breakdown products of lipid peroxidation.

#### **2.8. Preparation of Fatty Acids Methyl Esters (FAME)**

Lipids from retina, liposomes or liposomes exposed to peroxidation initiated by Fe2+, in absence or presence of BHT, were extracted according to the method of Folch et al (1957). A similar reaction mixture to that used in the analysis of conjugated dienes but scaled up 7.5 times was used to analyze the fatty acid composition of the samples

After one hour of incubation of liposomes with or without Fe2+ in the presence or absence of BHT, the samples were mixed with 15 ml of chloroform:methanol (2:1 v/v) containing 0.01 % BHT to stop the reaction. The mixture was stirred, gassed with nitrogen and kept in refrigerator overnight to achieve separation of phases. The lower chloroform phase was filtered through paper filter containing anhydrous sodium sulphate. The solvent was evaporated to dryness under nitrogen. Dry lipids of retina and/or liposomes were transmethylated with 300 μl of 1.3 M BF3 in methanol at 65C during 180 min. After incubation 1 ml of 0.15 M NaCl was added and the fatty acid methyl esters were extracted with 1 ml of hexane. This phase was injected onto the chromatograph.

#### **2.9. Gas chromatography – Mass spectrometry analyses**

GC–MS analyses were done using a Perkin Elmer Clarus 560D MS - gas chromatograph equipped with a mass selective detector with quadrupole analyzer and photomultiplier detector and a split/splitless injector. In the gas chromatographic system, a Elite 5MS (Perkin Elmer) capillary column (30 m, 0.25 mm ID, 0.25 μm df) was used. Column temperature was programmed from 130 to 250 °C at a rate of 5 °C/min and 250 °C for 6 min. Injector temperature was set to 260 ºC and inlet temperature was kept at 250 °C. Split injections were performed with a 10:1 split ratio. Helium carrier gas was used at a constant flow rate of 1 ml/min. In the mass spectrometer, electron ionization (EI+) mass spectra was recorded at 70 eV ionization energy, in full scan mode (50-400) unit mass range. The ionization source temperature was set at 180 °C. The fatty acid composition of the lipid extracts was determined by comparing their methyl derivatives mass fragmentation patterns with those of mass spectra from the NIST databases.

### **3. Results**

#### **3.1. Size of sonicated and non-sonicated liposomes made of retinal lipids in different aqueous media**

Average hydrodynamic radii of liposomes determined by DLS studies are presented in Table 1. We noted that NSL display a multimodal size distribution when analyzed by inverse Laplace transform (CONTIN), a result that is compatible with the high polydispersity index (PI > 0.4) from cumulants fit. Thus, hydrodynamic radii for NSL, at room temperature in different aqueous media, cover a broad range with intensity weighted maxima centered between 190 and 320 nm. On the other hand, results for liposomes formed by sonication gave, through cumulant method, hydrodynamic radii in the order of 76.4-83.3 nm, showing as expected significant influence of sonication on size and distribution. It is clear that NSL possessed higher hydrodynamic radii than SL. Either NSL or SL in water were slightly smaller than that in 0.15 M NaCl and Tris-buffer.

184 Lipid Peroxidation

**2.8. Preparation of Fatty Acids Methyl Esters (FAME)** 

times was used to analyze the fatty acid composition of the samples

with 1 ml of hexane. This phase was injected onto the chromatograph.

**2.9. Gas chromatography – Mass spectrometry analyses** 

of mass spectra from the NIST databases.

**different aqueous media** 

**3. Results** 

Lipids from retina, liposomes or liposomes exposed to peroxidation initiated by Fe2+, in absence or presence of BHT, were extracted according to the method of Folch et al (1957). A similar reaction mixture to that used in the analysis of conjugated dienes but scaled up 7.5

After one hour of incubation of liposomes with or without Fe2+ in the presence or absence of BHT, the samples were mixed with 15 ml of chloroform:methanol (2:1 v/v) containing 0.01 % BHT to stop the reaction. The mixture was stirred, gassed with nitrogen and kept in refrigerator overnight to achieve separation of phases. The lower chloroform phase was filtered through paper filter containing anhydrous sodium sulphate. The solvent was evaporated to dryness under nitrogen. Dry lipids of retina and/or liposomes were transmethylated with 300 μl of 1.3 M BF3 in methanol at 65C during 180 min. After incubation 1 ml of 0.15 M NaCl was added and the fatty acid methyl esters were extracted

GC–MS analyses were done using a Perkin Elmer Clarus 560D MS - gas chromatograph equipped with a mass selective detector with quadrupole analyzer and photomultiplier detector and a split/splitless injector. In the gas chromatographic system, a Elite 5MS (Perkin Elmer) capillary column (30 m, 0.25 mm ID, 0.25 μm df) was used. Column temperature was programmed from 130 to 250 °C at a rate of 5 °C/min and 250 °C for 6 min. Injector temperature was set to 260 ºC and inlet temperature was kept at 250 °C. Split injections were performed with a 10:1 split ratio. Helium carrier gas was used at a constant flow rate of 1 ml/min. In the mass spectrometer, electron ionization (EI+) mass spectra was recorded at 70 eV ionization energy, in full scan mode (50-400) unit mass range. The ionization source temperature was set at 180 °C. The fatty acid composition of the lipid extracts was determined by comparing their methyl derivatives mass fragmentation patterns with those

**3.1. Size of sonicated and non-sonicated liposomes made of retinal lipids in** 

Average hydrodynamic radii of liposomes determined by DLS studies are presented in Table 1. We noted that NSL display a multimodal size distribution when analyzed by inverse Laplace transform (CONTIN), a result that is compatible with the high polydispersity index (PI > 0.4) from cumulants fit. Thus, hydrodynamic radii for NSL, at room temperature in different aqueous media, cover a broad range with intensity weighted maxima centered between 190 and 320 nm. On the other hand, results for liposomes formed by sonication gave, through cumulant method, hydrodynamic radii in the order of 76.4-83.3


**Table 1.** Summary of values obtained by dynamic light scattering of SL and NSL made of retinal lipids in different aqueous media. Hydrodynamic radii values are the average of at least three representative determinations in each media.

## **3.2. Evolution of UV spectra as a function of time for Fe2+ initiated lipid peroxidation of SL and NSL in different aqueous media**

Figure 1 shows evolution of UV spectra as a function of time, for Fe2+ initiated lipid peroxidation of SL and NSL, in different aqueous media. This figure showed increases in UV absorption with a maximum at 234 nm and at 270 nm, due to conjugated dienes and trienes respectively, and a decrease of absorbance at 200-215 nm, due to loss of methylene interrupted double bonds (unoxidized lipids). When lipid peroxidation was carried out in water or 0.15 M NaCl decreases at 200-215 nm were more notorious than in reactions carried out in Tris-buffer.

## **3.3. Conjugated dienes, trienes and TBARS are excellent markers of lipid peroxidation of liposomes made of retinal lipids**

Figure 2 shows changes in TBARS production and variation of absorbance at 234 nm and 270 nm as a function of time.

When SL were peroxidized in water (**Figure 2A**) a lag phase of 30 min, followed by a fast rate, was observed in TBARS production. Absorbance final value at 532 nm reached was 0.24. Increase of absorbance at 234 nm showed a small lag phase followed by a fast initial phase until 40 min, since then speed of reaction became slighter. This behaviour was also observed in measured absorbance at 270 nm, but all absorbance values were lower than that at 234 nm in the range of time studied.

Lipid peroxidation of SL in 0.15 M NaCl (**Figure 2B**). showed an immediate and fast production of TBARS without lag phase, reaching a final value (Absf≈ 0.23) similar to that obtained in water. The absorbance at 234 nm increased with an initial speed greater than

**Figure 1.** Time evolution (0, 90 and 180 min) of UV spectra of liposomes peroxidized with Fe2+ as an initiator of the reaction. SL in A) water, B) 0.15 M NaCl, C) buffer Tris. NSL in D) water and) 0.15 M NaCl, F) buffer Tris

**Figure 2.** TBARS production (─•─) and variation of absorbance at 234 nm (─) and 270 nm (−−) as a function of time, during Fe2+-catalyzed lipid peroxidation of SL (top) and NSL (bottom). TBARS were determined at 0, 15, 30, 60, 120 and 180 min after addition of Fe2+. Aqueous media where reactions were carried out: A, D: water; B, E: 0.15 M NaCl; C, F: 20 mM Tris-HCl pH 7.4.

that observed in water, became the highest to 30 minutes and, then, diminished slowly. The absorbance at 270 nm increased with an initial speed greater than that observed in water, became the highest around the 30 min and then remained constant.

186 Lipid Peroxidation

NaCl, F) buffer Tris

**Figure 1.** Time evolution (0, 90 and 180 min) of UV spectra of liposomes peroxidized with Fe2+ as an initiator of the reaction. SL in A) water, B) 0.15 M NaCl, C) buffer Tris. NSL in D) water and) 0.15 M

**Figure 2.** TBARS production (─•─) and variation of absorbance at 234 nm (─) and 270 nm (−−) as a function of time, during Fe2+-catalyzed lipid peroxidation of SL (top) and NSL (bottom). TBARS were determined at 0, 15, 30, 60, 120 and 180 min after addition of Fe2+. Aqueous media where reactions were

carried out: A, D: water; B, E: 0.15 M NaCl; C, F: 20 mM Tris-HCl pH 7.4.

Lipid peroxidation of SL in Tris- buffer (**Figure 2C**) showed the largest lag phase and the lowest final value of absorbance (Absf= 0.09) for TBARS formation. The initial speed of TBARS production was also the lowest. Initial speed of reaction observed, by increase of absorbance at 234 nm, was lower than that measured on water and 0.15 M NaCl. Absorbance reached the maximum at 30 minutes and then remained constant. Conjugated trienes production was very similar to that of conjugated dienes.

Lipid peroxidation of NSL in water (**Figure 2D**) showed a lag phase of 30 min for the TBARS production and a final value of absorbance of 0.21. Changes of absorbance at 234 nm displayed a lag of 16 min, increased quickly from this time to 60 min and since then continued increasing with lower speed. Values of absorbance at 270 nm were below than those observed at 234 nm, although the behavior was similar.

Lipid peroxidation of NSL in 0.15 M NaCl (**Figure 2E**) showed an initial speed of TBARS production greater than that observed in water, but with a very similar final value (Absf= 0.21). Changes in absorbance at 270 nm and 234 nm showed greater initial speeds than the corresponding ones in water. These speeds stayed until 30 minutes and since then, absorbance values did not change. Final values of absorbance in 0.15 M NaCl were smaller than the water ones.

Lipid peroxidation of NSL in Tris-buffer (**Figure 2F**) showed the greatest lag phase (60 min) in TBARS production and the smallest initial reaction rate. The final value was 0.08, a result much smaller than those obtained in water and 0.15 M NaCl. Values of change of absorbance determined at 270 nm and 234 nm were practically the same. Initial speeds were similar to those obtained in water and slower to those observed in 0.15 M NaCl. The reached final values were below to those obtained in water and 0.15 M NaCl.

SL were more susceptible to lipid peroxidation than NSL both in water as in 0.15 M NaCl. Nevertheless, both types of liposomes were equally peroxidized in Tris-buffer.

### **3.4. Fatty acid composition of retinal lipids and liposomes made of these retinal lipids**

Figure 3 shows the fatty acid composition (area %) of retinal lipids and of liposomes made of these retinal lipids (SL-Fe, control). This table also compares fatty acid profiles of control with liposomes incubated with Fe2+ for 1 h, in absence and in presence of BHT. Retinal lipids show a high percent (25.8 ± 0.6 %) of docosahexaenoic acid (22:6 n-3), characteristic of this tissue. The retina has approximately 40 percent of PUFAs and 60 percent of saturated and monounsaturated fatty acids. SL prepared with these lipids show a decrease of 22:6 n-3. The PUFAs diminished significantly after incubation with Fe2+. This produce a relative increase of saturated and monounsaturated fatty acids. 5 μM BHT protected PUFAs avoiding lipid peroxidation effects and the fatty acid profile there was not significant differences with control.

**Figure 3.** Fatty acid composition (area %) of retinal lipids, liposomes made of retinal lipids (SL -Fe, control), liposomes incubated with Fe2+ (SL + Fe) and liposomes incubated with Fe2+ in the presence of BHT. Results are expressed as ݔ േ ܵܦ. ݔ: Average of area % of 3 assays, SD: standard deviation. Significant differences analyzed by ANOVA with control are marked with (\*).

### **4. Conclusion**

In summary, the presented results are indicative that liposomes made of retinal lipids by their structural similarities with the biomembranes constitute a very useful analytical system and can mimic the cellular membranes, providing additional information to that obtained with the whole retina. In addition, SL prepared with phospholipids obtained from selected tissues should be used in order to measure lipid peroxidation and the effect of different antioxidants. Additionally, we presented some simple techniques of many possibles that can be applied to study the lipid peroxidation process, different reaction initiators and the antioxidant effect of new compounds.

### **Abbreviations**

16:0: palmitic acid, 18:1 n-9: oleic acid, 18:2 n-6: linoleic acid, 20:4 n-6: arachidonic acid, 22:6 n-3: docosahexaenoic acid, BHT: butylated hydroxitoluene, GC-MS: gas chromatography–mass spectrometry, PUFAs: polyunsaturated fatty acids, TBARS: thiobarbituric acid reactive substances, RNS: reactive nitrogen species, ROS: reactive oxygen species, SL: sonicated liposomes.

### **Author details**

188 Lipid Peroxidation

**4. Conclusion** 

**Abbreviations** 

liposomes.

**Figure 3.** Fatty acid composition (area %) of retinal lipids, liposomes made of retinal lipids (SL -Fe, control), liposomes incubated with Fe2+ (SL + Fe) and liposomes incubated with Fe2+ in the presence of BHT. Results are expressed as ݔ േ ܵܦ. ݔ: Average of area % of 3 assays, SD: standard deviation.

In summary, the presented results are indicative that liposomes made of retinal lipids by their structural similarities with the biomembranes constitute a very useful analytical system and can mimic the cellular membranes, providing additional information to that obtained with the whole retina. In addition, SL prepared with phospholipids obtained from selected tissues should be used in order to measure lipid peroxidation and the effect of different antioxidants. Additionally, we presented some simple techniques of many possibles that can be applied to study the lipid peroxidation process, different reaction

16:0: palmitic acid, 18:1 n-9: oleic acid, 18:2 n-6: linoleic acid, 20:4 n-6: arachidonic acid, 22:6 n-3: docosahexaenoic acid, BHT: butylated hydroxitoluene, GC-MS: gas chromatography–mass spectrometry, PUFAs: polyunsaturated fatty acids, TBARS: thiobarbituric acid reactive substances, RNS: reactive nitrogen species, ROS: reactive oxygen species, SL: sonicated

Significant differences analyzed by ANOVA with control are marked with (\*).

initiators and the antioxidant effect of new compounds.

Natalia Fagali\* and Angel Catalá

*Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas, (INIFTA-CCT La Plata-CONICET), Facultad de Ciencias Exactas, Universidad Nacional de La Plata. Casilla de Correo 16, La Plata, Argentina* 

## **Acknowledgement**

The financial support of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina, Grant PIP-0157, is gratefully acknowledged. The authors thank at the Departamento de Física, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina for providing light scattering measurements facilities. Special thanks to Prof. J.L. Alessandrini for fitting experimental data.

## **5. References**


<sup>\*</sup> Corresponding Author

Ueda, T., Ueda, T., Armstrong, D. (1996). Preventive effect of natural and synthetic antioxidants on lipid peroxidation in the mammalian eye. *Ophthalmic Res*, 28, 184– 192.

**Lipid Peroxidation in Vegetables, Oils, Plants and Meats** 

190 Lipid Peroxidation

192.

Ueda, T., Ueda, T., Armstrong, D. (1996). Preventive effect of natural and synthetic antioxidants on lipid peroxidation in the mammalian eye. *Ophthalmic Res*, 28, 184–

## **The Effect of Plant Secondary Metabolites on Lipid Peroxidation and Eicosanoid Pathway**

Neda Mimica-Dukić, Nataša Simin, Emilija Svirčev, Dejan Orčić, Ivana Beara, Marija Lesjak and Biljana Božin

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/48193

## **1. Introduction**

Inflammation, free radical damage and oxidative stress have become major health issues in recent years and the subject of plenty of research. These processes are implicated in cancer [1], cardiovascular diseases [2], multiple sclerosis [3], diabetes mellitus [4], Alzheimer's and Parkinson's diseases [5], rheumatoid arthritis [6], premature aging [7] and almost any other degenerative condition. Reactive oxygen species (ROS), which are involved in these physiological functional changes, are often either by-products of the normal cellular processes or are formed by action of exogenous factors - xenobiotics, ionizing radiation, stress, pathogens etc. Overproduction of ROS leads to oxidative stress, with biomolecules, including lipids, proteins and nucleic acids undergoing oxidative alterations.

#### **1.1. Lipid peroxidation**

Lipid peroxidation (LP) – oxidative degradation of polyunsaturated fatty acids caused by ROS – is responsible for degradation of membrane lipids resulting in cell damage and formation of many toxic products. LP is a free radical chain reaction where three major steps - the initiation, propagation and termination – can be recognised (Figure 1.).

In initiation phase, highly reactive hydroxyl radical, formed in Fenton reaction, abstracts hydrogen atom in α position relative to the polyunsaturated fatty acid double bond. This results in the formation of fatty acid radical, highly unstable, short-lived intermediate that stabilises by abstracting hydrogen from another chemical species, or reacts with triplet oxygen to generate different radical species, including fatty acid peroxyl radical. In the termination step peroxyl radicals transform into nonradical compounds – hydrocarbons, aldehydes, alcohols, volatile ketones and lipid polymers, some of which are harmful (Figure 1.) [8, 9].

**Figure 1.** Steps in lipid peroxidation process

Organism uses a number of endogenous antioxidants, such as glutathione, α-lipoic acid, coenzyme Q10, bilirubin and antioxidant enzymes (glutathione peroxidase, catalase, superoxide dismutase), to protect itself from oxidative stress. When they are insufficient, it becomes necessary to introduce exogenous antioxidants. Most of these compounds are primarily taken into the body by food and are predominantly of herbal origin (phenolics, carotenoids, terpenoids and vitamins – ascorbic acid and tocopherol). Herbal antioxidants exhibit their activity through a wide variety of mechanisms, such as inhibition of oxidising enzymes, chelation of transition metals, transfer of hydrogen or single electron to radicals, singlet oxygen deactivation, or enzymatic detoxification of ROS [8]. They can stop LP process in either initiation or propagation step. Herbal antioxidants have become subjects of growing interest and targets of numerous scientific research. However, enormous number of plant species is still waiting to be investigated in this manner and explored as potential medical drugs or dietary supplements.

#### **1.2. Eicosanoid pathway**

In addition to direct detrimental effects on biomolecules and cellular structures, lipid peroxidation is also involved in biosynthesis of eicosanoids – arachidonic acid metabolites serving as inflammatory mediators. Arachidonic acid, released from phospholipids by action of phospholipase A, can be converted into these products by three different pathways: cyclooxygenase, leading to the formation of prostanoids (prostaglandins and thromboxanes), lipoxygenase, where leukotrienes and certain mono-, di- and trihydroxy acids are synthesised, and epoxygenase pathway, which includes cytochrome P-450 and gives epoxides as final products [10].

Cyclooxygenase (COX), key enzyme in cyclooxygenase pathway (Figure 2.), exists in three forms, COX-1, COX-2, and recently discovered COX-3. Despite the differences in structure, localisation and regulation, reactions catalysed by COX isoforms follow the same mechanism. All of them transform arachidonic acid into prostanoids: prostaglandins (PGH2, PGE2, PGF2α), thromboxanes (TXA2, TXB2), and 12(*S*)-hydroxyheptadeca-5*Z*,8*E*,10*E*-trienoic acid (12(*S*)-HHT) as a co-product.

**Figure 2.** COX and 12-LOX branches of eicosanoid pathway

194 Lipid Peroxidation

**Figure 1.** Steps in lipid peroxidation process

medical drugs or dietary supplements.

**1.2. Eicosanoid pathway** 

Organism uses a number of endogenous antioxidants, such as glutathione, α-lipoic acid, coenzyme Q10, bilirubin and antioxidant enzymes (glutathione peroxidase, catalase, superoxide dismutase), to protect itself from oxidative stress. When they are insufficient, it becomes necessary to introduce exogenous antioxidants. Most of these compounds are primarily taken into the body by food and are predominantly of herbal origin (phenolics, carotenoids, terpenoids and vitamins – ascorbic acid and tocopherol). Herbal antioxidants exhibit their activity through a wide variety of mechanisms, such as inhibition of oxidising enzymes, chelation of transition metals, transfer of hydrogen or single electron to radicals, singlet oxygen deactivation, or enzymatic detoxification of ROS [8]. They can stop LP process in either initiation or propagation step. Herbal antioxidants have become subjects of growing interest and targets of numerous scientific research. However, enormous number of plant species is still waiting to be investigated in this manner and explored as potential

In addition to direct detrimental effects on biomolecules and cellular structures, lipid peroxidation is also involved in biosynthesis of eicosanoids – arachidonic acid metabolites serving as inflammatory mediators. Arachidonic acid, released from phospholipids by action of phospholipase A, can be converted into these products by three different pathways: cyclooxygenase, leading to the formation of prostanoids (prostaglandins and thromboxanes), lipoxygenase, where leukotrienes and certain mono-, di- and trihydroxy COX-catalysed transformation of arachidonic acid starts with tyrosyl radical generation through Tyr385 oxidation by heme in COX active site. Formed tyrosyl radical abstracts hydrogen from C-13 of arachidonic acid. In subsequent steps, free electron migration, reaction with oxygen yielding peroxyl radical, and cyclisation reactions give PGG2 which is converted to PGH2 through the action of peroxidase (Px) (Figure 3.) [11].

COX-1 is expressed constitutively in different tissues, blood monocytes and platelets, and is involved in normal cellular homeostasis. In contrast, COX-2 may be induced by a series of pro-inflammatory stimuli and its role in the progress of inflammation, fever and pain has been known [12]. Furthermore, COX-2 has been targeted in many cancers including: colon cancer, colorectal cancer, breast cancer, gliomas, prostate cancer, esophageal carcinoma, pancreatic cancer, lung carcinoma, gastric carcinoma, ovarian cancer, Kaposi's sarcoma and melanoma [13].

In lipoxygenase branch of arachidonic acid metabolism, there are three types of lipoxygenases, termed 5-, 12- and 15-lipoxygenase. In 12-lipooxigenase (12-LOX) pathway, the first reaction is abstraction of hydrogen from C-10 of arachidonic acid, which includes reduction of Fe3+ to Fe2+ in enzyme active site. This results in the formation of arachidonic acid radical, which than reacts with oxygen and generates 12-hydroperoxyeicosatetraenoic acid (12-HPETE). Finally, formation of 12(*S*)-hydroxy-(5*Z*,8*Z*,10*E*,14*Z*)-eicosatetraenoic acid (12-HETE) is catalysed by glutathione peroxidase (GPx), whereby glutathione (GSH) is oxidised to GS-SG (Figure 3.) [14, 15].

**Figure 3.** Mechanism of COX and 12-LOX-catalysed arachidonic acid transformation.

12-HETE is implicated in regulation of platelet aggregation, angiogenesis, as well as the progression of several human diseases like various cancers, rheumatoid arthritis and psoriasis [13, 16, 17]. Also, 12-HETE is known to take part in the metastatic cascade as a crucial intracellular signalling molecule which activates protein kinase C and mediates the biological functions of growth factors and cytokines.

Nowadays, there is a great need for a new anti-inflammatory compounds with minimal side effects. Natural products, especially phenolics frequently consumed in a diet of plant origin, are known to have great anti-inflammatory potential considering inhibition of COX and LOX enzymes [18, 19]. Thus, screening of plants for COX/LOX inhibitory activity, followed by effect-guided fractionation, can be a useful tool for discovering new secondary biomolecules with anti-inflammatory potential. Although plant species widely used in traditional medicine may be a good starting point, there is also a vast number of currently unexplored species of unknown composition and activity. Therefore, this study included the poorly investigated plant species classified into four families – Alliaceae, Cupressaceae, Plantaginaceae and Polygonaceae – wild growing in Serbia, that are part of our continuing research [20-29]. Most of these species are used in diet or used in folk medicine for healing various disorders.

Considering the fact that lipid peroxidation is involved in an inflammation process, the aim of this study was to compare antioxidant activity (more specifically, the ability to inhibit lipid peroxidation) of the selected plant extracts with their ability to inhibit production of particular arachidonic acid metabolites and to correlate these activities with the phenolic and flavonoid content.

## **2. Methods**

196 Lipid Peroxidation

**Figure 3.** Mechanism of COX and 12-LOX-catalysed arachidonic acid transformation.

#### **2.1. Plant material and extract preparation**

Plant material used in this study was collected from different locations in Serbia during the period between 2008–2010, during a flowering phase or as ripe specimens. Species belonging to four families: Alliaceae (genus Allium – *A. flavum* L., *A. carinatum* L., *A. melanantherum*  Panč., *A. pallens* L., *A. rhodopeum* Velen., *A. paniculatum* L.), Cupressaceae (genus *Juniperus* L. – *J. communis* L., *J. sibirica* Burgsdorf., *J. foetidissima* Willd.), Plantaginaceae (genus *Plantago* L. – *P*. *major* L., *P*. *maritima* L., *P*. *media* L., *P*. *lanceolata* L., *P*. *altisima* L.) and Polygonaceae (genus *Rumex* L. – *R. patientia* L., *R. crispus* L., *R. obtusifolius* L.) were investigated. The voucher specimens were deposited in the Herbarium of the Department of Biology and Ecology (BUNS Herbarium), University of Novi Sad Faculty of Sciences.

Air-dried and grounded plant material (30 g) (whole plants of *Allium* sp., needles and cones of *Juniperus* sp., aerial parts of *Plantago* sp. and herbs and rhizomes of *Rumex* sp.) was extracted by maceration with 80% aqueous methanol (8 mL per 1 g of drug) during 72 h at room temperature. After filtration, the solvent was evaporated to dryness under reduced pressure. All raw extracts except those of *Allium* sp. and of *Rumex* sp. rhizomes were resuspended in hot distilled water (to a final concentration of approx. 1 g/mL), washed exhaustively with petroleum ether (fraction 40–60 °C) to remove nonpolar pigments, and evaporated to dryness under vacuum.

Dried extracts were dissolved in 80% aqueous methanol and DMSO for evaluation of the antioxidant and anti-inflammatory activity, respectively, to obtain 300 mg/mL stock solutions.

### **2.2. Determination of total phenolic content**

Total phenolic content was determined according to method of Singleton et al. [30], modified for 96-well microplates. Gallic acid was used as a standard for calibration curve construction. Thirty microliters of each extract or standard solution was added to 150 μL of 0.1 mol/L Folin-Ciocalteu reagent and after 10 min mixed with 120 μL of sodium carbonate (7.5%). The same mixture, with solvent instead of extract, was used as a blank. Absorbance at 760 nm was read after 2 h. The phenolics concentration was determined by using the calibration curve of gallic acid. The total phenolics value was expressed as milligrams of gallic acid equivalents per gram of dry weight (dw).

#### **2.3. Determination of total flavonoid content**

The aluminium chloride spectrophotometric method described by Chang et al. [31] and modified for 96-well microplates, was used for determination of of the total flavonoid content. Quercetin was used as a standard to prepare a calibration curve. The reaction mixture was comprised of 30 μL of the extract or standard solution, 90 μL of methanol, 6 μL of 10% aluminium chloride (substituted with distilled water in blank probe), 6 μL of 1 mol/L potassium acetate and 170 μL of distilled water. Absorbance at 415 nm was measured after 30 min. Flavonoid content was calculated according to the standard calibration curve and are expressed in milligrams of quercetin equivalents per gram of dw.

#### **2.4. Lipid peroxidation**

There are several methods for measuring the ability of plant extracts to inhibit lipid peroxidation [8]. Some of the methods are based on monitoring of malondialdehyde (MDA), a degradation product of polyunsaturated fatty acids peroxidation. Possible ways for quantification of MDA are GC-FID after derivatisation, HPLC with DAD or fluorimetric detector and spectrophotometric method. The latter is based on the reaction of MDA with thiobarbituric acid (TBA) and it is commonly used both in *in vitro* and *in vivo* studies. Formation of the red-coloured MDA-TBA adduct is measured at 532 nm. Different substrates can be used in this test: lecithin liposomes, free fatty acids, LDL and body fluids [8, 32]. Also, a few different initiators of LP, such as ionizing radiation, chemical agents – metal ions, free radicals and metalloproteins may be used [8].

In this study, we used spectrophotometric TBA assay [28, 33] for evaluation the ability of extracts to inhibit LP. Linseed oil, used as a source of polyunsaturated fatty acids (69.7 % linolenic, 13.5 % linoleic acid, as determined by GC-MS), was obtained from linseed by Soxhlet extraction. Oil was added to 0.067 mol/L phosphate buffer, pH 7.4, in the presence of 0.25% Tween-80 to obtain a 0.035% suspension and sonicated for 1 hour. This suspension (3.0 mL) was mixed with 20 μL of FeSO4 (4.58 mmol/L), 20 μL of ascorbic acid (87 μmol/L), and 20 μL of extract (or solvent in control); 3.0 mL of phosphate buffer and 20 μL of extract were added in the blank probe. After incubation at 37 °C for 1 hour, 0.2 mL 3.72% EDTA was added to all samples followed by 2 mL of an aqueous mixture containing TBA (3.75 mg/mL), HClO4 (1.3%), and trichloroacetic acid (0.15 g/mL). Reaction mixtures were heated at 100 °C for 15 min, cooled, centrifuged at 1600 g for 15 min, and absorbance was measured at 532 nm. All samples and control were made in triplicate. IC50 values were determined from inhibition vs. concentration plots.

#### **2.5. Anti-inflammatory activity**

198 Lipid Peroxidation

solutions.

evaporated to dryness under vacuum.

**2.2. Determination of total phenolic content** 

gallic acid equivalents per gram of dry weight (dw).

**2.3. Determination of total flavonoid content** 

**2.4. Lipid peroxidation** 

are expressed in milligrams of quercetin equivalents per gram of dw.

metal ions, free radicals and metalloproteins may be used [8].

exhaustively with petroleum ether (fraction 40–60 °C) to remove nonpolar pigments, and

Dried extracts were dissolved in 80% aqueous methanol and DMSO for evaluation of the antioxidant and anti-inflammatory activity, respectively, to obtain 300 mg/mL stock

Total phenolic content was determined according to method of Singleton et al. [30], modified for 96-well microplates. Gallic acid was used as a standard for calibration curve construction. Thirty microliters of each extract or standard solution was added to 150 μL of 0.1 mol/L Folin-Ciocalteu reagent and after 10 min mixed with 120 μL of sodium carbonate (7.5%). The same mixture, with solvent instead of extract, was used as a blank. Absorbance at 760 nm was read after 2 h. The phenolics concentration was determined by using the calibration curve of gallic acid. The total phenolics value was expressed as milligrams of

The aluminium chloride spectrophotometric method described by Chang et al. [31] and modified for 96-well microplates, was used for determination of of the total flavonoid content. Quercetin was used as a standard to prepare a calibration curve. The reaction mixture was comprised of 30 μL of the extract or standard solution, 90 μL of methanol, 6 μL of 10% aluminium chloride (substituted with distilled water in blank probe), 6 μL of 1 mol/L potassium acetate and 170 μL of distilled water. Absorbance at 415 nm was measured after 30 min. Flavonoid content was calculated according to the standard calibration curve and

There are several methods for measuring the ability of plant extracts to inhibit lipid peroxidation [8]. Some of the methods are based on monitoring of malondialdehyde (MDA), a degradation product of polyunsaturated fatty acids peroxidation. Possible ways for quantification of MDA are GC-FID after derivatisation, HPLC with DAD or fluorimetric detector and spectrophotometric method. The latter is based on the reaction of MDA with thiobarbituric acid (TBA) and it is commonly used both in *in vitro* and *in vivo* studies. Formation of the red-coloured MDA-TBA adduct is measured at 532 nm. Different substrates can be used in this test: lecithin liposomes, free fatty acids, LDL and body fluids [8, 32]. Also, a few different initiators of LP, such as ionizing radiation, chemical agents –

In this study, we used spectrophotometric TBA assay [28, 33] for evaluation the ability of extracts to inhibit LP. Linseed oil, used as a source of polyunsaturated fatty acids (69.7 % linolenic, 13.5 % linoleic acid, as determined by GC-MS), was obtained from linseed by There are a great number of different *in vitro* methods used for estimation of inhibitory activity of COX and LOX enzymes including a number of commercial kits. Some assays, as a source of enzymatic activity, include native or recombinant enzymes, animals or human origin, while others use different cell lines that express desirable activities. Arachidonic acid is often added in reaction medium and in some cases is radio labelled. Induction of inflammatory respond also differs, and in most cases is performed by bacterial lipopolysaccharide, various cytokines and tumour necrosis factor. Different techniques are used for detection of enzymatic activity, such as different chromatographic techniques (TLC, HPLC-UV), as well as EIA [19, 34].

In this study, COX-1 and 12-LOX inhibitory activity was investigated using *ex vivo* assay according to modified method of Safayhi *et al*. [29, 35]. Intact cells (human platelets) were used as a source of COX-1 and 12-LOX enzymes. Arachidonic acid metabolites (12-HHT and 12-HETE) were determined by use of LC–MS/MS technique [29].

An aliquot of human platelet concentrate (viable but outdated for medical use) containing 4·108 cells was suspended in buffer (0.137 mol/L NaCl, 2.7 mmol/L KCl, 2.0 mmol/L KH2PO4, 5.0 mmol/L Na2HPO4 and 5.0 mmol/L glucose, pH 7.2) to obtain final volume of 2 mL. This mixture was slowly stirred at 37 °C for 5 min. Subsequently, 0.1 mL of extracts or standard compounds solutions in DMSO (concentration ranging from 10.0 to 200.0, 0.156 to 5.0 and 0.01 to 0.6 mg/ml for extracts, quercetin and aspirin, respectively) and 0.1 mL of calcimycin (Calcium Ionophore A23187, 125 μmol/L in DMSO) were added and incubated for 2 min at 37 °C, with moderate shaking. The exact volume of extract in control and calcimycin in blank probe were substituted with solvent (DMSO). Thereafter, 0.3 mL of CaCl2 aqueous solution (16.7 mmol/L), substituted with water in blank probe, was added and the mixture was incubated for further 5 min at 37 °C with shaking. Acidification with cold 1% aqueous formic acid (5.8 mL) to pH 3 terminated the reaction. If gel formation occurred, vortexing was applied before mixing with the acid. Prostaglandin B2 (50 μL of 6 μg/mL solution in DMSO) was added as internal standard, and extraction of products was done with mixture of chloroform and methanol (1:1, 8.0 mL) with vigorous vortexing for 15 min. After centrifugation at 7012 × g for 15 min at 4 °C, organic layer was separated, evaporated to dryness, dissolved in methanol (0.5 mL), filtered and used for further LC–MS/MS analysis. All samples and control were made in triplicate.

Test for estimation of the anti-inflammatory activity, applied in our research, has a lot of advantages. Firstly, the advantage is avoidance of the undesirable *in vivo* tests on experimental animals. Even though the exact anti-inflammatory activity can be validated only through *in vivo* tests, creating *in vitro* assays, in which physiological conditions similar to *in vivo* assays are used, can provide valuable information about inhibitory potential of the compounds tested. Platelets are a suitable cell system for testing inhibitory activity, because they can provide physiological cell conditions and possibility to examine the inhibition of both enzymes at the same time. Secondly, for determination of the formed metabolites as a measure of level of inhibition of COX and LOX activity, LC-MS/MS technique was used. LC-MS/MS provided a highly sensitive and specific detection of desirable metabolites within a short analysis time [29].

#### **2.6. Statistical analysis**

Percent of lipid peroxidation inhibition achieved by different concentration of extracts was calculated by the following equation: *I*(%) = (*A*0−*A*)/*A*0 × 100, where *A*0 was the absorbance of the control reaction and *A* was the absorbance of the examined samples, corrected for the value of blank probe. Percent of COX-1 and 12-LOX inhibition achieved by different concentrations of extracts was calculated by the following equation: *I*(%) = 100 × (*R*0−*R*)/*R*0, where *R*0 and *R* were response ratios (metabolite peak area divided by internal standard peak area) in the control reaction and in the examined samples, respectively. Both *R* and *R*<sup>0</sup> were corrected for the value of blank probe. For both assays, corresponding inhibitionconcentration curves were drawn using Origin software, version 8.0 (Origin Labs) and IC50 values (concentration of extract that inhibited lipid peroxidation and COX-1 and 12-LOX metabolites formation by 50%) were determined. All of the results were expressed as mean ± SD of three replicates. Correlation analyses were done using Statistica software version 6 (StatSoft). Concentrations of total phenolics and the total flavonoids were used as independent variables, while inhibitory activities towards LP and 12-HETE synthesis (expressed as 1/IC50) were used as dependent variables. Due to a wide range of values, loglog plots were applied. Pearson's correlation coefficients were calculated.

## **3. Results**

In this study, we tested the effect of 17 taxa from four families (*Alliaceae, Cupressaceae, Plantaginaceae* and *Polygonaceae*) on lipid peroxidation and metabolism of arachidonic acid. The total phenolic and flavonoid contents were determined in the plant extracts, as well. All results are presented in Figures 4, 5, 6 and 9.

Among all tested samples, herb and rhizome extracts of *Rumex* species were the most potent inhibitors of LP (0.009-0.047 mg/mL). Slightly lower activity was shown by herb extracts of *Plantago* species (0.025-0.178 mg/mL), while extracts of cones and needles of *Juniperus* species, as well as the whole plant extracts of *Allium* species expressed a much lower activity (0.117-0.887 and 0.68-1.986 mg/mL, respectively).

Legend: H – herb, Rh – rhizome, C – cones, N – needles, W – whole plant, n.a. - not achieved.

**Figure 4.** Results of lipid peroxidation inhibition assay.

**2.6. Statistical analysis** 

**3. Results** 

All samples and control were made in triplicate.

dryness, dissolved in methanol (0.5 mL), filtered and used for further LC–MS/MS analysis.

Test for estimation of the anti-inflammatory activity, applied in our research, has a lot of advantages. Firstly, the advantage is avoidance of the undesirable *in vivo* tests on experimental animals. Even though the exact anti-inflammatory activity can be validated only through *in vivo* tests, creating *in vitro* assays, in which physiological conditions similar to *in vivo* assays are used, can provide valuable information about inhibitory potential of the compounds tested. Platelets are a suitable cell system for testing inhibitory activity, because they can provide physiological cell conditions and possibility to examine the inhibition of both enzymes at the same time. Secondly, for determination of the formed metabolites as a measure of level of inhibition of COX and LOX activity, LC-MS/MS technique was used. LC-MS/MS provided a highly sensitive

Percent of lipid peroxidation inhibition achieved by different concentration of extracts was calculated by the following equation: *I*(%) = (*A*0−*A*)/*A*0 × 100, where *A*0 was the absorbance of the control reaction and *A* was the absorbance of the examined samples, corrected for the value of blank probe. Percent of COX-1 and 12-LOX inhibition achieved by different concentrations of extracts was calculated by the following equation: *I*(%) = 100 × (*R*0−*R*)/*R*0, where *R*0 and *R* were response ratios (metabolite peak area divided by internal standard peak area) in the control reaction and in the examined samples, respectively. Both *R* and *R*<sup>0</sup> were corrected for the value of blank probe. For both assays, corresponding inhibitionconcentration curves were drawn using Origin software, version 8.0 (Origin Labs) and IC50 values (concentration of extract that inhibited lipid peroxidation and COX-1 and 12-LOX metabolites formation by 50%) were determined. All of the results were expressed as mean ± SD of three replicates. Correlation analyses were done using Statistica software version 6 (StatSoft). Concentrations of total phenolics and the total flavonoids were used as independent variables, while inhibitory activities towards LP and 12-HETE synthesis (expressed as 1/IC50) were used as dependent variables. Due to a wide range of values, log-

In this study, we tested the effect of 17 taxa from four families (*Alliaceae, Cupressaceae, Plantaginaceae* and *Polygonaceae*) on lipid peroxidation and metabolism of arachidonic acid. The total phenolic and flavonoid contents were determined in the plant extracts, as well. All

Among all tested samples, herb and rhizome extracts of *Rumex* species were the most potent inhibitors of LP (0.009-0.047 mg/mL). Slightly lower activity was shown by herb extracts of *Plantago* species (0.025-0.178 mg/mL), while extracts of cones and needles of *Juniperus* species, as well as the whole plant extracts of *Allium* species expressed a much lower activity

and specific detection of desirable metabolites within a short analysis time [29].

log plots were applied. Pearson's correlation coefficients were calculated.

results are presented in Figures 4, 5, 6 and 9.

(0.117-0.887 and 0.68-1.986 mg/mL, respectively).

Legend: H – herb, Rh – rhizome, C – cones, N – needles, W – whole plant.

**Figure 5.** Results of total phenolic content assay (expressed as mg eq. gallic acid / 1 g dw).

Total phenolic content in examined genera decreased in following order: *Rumex*, *Juniperus*, *Plantago* and *Allium*, with *R. patentia* and *R. crispus* extracts being by far the richest in phenolics (550 and 527 mg eq. gallic acid per 1 g dw, respectively). Regarding the content of the total flavonoids, significant intrageneric variations were observed, hence it was not

possible to point out a genus with the highest content. Flavonoids were the most abundant in *J. foetidissima* needles extract (60 mg quercetin eq per 1 g dw), and scarcest in *A. rhodopeum*  (0.2 mg quercetin eq per 1 g dw).

Legend: H – herb, Rh – rhizome, C – cones, N – needles, W – whole plant.

**Figure 6.** Results of total flavonoid content assay (expressed as mg eq. quercetin / 1 g dw).

Plant phenolics present in extracts can counteract lipid peroxidation in either initiation or propagation step. Possible mechanisms are chelation of transition metals (Fe2+), reduction of Fe3+ and neutralisation of lipid peroxidation radical intermediates by transfer of hydrogen or single electron [8]. An attempt was made to correlate the observed lipid peroxidation inhibitory activity (given as 1/IC50) with the content of the total phenolics and total flavonoids. The corresponding plots are given in Figure 7. and Figure 8.

A high degree of correlation was observed between the lipid peroxidation inhibitory activity and the total phenolic content (*r* = 0.7713), while correlation with the total flavonoids content was not established (*r* = 0.1410). Thus, flavonoids do not contribute to the total observed activity to a significant extent. The lower activity of flavonoids, compared to other phenolics, can be explained through Porter's polar paradox. Namely, flavonoids present in the extracts investigated were predominantly in glycosylated form and thus highly polar. It is demonstrated that compounds of lower polarity are more effective in polar reaction media, such as oil-in-water emulsion used in our experiments, since they exhibit their activity at oil-water interface [36]. In addition, hydrogen atom donation ability of flavonoids decrease with glycosylation, especially if the most active hydroxyl groups (C-3, C-3' or C-4') are occupied by carbohydrate moiety [37, 38].

Regarding the eicosanoid pathway, *Rumex* and *Plantago* species showed, on average, very high 12-LOX inhibitory effect, with IC50 in range 0.75–3.59 mg/mL (Figure 9.). While *Juniperus* and *Allium* also exhibited dose-dependent inhibition of 12-HETE production, their IC50 values were higher, ranging from 1.45 mg/mL to 11.04 mg/mL.

202 Lipid Peroxidation

(0.2 mg quercetin eq per 1 g dw).

Legend: H – herb, Rh – rhizome, C – cones, N – needles, W – whole plant.

**Figure 6.** Results of total flavonoid content assay (expressed as mg eq. quercetin / 1 g dw).

flavonoids. The corresponding plots are given in Figure 7. and Figure 8.

Plant phenolics present in extracts can counteract lipid peroxidation in either initiation or propagation step. Possible mechanisms are chelation of transition metals (Fe2+), reduction of Fe3+ and neutralisation of lipid peroxidation radical intermediates by transfer of hydrogen or single electron [8]. An attempt was made to correlate the observed lipid peroxidation inhibitory activity (given as 1/IC50) with the content of the total phenolics and total

A high degree of correlation was observed between the lipid peroxidation inhibitory activity and the total phenolic content (*r* = 0.7713), while correlation with the total flavonoids content was not established (*r* = 0.1410). Thus, flavonoids do not contribute to the total observed activity to a significant extent. The lower activity of flavonoids, compared to other phenolics, can be explained through Porter's polar paradox. Namely, flavonoids present in the extracts investigated were predominantly in glycosylated form and thus highly polar. It is demonstrated that compounds of lower polarity are more effective in polar reaction media, such as oil-in-water emulsion used in our experiments, since they exhibit their activity at oil-water interface [36]. In addition, hydrogen atom donation ability of flavonoids decrease with glycosylation, especially if the most active hydroxyl groups (C-3, C-3' or C-4') are occupied by carbohydrate moiety [37, 38]. Regarding the eicosanoid pathway, *Rumex* and *Plantago* species showed, on average, very high 12-LOX inhibitory effect, with IC50 in range 0.75–3.59 mg/mL (Figure 9.). While

possible to point out a genus with the highest content. Flavonoids were the most abundant in *J. foetidissima* needles extract (60 mg quercetin eq per 1 g dw), and scarcest in *A. rhodopeum* 

**Figure 7.** Correlation between total phenolic content and ability of extracts to inhibit lipid peroxidation

**Figure 8.** Correlation between total flavonoids content and ability of extracts to inhibit lipid peroxidation

Legend: H – herb, Rh – rhizome, C – cones, N – needles, W – whole plant.

**Figure 9.** Results of 12-LOX inhibition assay.

Moreover, certain inhibitory activity of the examined extracts towards COX-1 was also confirmed in the anti-inflammatory assay applied in this research. Determined IC50 values ranged from 0.34 to 8.00 mg/mL, with no genus exhibiting significantly higher activity than others. However, the results are not shown due to lack of correlation with the total phenolics and flavonoid content. This is in agreement with our previous findings [27].

The exact mechanism of COX and LOX enzymes inhibition by natural products is still not fully elucidated. However, bearing in mind their structures and chemical properties, several mechanisms could be speculated. Both pathways of arachidonic acid metabolism include free radical reactions (Figure 3.). Due to their radical scavenging activity or reducing properties, many plant natural products can interfere with reactions catalysed by COX and LOX [39]. They can neutralise radical intermediates, thus terminating the reaction. In addition, they can reduce Fe3+ ion that is a part of active site of both enzymes and is necessary for initiation reaction. Some natural products, including acetylenes, can bind covalently to enzymes and inhibit them irreversibly [19]. Also, in inhibition of LOX, isoprenyl moiety of some phenolics and terpenoid backbone structure could play an important role. Prenylated phenolics are usually more hydrophobic than conventional ones. Terpenoids are also mostly non-polar compounds. These characteristics suggest an easy penetration through the cell membrane and their good 12-lipoxygenase inhibitory properties [19]. Finally, the COX or LOX activity can be decreased by suppressing their transcription by phenolics [18], although this effect is observable only in long-duration experiments.

To identify the compound class responsible for the observed 12-LOX-inhibitory activity, 1/IC50 was correlated with the content of the total phenolics and flavonoids. The corresponding plots are given in Figure 10. and Figure 11. As with the lipid peroxidation, the correlation between the content of the total phenolics and 12-LOX-inhibitory activity was also observed, although slightly weaker, with Pearson's correlation coefficient *r* = 0.6037. At the same time, no relationship was found between the total flavonoids content and 12-HETE production (*r* = 0.2825). Bearing in mind a good correlation of 12-LOX inhibition with the phenolic content, and the lack thereof with the flavonoid content, it is possible that only small phenolic molecules, but not the voluminous flavonoid glycosides, can enter 12-LOX active site and exhibit the inhibitory effect there. Thus, the observed differences in the total phenolics and the total flavonoids effects can at least partially be explained by steric hindrance.

204 Lipid Peroxidation

Legend: H – herb, Rh – rhizome, C – cones, N – needles, W – whole plant.

Moreover, certain inhibitory activity of the examined extracts towards COX-1 was also confirmed in the anti-inflammatory assay applied in this research. Determined IC50 values ranged from 0.34 to 8.00 mg/mL, with no genus exhibiting significantly higher activity than others. However, the results are not shown due to lack of correlation with the total phenolics

The exact mechanism of COX and LOX enzymes inhibition by natural products is still not fully elucidated. However, bearing in mind their structures and chemical properties, several mechanisms could be speculated. Both pathways of arachidonic acid metabolism include free radical reactions (Figure 3.). Due to their radical scavenging activity or reducing properties, many plant natural products can interfere with reactions catalysed by COX and LOX [39]. They can neutralise radical intermediates, thus terminating the reaction. In addition, they can reduce Fe3+ ion that is a part of active site of both enzymes and is necessary for initiation reaction. Some natural products, including acetylenes, can bind covalently to enzymes and inhibit them irreversibly [19]. Also, in inhibition of LOX, isoprenyl moiety of some phenolics and terpenoid backbone structure could play an important role. Prenylated phenolics are usually more hydrophobic than conventional ones. Terpenoids are also mostly non-polar compounds. These characteristics suggest an easy penetration through the cell membrane and their good 12-lipoxygenase inhibitory properties [19]. Finally, the COX or LOX activity can be decreased by suppressing their transcription by phenolics [18], although this effect is

To identify the compound class responsible for the observed 12-LOX-inhibitory activity, 1/IC50 was correlated with the content of the total phenolics and flavonoids. The corresponding plots

and flavonoid content. This is in agreement with our previous findings [27].

**Figure 9.** Results of 12-LOX inhibition assay.

observable only in long-duration experiments.

**Figure 10.** Correlation between total phenolic content and 12-LOX inhibitory activity

The differences in COX and LOX inhibition (LOX inhibition being correlated with phenolic content) can be attributed to the differences in reaction mechanism and active site threedimensional structure. Namely, both mechanisms include abstraction of hydrogen from arachidonic acid leading to formation of radical species. However, hydrogen acceptor in COX is tyrosyl radical (formed through tyrosine oxidation by Fe3+) while in LOX, electron is transferred directly to Fe3+. Phenolics from plant extracts could reduce Fe3+ to Fe2+, thus inactivating both enzymes. However, the presence of tyrosyl residue in COX active site could provide steric protection and prevent phenolics from approaching the Fe3+ ion.

Finally, the correlation between the ability of extracts to counteract lipid peroxidation and inhibit production of 12-HETE is shown in Figure 12. High correlation coefficient (*r* = 0.6819) suggests that extracts with a high inhibitory effect on LP, also represent potent inhibitors of 12-LOX pathway. Methods for examination of anti-inflammatory activity are labourintensive, expensive and sometimes involve ethical issues due to the usage of laboratory animals. Therefore, *in vitro* measurement of lipid peroxidation inhibition and the total phenolic content could be a useful tool for screening of plant extracts with a potential antiinflammatory activity.

**Figure 11.** Correlation between total flavonoid content and 12-LOX inhibitory activity

**Figure 12.** Correlation between the ability of extracts to inhibit 12-HETE production and lipid peroxidation

To summarise, the most of the examined species expressed high lipid peroxidation and antiinflammatory activity, especially *Rumex* and *Plantago* species. High inhibitory activity of these species towards 12-LOX pathway makes them good candidates for further research taking into account role of this enzyme in cancer development. A good correlation was found between total phenolic content of 23 investigated plant extracts and their ability to inhibit lipid peroxidation and 12-LOX pathway. Consequently, LP inhibitory activity was also highly correlated with 12-LOX inhibition. Thus it can serve as indicator for preliminary selection of plant extracts further to be tested for anti-inflammatory activity by expensive and time-consuming methods.

## **Author details**

206 Lipid Peroxidation

peroxidation

inflammatory activity.

animals. Therefore, *in vitro* measurement of lipid peroxidation inhibition and the total phenolic content could be a useful tool for screening of plant extracts with a potential anti-

**Figure 11.** Correlation between total flavonoid content and 12-LOX inhibitory activity

**Figure 12.** Correlation between the ability of extracts to inhibit 12-HETE production and lipid

Neda Mimica-Dukić, Nataša Simin, Emilija Svirčev, Dejan Orčić, Ivana Beara and Marija Lesjak *Department of Chemistry, Biochemistry and Environmental Protection, University of Novi Sad,* 

Biljana Božin *Department of Pharmacy, Faculty of Medicine, University of Novi Sad, Novi Sad, Republic of Serbia* 

## **Acknowledgement**

*Faculty of Sciences, Novi Sad, Republic of Serbia* 

The Ministry of Education and Science, Republic of Serbia (Grant No. 172058) funded this research. We wish to thank to Goran Anačkov, PhD, for the providing voucher specimens, and Gordana Vlahović, MSc, for language corrections. Platelet concentrate was kindly provided by The Institute for Blood Transfusion of Vojvodina, Novi Sad, Republic of Serbia.

## **4. References**


in rheumatoid arthritis: insights from the Nrf2-knockout mice. Annals of the Rheumatic Diseases 2010;doi:10.1136/ard.2010.132720


[22] Orčić DZ., Mimica-Dukić NM., Francišković MM., Petrović SS., Jovin ED. Antioxidant activity relationship of phenolic compounds in *Hypericum perforatum* L. Chemistry Central Journal. 2011;5(1) 34-41.

208 Lipid Peroxidation

61.

Diseases 2010;doi:10.1136/ard.2010.132720

antioxidants. Amsterdam: Elsevier; 1990. p1-18.

of Biological Chemistry 1999;274(33) 22903-22906.

[12] Hawkey CJ. COX-2 inhibitors. The Lancet 1999;353(9149) 307-314.

Lipid Research 2007;46(5) 244-282.

Journal 1989;259(2) 315-324.

Wiley-VCH; 1999. p65-83.

1997;414(1) 159-164.

2010;75(7) H212-H217.

263-272.

in rheumatoid arthritis: insights from the Nrf2-knockout mice. Annals of the Rheumatic

[7] Alvarado C., Alvarez P., Jimenez L., De la Fuente M. Oxidative stress in leukocytes from young prematurely aging mice is reversed by supplementation with biscuits rich in antioxidants. Developmental & Comparative Immunology 2006;30(12) 1168-1180. [8] Laguerre M., Lecomte J., Villeneuve P. Evaluation of the ability of antioxidants to counteract lipid oxidation: Existing methods, new trends and challenges. Progress in

[9] Gordon MH. The mechanism of antioxidant action in vitro. In: Hudson BJF. (ed.) Food

[10] Smith WL. The eicosanoids and their biochemical mechanisms of action. Biochemical

[11] Marnett LJ., Rowlinson SW., Goodwin DC., Kalgutkar AS., Lanzo CA. Arachidonic acid oxygenation by COX-1 and COX-2. Mechanisms of catalysis and inhibition. The Journal

[13] Greene ER., Huang S., Serhan CN., Panigrahy D. Regulation of inflammation in cancer by eicosanoids. Prostaglandins and Other Lipid Mediators 2011;96(1-4): 27-36. [14] Yamamoto S., Suzuki H., Ueda N., Takahashi Y., Zoshimoto T. Mammalian Lipoxygenases. In: Curtis-Prior PB. (ed.) The Eicosanoids. Cambridge: Wiley; 2004. p53-

[15] Müller-Decker K. Cyclooxygenases. In: Marks F., Fürstenberger G. (ed.) Prostaglandins, leukotrienes, and other eicosanoids: from biogenesis to clinical applications. Weinheim:

[16] Liagre B., Vergne P., Rigaud M., Beneytout JL. Expression of arachidonate platelet-type 12-lipoxygenase in human rheumatoid arthritis type B synoviocytes. FEBS Letters

[17] Müller K. 5-lipoxygenase and 12-lipoxygenase: attractive targets for the development of

[18] Lee JH., Kim GH. Evaluation of Antioxidant and Inhibitory Activities for Different Subclasses Flavonoids on Enzymes for Rheumatoid Arthritis. Journal of Food Science*.* 

[19] Schneider I., Bucar F. Lipoxygenase Inhibitors from Natural Plant Sources. Part 2: Medicinal Plants with Inhibitory Activity on Arachidonate 12-lipoxygenase, 15 lipoxygenase and Leukotriene Receptor Antagonists. Phytotherapy Research*.* 2005;19(4)

[20] Beara IN., Lesjak MM., Orčić DZ., Simin NĐ., Četojević-Simin DD., Božin BN., Mimica-Dukić NM. Comparative analysis of phenolic profile, antioxidant, anti-inflammatory and cytotoxic activity of two closely-related Plantain species: *Plantago altissima* L. and

[21] Lesjak MM., Beara IN., Orčić DZ., Anačkov GT., Balog KJ., Francišković MM., Mimica-Dukić NM. *Juniperus sibirica* Burgsdorf. as a novel source of antioxidant and anti-

*Plantago lanceolata* L. LWT - Food Science and Technology. 2012;47(1) 64-70.

inflammatory agents. Food Chemistry. 2011;124(3) 850-856.

novel antipsoriatic drugs. Archiv der Pharmazie. 1994;327(1) 3-19.


**Chapter 10** 

## **Repeatedly Heated Vegetable Oils and Lipid Peroxidation**

Kamsiah Jaarin and Yusof Kamisah

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46076

## **1. Introduction**

210 Lipid Peroxidation

[36] Porter WL. Paradoxical behavior of antioxidants in food and biological systems.

[37] Rice-Evans CA., Miller NJ., Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine. 1996;20(7) 933-56. [38] Rice-Evans CA., Miller NJ., Paganga G. Antioxidant properties of phenolic compounds.

[39] Hostettmann K. Assays for immunomodulation and effects on mediators of inflammation. In: Dey PM., Harborne JB. (ed.) Methods in Plant Biochemistry vol. 6,

Toxicology and Industrial Health. 1993;9(1-2) 93-122.

Trends in Plant Science. 1997;2(4) 152-159.

London: Academic Press Limited; 1991. p207-211.

Deep frying is the most common and one of the oldest methods of food preparation worldwide. It involves heat and mass transfer. To reduce the expenses, the oils tend to be used repeatedly for frying. When heated repeatedly, changes in physical appearance of the oil will occur such as increased viscosity and darkening in colour [1], which may alter the fatty acid composition of the oil. Heating causes the oil to undergo a series of chemical reactions like oxidation, hydrolysis and polymerization [2]. During this process, many oxidative products such as hydroperoxide and aldehydes are produced, which can be absorbed into the fried food [3].

Palm and soy oil are the most commonly used vegetable oils in the household and industry in Malaysia for deep frying purposes. Both palm and soy oils are rich in tocopherols [4-5]. In addition to the tocopherols, palm oil also contains an abundant amount of tocotrienols. The latter form of vitamin E was consistently shown to possess better antioxidant activity than the former form [6]. The soy oil has bigger proportion of polyunsaturated fatty acid compared to the palm oil. Whereas in the palm oil, the major fatty acids present are the monounsaturated and saturated fatty acids [7].

A survey conducted in Kuala Lumpur recently had revealed that majority of the respondents admitted using repeatedly heated cooking oil [8]. The public level of awareness regarding such usage is influenced by the socioeconomic status. Respondents with higher income and education level had higher level of awareness [9]. Chronic consumption of repeatedly heated vegetable oils could be detrimental to health. It was shown to demonstrate genotoxic and preneoplastic change in the rat liver [10]. It also impaired fluid and glucose intestinal absorption in rats [11]. In rats given alcohol plus heated sunflower, an apparent liver damage as well as increased cholesterol level was observed [12]. Soriguer et al. [13] found an independent positive association between the risk of hypertension and

© 2012 Jaarin and Kamisah, 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. © 2012 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.

intake of heated cooking oil. These accumulating data suggest chronic intake of heated cooking oils increases the risk of cancer and cardiovascular diseases.

The incidence of cardiovascular disease is higher in men compared to women of similar age [14]. However, after menopause, the risk increases in women due to deficiency of estrogen level. The estrogen was shown to be cardioprotective in ovariectomized female rats [15]. It also possesses antioxidant activity which can lower the risk of lipid peroxidation [16]. Ovariectomized female rats have been used widely as a postmenopausal model [17-18].

In this study, we used two models, normal male rats and ovariectomized female rats to compare the effects of prolonged consumption of repeatedly heated palm oil and soy oil on in vivo plasma lipid peroxidation content. The oxidative stability of these repeatedly heated palm oil and soy oil were also compared.

## **2. Materials and methods**

#### **2.1. Animals, materials and chemicals**

Male and female Sprague Dawley rats (180-200 gram) were obtained from the Laboratory Animal Resource Unit of Universiti Kebangsaan Malaysia. They were kept in polyethylene cages in a well ventilated room at room temperature. Food and water were provided *ad libitum*. Palm oil (Lam Soon Edible Oil, Malaysia) and soy oil (Yee Lee Edible Oil, Malaysia) were used in this study. Sweet potatoes were purchased from the same source at a local market. All chemicals and enzymes were obtained from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated.

Ethical approval regarding the experimental procedure and humane animal handling in the study was obtained from the Universiti Kebangsaan Malaysia Animal Ethical Commitee and Medical Research Ethics Committee.

### **2.2. Frying procedure**

The oils were heated according to the method of Owu et al. [19]. A kilogram of sweet potato slices were fried in a stainless steel wok containing two and half litres of palm oil or soy oil for 10 minutes at 180C. Upon completion of the frying process, once heated oil was obtained. The process was repeated four times to obtain five times heated oil with a cooling interval of at least five hours. The food quantity was proportionately adjusted with the amount of vegetable oil left. No fresh oil was added between the frying processes to make up for the loss due to uptake by the frying materials. After being heated, small quantity of the oils was extracted for the peroxide value, fatty acid composition and vitamin E content measurements.

#### **2.3. Diet preparation**

Diets enriched with heated palm or soy oils were prepared by mixing 850 g grinded standard mouse pellet with 150 g heated oil. While to prepare high cholesterol (2%) diets fortified with heated palm or soy oils, 150 g palm or soy oil were mixed with 20 g cholesterol (MP Biomedical Inc., Australia) with 830 g grinded standard mouse pellet. The pellets were then remolded and dried in an oven at 80C overnight.

#### **2.4. Effects of heated oils in male rats**

212 Lipid Peroxidation

intake of heated cooking oil. These accumulating data suggest chronic intake of heated

The incidence of cardiovascular disease is higher in men compared to women of similar age [14]. However, after menopause, the risk increases in women due to deficiency of estrogen level. The estrogen was shown to be cardioprotective in ovariectomized female rats [15]. It also possesses antioxidant activity which can lower the risk of lipid peroxidation [16]. Ovariectomized female rats have been used widely as a postmenopausal model [17-18].

In this study, we used two models, normal male rats and ovariectomized female rats to compare the effects of prolonged consumption of repeatedly heated palm oil and soy oil on in vivo plasma lipid peroxidation content. The oxidative stability of these repeatedly heated

Male and female Sprague Dawley rats (180-200 gram) were obtained from the Laboratory Animal Resource Unit of Universiti Kebangsaan Malaysia. They were kept in polyethylene cages in a well ventilated room at room temperature. Food and water were provided *ad libitum*. Palm oil (Lam Soon Edible Oil, Malaysia) and soy oil (Yee Lee Edible Oil, Malaysia) were used in this study. Sweet potatoes were purchased from the same source at a local market. All chemicals and enzymes were obtained from Sigma-Aldrich (St. Louis, MO,

Ethical approval regarding the experimental procedure and humane animal handling in the study was obtained from the Universiti Kebangsaan Malaysia Animal Ethical Commitee

The oils were heated according to the method of Owu et al. [19]. A kilogram of sweet potato slices were fried in a stainless steel wok containing two and half litres of palm oil or soy oil for 10 minutes at 180C. Upon completion of the frying process, once heated oil was obtained. The process was repeated four times to obtain five times heated oil with a cooling interval of at least five hours. The food quantity was proportionately adjusted with the amount of vegetable oil left. No fresh oil was added between the frying processes to make up for the loss due to uptake by the frying materials. After being heated, small quantity of the oils was extracted for

Diets enriched with heated palm or soy oils were prepared by mixing 850 g grinded standard mouse pellet with 150 g heated oil. While to prepare high cholesterol (2%) diets fortified with heated palm or soy oils, 150 g palm or soy oil were mixed with 20 g cholesterol

the peroxide value, fatty acid composition and vitamin E content measurements.

cooking oils increases the risk of cancer and cardiovascular diseases.

palm oil and soy oil were also compared.

**2.1. Animals, materials and chemicals** 

**2. Materials and methods** 

USA), unless otherwise stated.

**2.2. Frying procedure** 

**2.3. Diet preparation** 

and Medical Research Ethics Committee.

After one week of acclimatization period, forty-two male Sprague Dawley rats were randomly divided into seven groups. The first group was given standard mouse pellet (control). The second, third and fourth groups were given fresh, once and five times heated palm oil diets, while the fifth, sixth and the last groups were given fresh, once and five times heated soy oil diets, respectively for four months. Blood was sampled before and at the end of treatment duration.

#### **2.5. Effects of heated oils in ovariectomized female rats fed high cholesterol diet**

Forty-two female Sprague Dawley rats were allowed to acclimatize for a week before the treatment was started and were ovariectomized ahead of the study. They were randomly divided into seven groups. Group 1 was fed 2% cholesterol diet (control), while groups 2, 3 and 4 were respectively fed 2% cholesterol diet added with fresh, once and five times heated palm oil. Groups 5, 6 and 7 were respectively given 2% cholesterol diet added with fresh, once and five times heated soy oil. The treatment duration was four months, after which the rats were sacrificed and blood samples were taken. Blood sample was also taken prior to the treatment.

#### **2.6. Peroxide content measurement**

Measurement of peroxide values of the heated oils was done according to the American Oil Chemists' Society (AOCS) Official Methods Cd 8-53 [20]. Briefly, five grams of the oil sample were added with 30 ml of acetic acid-chloroform (3:2) in a flask. The flask was then swirled before the addition of 0.5 ml saturated potassium iodide. The solution was swirled again for a minute. An amount of 30 ml distilled water and a few drops of starch solution (10%) were added. The solution was titrated against 0.01 N sodium thiosulphate solution which was priorly standardised using potassium dichromate and potassium iodide, until blue colour disappeared. The peroxide value in the oils was calculated as the difference in the volume of sodium thiosulphate solution (ml) used for samples and blank, divided by its normality. The values were expressed in miliequivalents of peroxide per kilogram of the sample.

#### **2.7. Fatty acid composition measurement**

Fatty acid composition in the fresh and heated oils was analysed using gas chromatography (GC-17A, Shimadzu, Japan), which consisted of a flame ionisation detector, a BPX 70 capillary column (30 m x 0.25 mm x 0.25 m), programmable injector temperature, set at 250C and detector temperature, set at 280C. The oil samples (100 l) were first

transesterified to fatty acid methyl ester using 1 ml of 1 M sodium methoxide in 1 ml hexane before injected into the gas chromatographic system. The injection volume was 1 l. Nitrogen at a flow rate of 0.40 ml/min was used as carrier gas in the analysis. Identification of fatty acid methyl ester peaks was carried out by comparing their retention times with their authentic standards. The fatty acid composition was expressed as the percentage of the total fatty acids.

#### **2.8. Vitamin E content measurement**

The vitamin E content in the oil samples (20 l sample) was analysed using an analytical high performance liquid chromatography (HPLC) using a programmable fluorescence detector at excitation 295 nm and emission 330 nm (Hewlett Packard HP1100, USA). The chromatographic system consisted of an isocratic pump and the stationary phase was a 150 mm silica normal phase column (YMC 5U) with an internal diameter 6 mm. The mobile phase was 0.5% isopropanol in hexane at a flow rate of 1 ml/min. The oil samples were injected directly into the HPLC system without any processing, after being cooled from the heating process. The vitamin E standard was obtained from the Malaysian Palm Oil Board (Bangi, Malaysia). Vitamin E measurement in the oils was done for six samples (n=6) for each of the three corresponding groups; fresh, once and five times heated. The vitamin E content in the oils was expressed as part per million (ppm). The estimated percentage of difference compared to the respective fresh oils was also calculated.

#### **2.9. Plasma lipid peroxidation measurement**

Lipid peroxidation content in the plasma measured as thiobarbituric acid reactive substance (TBARS) was determined following a method described by Ledwozyw et al. [21] with some modification. Briefly, 2.5 ml trichloroacetic acid (1.22 M in 0.6 M HCl) was used to acidify 0.5 ml plasma and incubated at room temperature for 15 minutes. Next, 1.5 ml of 0.67% thiobarbituric acid (in 0.05 M NaOH) was added. The samples were then incubated at 100C for 30 minutes. After being cooled, the lipid peroxide content was extracted by the addition of 4 ml butanol using vigorous shaking. Later, the samples fluorescence unit was read at 515 nm (excitation wavelength) and 553 nm (emission wavelength) using a spectrofluorometer (Shimadzu RF500, Japan). 1,1,3,3-Tetraethoxypropane was used as the standard. The unit of the plasma lipid peroxidation was nmol malondialdehyde/mg protein. The results were shown as the difference percentage of post-treatment content compared to the pretreatment content.

The protein content in the plasma was carried out according to the method of Lowry et al. [22], using bovine serum albumin as the standard. A plasma sample (0.5 ml) was added with 5 ml mixture of sodium carbonate (2%), sodium potassium tartrate (2%) and copper sulphate (1%) solution at a ratio of 100 : 1 : 1, prior to incubation at room temperature for 15 minutes. Subsequently, 0.5 ml Folin-Ciocalteau phenol reagent (0.5 N) was added and then was left to stand at room temperature for 35 minutes. The absorbance of the samples was read at 700 nm with a spectrophotometer (Shimadzu UV-160A, Japan).

#### **2.10. Statistical analysis**

The results were expressed as the means ± standard error of mean (SEM). Normality of the data was analysed using Kolmogorov-Smirnov test. For normally distributed data, they were then analysed using one-way analysis of variance (ANOVA) followed by Tukey's HSD post-hoc test. While for not normally distributed data, the differences among the groups were determined using Kruskal-Wallis H and Mann-Whitney U test. Values of P<0.05 were considered statistically significant. All statistical analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago, IL, USA).

#### **3. Results**

214 Lipid Peroxidation

total fatty acids.

content.

**2.8. Vitamin E content measurement** 

transesterified to fatty acid methyl ester using 1 ml of 1 M sodium methoxide in 1 ml hexane before injected into the gas chromatographic system. The injection volume was 1 l. Nitrogen at a flow rate of 0.40 ml/min was used as carrier gas in the analysis. Identification of fatty acid methyl ester peaks was carried out by comparing their retention times with their authentic standards. The fatty acid composition was expressed as the percentage of the

The vitamin E content in the oil samples (20 l sample) was analysed using an analytical high performance liquid chromatography (HPLC) using a programmable fluorescence detector at excitation 295 nm and emission 330 nm (Hewlett Packard HP1100, USA). The chromatographic system consisted of an isocratic pump and the stationary phase was a 150 mm silica normal phase column (YMC 5U) with an internal diameter 6 mm. The mobile phase was 0.5% isopropanol in hexane at a flow rate of 1 ml/min. The oil samples were injected directly into the HPLC system without any processing, after being cooled from the heating process. The vitamin E standard was obtained from the Malaysian Palm Oil Board (Bangi, Malaysia). Vitamin E measurement in the oils was done for six samples (n=6) for each of the three corresponding groups; fresh, once and five times heated. The vitamin E content in the oils was expressed as part per million (ppm). The estimated percentage of

Lipid peroxidation content in the plasma measured as thiobarbituric acid reactive substance (TBARS) was determined following a method described by Ledwozyw et al. [21] with some modification. Briefly, 2.5 ml trichloroacetic acid (1.22 M in 0.6 M HCl) was used to acidify 0.5 ml plasma and incubated at room temperature for 15 minutes. Next, 1.5 ml of 0.67% thiobarbituric acid (in 0.05 M NaOH) was added. The samples were then incubated at 100C for 30 minutes. After being cooled, the lipid peroxide content was extracted by the addition of 4 ml butanol using vigorous shaking. Later, the samples fluorescence unit was read at 515 nm (excitation wavelength) and 553 nm (emission wavelength) using a spectrofluorometer (Shimadzu RF500, Japan). 1,1,3,3-Tetraethoxypropane was used as the standard. The unit of the plasma lipid peroxidation was nmol malondialdehyde/mg protein. The results were shown as the difference percentage of post-treatment content compared to the pretreatment

The protein content in the plasma was carried out according to the method of Lowry et al. [22], using bovine serum albumin as the standard. A plasma sample (0.5 ml) was added with 5 ml mixture of sodium carbonate (2%), sodium potassium tartrate (2%) and copper sulphate (1%) solution at a ratio of 100 : 1 : 1, prior to incubation at room temperature for 15 minutes. Subsequently, 0.5 ml Folin-Ciocalteau phenol reagent (0.5 N) was added and then was left to stand at room temperature for 35 minutes. The absorbance of the samples was

read at 700 nm with a spectrophotometer (Shimadzu UV-160A, Japan).

difference compared to the respective fresh oils was also calculated.

**2.9. Plasma lipid peroxidation measurement** 

#### **3.1. Peroxide value in the oils**

The peroxide values measured in the oils are shown in Figure 1. The values in the once and five times heated palm and soy oils were significantly elevated compared to the fresh oil respectively. In five times heated oils, the values were also significantly higher than the once heated oils. Fresh and five times heated soy oils had bigger peroxide values compared to those of palm oils. According to the American Oil Chemists' Society (AOCS) [23], only five times heated soy oil had peroxide value exceeded the maximum allowable peroxide value for edible oils (> 10 meq/kg).

**Figure 1.** The peroxide values (meq/kg) in fresh and heated palm and soy oils. Bars represent mean ± SEM (n=6). \*Significantly different from the fresh oils respectively (P<0.05). #Significantly different from the once heated oils respectively (P<0.05). §Significantly different from the palm oil groups respectively (P<0.05). Dashed horizontal line indicates maximum allowable peroxide value for edible oils according to the American Oil Chemists' Society (AOCS) [23].

### **3.2. Fatty acid composition of the oils**

The fatty acid composition in both palm and soy oils is tabulated in Table 1. All the main components of fatty acid were present in the oils regardless of the frequency of heating. Once and five times heated palm oils had similar percentages of saturated, monounsaturated and polyunsaturated fatty acids composition compared to the fresh palm oil. In heated soy oil, generally the fatty acid composition was somewhat similar to the fresh oil. However, its polyunsaturated fatty acid percentage seemed to be lower than once heated and fresh oils. Overall, heating did not affect saturated and monounsaturated and polyunsaturated fatty acids components of palm oil. However, the repeated heating reduced the percentage of polyunsaturated and monounsaturated fatty acids, and increased saturated fatty acids components of the soy oil.


**Table 1.** The percentage of saturated, monounsaturated and polyunsaturated fatty acids in the fresh, once and five times heated palm and soy oils.

#### **3.3. Vitamin E content in the oils**

The vitamin E isoforms, namely -, - and -tocopherols, -, - and -tocotrienols content in the oils is tabulated in Table 2. In palm oil, only -tocopherol, -, - and -tocotrienols were present. Whilst in soy oil, only tocopherol isoforms (, and ) were detected but none of tocotrienols. Fresh palm oil had larger content of total vitamin E compared to fresh soy oil. Once and five times heated palm oils had significantly lower content of all vitamin E isoforms than the fresh palm oil. In five times heated palm oil, all the isoforms content were also significantly reduced compared once heated palm oil. For soy oil, once and five times heating decreased the -tocopherol content significantly compared to the fresh oil. The contents of - and -tocopherols were only reduced significantly in five times heated soy oil compared to once heated and fresh soy oils.

The difference in vitamin E content relative to fresh palm oil is diagrammatically shown in Figure 2. The relative reductions in -tocopherol and all tocotrienol isoforms as well as total contents were greater in five times heated palm oil than once heated palm oil. The relative reduction was the least seen in the -tocotrienol content. In once heated soy oil, a big relative


Palm oil

Soy oil

**3.2. Fatty acid composition of the oils** 

saturated fatty acids components of the soy oil.

once and five times heated palm and soy oils.

compared to once heated and fresh soy oils.

**3.3. Vitamin E content in the oils** 

The fatty acid composition in both palm and soy oils is tabulated in Table 1. All the main components of fatty acid were present in the oils regardless of the frequency of heating. Once and five times heated palm oils had similar percentages of saturated, monounsaturated and polyunsaturated fatty acids composition compared to the fresh palm oil. In heated soy oil, generally the fatty acid composition was somewhat similar to the fresh oil. However, its polyunsaturated fatty acid percentage seemed to be lower than once heated and fresh oils. Overall, heating did not affect saturated and monounsaturated and polyunsaturated fatty acids components of palm oil. However, the repeated heating reduced the percentage of polyunsaturated and monounsaturated fatty acids, and increased

Saturated Monounsaturated Polyunsaturated

 Fresh 42.87 48.94 8.18 Once heated 42.64 49.24 8.52 Five times heated 43.25 48.21 7.97

 Fresh 16.69 25.00 52.48 Once heated 17.14 26.10 51.78 Five times heated 18.10 24.21 41.72

**Table 1.** The percentage of saturated, monounsaturated and polyunsaturated fatty acids in the fresh,

The vitamin E isoforms, namely -, - and -tocopherols, -, - and -tocotrienols content in the oils is tabulated in Table 2. In palm oil, only -tocopherol, -, - and -tocotrienols were present. Whilst in soy oil, only tocopherol isoforms (, and ) were detected but none of tocotrienols. Fresh palm oil had larger content of total vitamin E compared to fresh soy oil. Once and five times heated palm oils had significantly lower content of all vitamin E isoforms than the fresh palm oil. In five times heated palm oil, all the isoforms content were also significantly reduced compared once heated palm oil. For soy oil, once and five times heating decreased the -tocopherol content significantly compared to the fresh oil. The contents of - and -tocopherols were only reduced significantly in five times heated soy oil

The difference in vitamin E content relative to fresh palm oil is diagrammatically shown in Figure 2. The relative reductions in -tocopherol and all tocotrienol isoforms as well as total contents were greater in five times heated palm oil than once heated palm oil. The relative reduction was the least seen in the -tocotrienol content. In once heated soy oil, a big relative

Repeatedly Heated Vegetable Oils and Lipid Peroxidation 217

**Table 2.** Composition of tocopherols and tocotrienols (ppm) in the fresh, once and five times heated palm and soy oils. Values are mean ± SEM (n=6). \*Significantly different from the fresh oils respectively (P<0.05), #Significantly different from once heated oil respectively (P<0.05). ND, not detectable.

**Figure 2.** The percentage difference in -tocopherol (T), -tocotrienol (T3), -tocotrienol (T3) and tocotrienol (T3) contents in heated palm oils (once and five times) in comparison to fresh palm oil.

decrease was seen in -tocopherol content, whereas other isoforms content were not much affected. However, there was only a slight relative decrease in total vitamin E content. In five times heated soy oil, the relative reductions were seen in all tocopherol isoforms (, and isoforms) (Figure 3). However, the least relative reduction was noted in the tocopherol content. It thus appeared that -tocopherol and -tocotrienol were more resistant to heat compared to and isoforms.

**Figure 3.** The percentage difference in -tocopherol (T), -tocopherol (T) and -tocopherol (T) contents in heated soy oils (once and five times) in comparison to the fresh soy oil.

#### **3.4. Plasma lipid peroxidation in male rats**

Relative plasma lipid peroxidation, measured as TBARS was increased significantly in male rats that were given diet containing 15% once and five times heated palm or soy oil for 4 months compared to the control and fresh oils, respectively (Figure 4). The five times heated groups also had significantly higher TBARS content than the once heated groups, respectively. In the once and five times heated groups, the TBARS content was significantly elevated in the soy oil-fed group compared to the palm oil-fed group, respectively. Both fresh palm and soy oil groups had significantly lower relative plasma TBARS content than the control.

#### **3.5. Plasma lipid peroxidation in ovariectomized female rats**

Ovariectomized female rats that were fed once and five times heated palm and soy oils in addition to 2% cholesterol for four months had significantly elevated relative plasma TBARS compared to both groups that were given either control diet or fresh oil respectively. The rats that ingested five times heated oils had significantly higher plasma TBARS than the

to heat compared to and isoforms.

decrease was seen in -tocopherol content, whereas other isoforms content were not much affected. However, there was only a slight relative decrease in total vitamin E content. In five times heated soy oil, the relative reductions were seen in all tocopherol isoforms (, and isoforms) (Figure 3). However, the least relative reduction was noted in the tocopherol content. It thus appeared that -tocopherol and -tocotrienol were more resistant

**Figure 3.** The percentage difference in -tocopherol (T), -tocopherol (T) and -tocopherol (T)

Relative plasma lipid peroxidation, measured as TBARS was increased significantly in male rats that were given diet containing 15% once and five times heated palm or soy oil for 4 months compared to the control and fresh oils, respectively (Figure 4). The five times heated groups also had significantly higher TBARS content than the once heated groups, respectively. In the once and five times heated groups, the TBARS content was significantly elevated in the soy oil-fed group compared to the palm oil-fed group, respectively. Both fresh palm and soy oil groups had significantly lower relative plasma TBARS content than

1HSO

5HSO

Total

Ovariectomized female rats that were fed once and five times heated palm and soy oils in addition to 2% cholesterol for four months had significantly elevated relative plasma TBARS compared to both groups that were given either control diet or fresh oil respectively. The rats that ingested five times heated oils had significantly higher plasma TBARS than the

contents in heated soy oils (once and five times) in comparison to the fresh soy oil.

**3.5. Plasma lipid peroxidation in ovariectomized female rats** 

**3.4. Plasma lipid peroxidation in male rats** 

the control.


**Percentage difference**

**Figure 4.** The change percentage of thiobarbituric acid reactive substance (TBARS), a lipid peroxidation product in male rats that were fed 15% once or five times heated (w/w) palm or soy oils for 4 months. Bars represent mean ± SEM (n=6). \*Significantly different from the control and fresh oil respectively (P<0.05). #Significantly different from once heated soy oil (P<0.05). ¥Significantly different from the heated palm oil, respectively (P<0.05). §Significantly different from the control (P<0.05).

**Figure 5.** The percentage of change of lipid peroxidation product measured as thiobarbituric acid reactive substance (TBARS) in ovariectomised female rats that were fed 2% cholesterol together with 15% once or five times heated (w/w) palm or soy oils for 4 months. Bars represent mean ± SEM (n=6). \*Significantly different from the control and fresh oil respectively (P<0.05). #Significantly different from once heated soy oil (P<0.05).

once heated groups respectively. The plasma TBARS in palm oil-fed groups were similar to the soy oil-fed groups except in five times heated groups which the palm oil group had a lower plasma TBARS (P<0.05). The plasma TBARS of the fresh oil groups was not different from that of the control.

## **4. Discussion**

Repeatedly heated cooking oil is often used interchangeably with thermoxidized or recycled cooking oil. Repeated use of this oil has become a common practice due to low level of awareness among the public about the bad effect of this practice [9]. Nowadays, the consumption of deep-fried food has gained popularity which may cause increased risk of obesity [24].

During frying, food is immersed in hot oil at a high temperature of 150 °C to 190 °C. The heat and mass transfer of oil, food and air that occurs during deep frying produces the unique and desirable quality of fried foods [2]. It was shown in the present study that the peroxide values were increased with the increasing frequency of heating in both types of oil. Increased values indicate increased lipid peroxidation byproduct content, mainly the peroxides that were formed in the oil during heating process. The extent of oxidation in the oils was affected by the number of frying. Other than the peroxides, there are other oxidized components that are formed during oil heating such as oxidative dimers and oxidized triacylglycerols [25]. Only five times heated soy oil exceeded the upper limit of peroxide value set by the American Oil Chemists' Society (AOCS) that is 10 meq/kg oil [23]. However, if the Food Sanitation Law of Japan guideline (peroxide value ≤ 30 meq/kg oil) is used instead, all fresh and heated oils can be considered safe for human ingestion. However, a recent study done by Awney [26] has demonstrated that in male rats fed thermally oxidized soy oil with peroxide value of 14 meq/kg, had significantly increased lipid peroxidation content in various organs such as liver, kidney, testes and brain. Chronic intake of repeatedly heated cooking could be harmful to health and increase risk of many diseases including hypertension [13] and cancer [10].

During heating at high temperature, several complex chemical reactions take place in the oil. It is dependent on the temperature, duration of heating, type of frying materials, type of oils, presence of antioxidant and prooxidant as well as the amount of oxygen [2,27]. Repeatedly heated oils at high temperature will undergo chemical conversion of fatty acid configuration from cis to trans isomer [28]. Diets containing trans fatty acid could be detrimental to cardiovascular health because it was reported that this fatty acid isomer could induce inflammation of the blood vessels and decrease its nitric oxide production [29-30]. The polymer and polar compounds content are also increased more than 37% and 47% respectively when the oil is used to fry [31]. The repeatedly heating would reduce the quality of cooking oil by darkening its color and changing the smell as well as the taste [1].

We found that the fresh and repeatedly heated soy oil had greater peroxide value than palm oil. The peroxide value is often used as an indicator for oxidative stability or the extent of degradation for fats and oils [32]. Therefore, greater values found in soy oil suggest that the soy oil were more susceptible to oxidative modification than palm oil. Vegetable oils rich in polyunsaturated fatty acid are more prone to oxidation compared to the oils which are rich in monounsaturated fatty acids [33]. Fats are usually oxidized by prooxidants or free radicals at the unsaturated bonds of the fatty acids, which are abundantly present in the polyunsaturated fatty acids such as linoleic (C18:2) and linolenic (C18:3) acids. The bigger the numbers of unsaturated bonds, the more prone the fatty acids are to oxidation. Intense heating of oils decreases unsaturation of the fatty acids [34].

220 Lipid Peroxidation

from that of the control.

including hypertension [13] and cancer [10].

**4. Discussion** 

obesity [24].

once heated groups respectively. The plasma TBARS in palm oil-fed groups were similar to the soy oil-fed groups except in five times heated groups which the palm oil group had a lower plasma TBARS (P<0.05). The plasma TBARS of the fresh oil groups was not different

Repeatedly heated cooking oil is often used interchangeably with thermoxidized or recycled cooking oil. Repeated use of this oil has become a common practice due to low level of awareness among the public about the bad effect of this practice [9]. Nowadays, the consumption of deep-fried food has gained popularity which may cause increased risk of

During frying, food is immersed in hot oil at a high temperature of 150 °C to 190 °C. The heat and mass transfer of oil, food and air that occurs during deep frying produces the unique and desirable quality of fried foods [2]. It was shown in the present study that the peroxide values were increased with the increasing frequency of heating in both types of oil. Increased values indicate increased lipid peroxidation byproduct content, mainly the peroxides that were formed in the oil during heating process. The extent of oxidation in the oils was affected by the number of frying. Other than the peroxides, there are other oxidized components that are formed during oil heating such as oxidative dimers and oxidized triacylglycerols [25]. Only five times heated soy oil exceeded the upper limit of peroxide value set by the American Oil Chemists' Society (AOCS) that is 10 meq/kg oil [23]. However, if the Food Sanitation Law of Japan guideline (peroxide value ≤ 30 meq/kg oil) is used instead, all fresh and heated oils can be considered safe for human ingestion. However, a recent study done by Awney [26] has demonstrated that in male rats fed thermally oxidized soy oil with peroxide value of 14 meq/kg, had significantly increased lipid peroxidation content in various organs such as liver, kidney, testes and brain. Chronic intake of repeatedly heated cooking could be harmful to health and increase risk of many diseases

During heating at high temperature, several complex chemical reactions take place in the oil. It is dependent on the temperature, duration of heating, type of frying materials, type of oils, presence of antioxidant and prooxidant as well as the amount of oxygen [2,27]. Repeatedly heated oils at high temperature will undergo chemical conversion of fatty acid configuration from cis to trans isomer [28]. Diets containing trans fatty acid could be detrimental to cardiovascular health because it was reported that this fatty acid isomer could induce inflammation of the blood vessels and decrease its nitric oxide production [29-30]. The polymer and polar compounds content are also increased more than 37% and 47% respectively when the oil is used to fry [31]. The repeatedly heating would reduce the quality of cooking oil by darkening its color and changing the smell as well as the taste [1].

We found that the fresh and repeatedly heated soy oil had greater peroxide value than palm oil. The peroxide value is often used as an indicator for oxidative stability or the extent of degradation for fats and oils [32]. Therefore, greater values found in soy oil suggest that the The fresh soy oil contained about five times more polyunsaturated fatty acid compared to the palm oil. It seemed that five times heating had reduced about 10% of the polyunsaturated fatty acid content in the soy oil. The content of monounsaturated fatty acid in the fresh palm oil was higher than that of the fresh soy oil. Palm oil had a quite balanced ratio of saturated and unsaturated fatty acids, whereas more than 70% of soy oil fatty acid was unsaturated (polyunsaturated and monounsaturated). This unique fatty acid composition of palm oil renders its stability against oxidative insult. The fatty acid composition of heated soy oil [26] and palm oil [35] was similarly obtained in previous studies.

The examples of the monounsaturated are oleic (C18:1) and palmitoleic (C16:1), whereas palmitic (C16:0) and stearic (C18:0) are saturated fatty acids [26]. Soy oil majorly contains linoleic (54%) and oleic (23%) acids. It also contains linolenic (8%), palmitic (11%) and stearic (4%) acids [26]. While palm oil has a bigger proportion of oleic (46%) and palmitic acids (36%), a smaller amount of linoleic (12%) and linolenic (< 1%) acids, and similar proportion of stearic acid (4.5%) compared to soy oil [36]. However in the present study, the individual fatty acid composition in the oil was not determined.

Most antioxidants are heat labile. Vitamin E is not an exception. When subjected to heat at high temperature, we found that all tocotrienol isoforms (, and ) as well as -tocopherol in the palm oil were lost. It became more prominent when the oil was heated five times. The fresh palm oil contained almost 700 ppm of total vitamin E, but once and five times heating had further reduced it to barely 400 and 75 ppm, respectively. -Tocotrienol content was the highest in fresh and once heated palm oil, followed by -tocotrienol, -tocopherol and the least being the -tocotrienol. Once heating caused almost 50% reduction in -tocopherol and -tocotrienol contents in palm oil. While the loss of other isoforms (- and -tocotrienols) were at smaller percentages. In five times heated palm oil, the percentage loss of all vitamin E components were almost 100% except -tocotrienol (almost 50%), compared to the fresh palm oil. It seemed that amongst the tocotrienol isoforms, -tocotrienol had the highest resistance to heat.

Different from palm oil, the soy oil exclusively contained -, - and -tocopherols. - Tocopherol was the largest constituent in the soy oil, followed by -tocopherol and lastly -tocopherol. The total content of vitamin E in fresh soy oil was about 60% of that of fresh palm oil. Compared to tocopherols, tocotrienols are less widespread in plants [37]. Other than palm oil, tocotrienols are found abundantly in barley [38], rice bran [39], grape seed oil [40], rye, oat, maize and wheat germ [41]. Tocopherols on the other hand, are

distributed at a wider range of food and vegetable oils such as soybean, corn, sunflower and cottonseed oils [40,42].

Once heating did not significantly affect - and -tocopherols contents in the soy oil, but the -tocopherol content was apparently reduced. However in five times heated, all tocopherol isoforms content were significantly decreased. When the percentage difference relative to the fresh soy oil calculated, the most prominent reduction was seen in -tocopherol content after once heating, followed by the -tocopherol and -tocopherol contents. Quite a similar finding was reported by Rennick & Warner [43] that the -tocopherol content of soy oil was significantly decreased when was heated for 5 hours and was further decreased after heated for 10 hours. The loss of the tocopherol content was accompanied by an increasing appearance of -tocopherolquinones.

From the findings obtained, it seemed that the loss of tocotrienol was more than the same isoform of tocopherol. The vitamin E chemical structure is consisted of a chromanol ring and a 15-carbon phytyl side chain. Tocotrienol differs from tocopherol by the presence of three unsaturated bonds in its phytyl chain [44]. The presence of unsaturations in the tocotrienol phytyl chain may explain the susceptibility of the compound to repeated heating. It was also noted that the isoform of both tocopherol and tocotrienol was the mostly susceptible to oxidative loss in both palm and soy oils. The second sensitive isoform was the isoform (both -tocopherol and tocotrienol), and the least sensitive being the isoform. The isoform has three methylated groups, the isoform has two while the isoform has only one methylated group on the chromanol ring [45]. However Miyagawa et al. [46] had reported that the decomposition rate of -tocopherol was the fastest, followed by - and -tocopherol when a mixture of soy and rapeseed oil was used to deep fry potato. The discrepancy could be due to the different type of oils used in their study. It can be postulated that the degree and position of the methylation on the chromanol ring also determine the susceptibility of the vitamer to oxidative loss in addition to the degree of saturation of the phytyl chain.

The higher vitamin E content in the palm oil than soy oil might contribute to lesser peroxide value in the former oil. Both tocopherols and tocotrienols possess antioxidant property, with the latter having greater antioxidant property [6]. Vitamin E could effectively protect the fatty acids in the oil from oxidation. During oil heating, the vitamin E is consumed by scavenging the lipid free radicals which are derived from the oxidation of unsaturated fatty acids in the oils. -Tocopherol addition to frying oil increased the stability and resistance of polyunsaturated fatty acid against oxidation [47]. Inclusion of antioxidants lemon seed extract and tert-butylhydroquinone was demonstrated to retard lipid oxidation and contribute to the -tocopherol retention in the soy oil heated at high temperatures for several hours [48].

Male rats that were fed heated oils had elevated plasma lipid peroxidation content. The elevation was more prominent with the five times heated oils. The increased peroxide value of the heated oils may be associated with the significant increase in plasma lipid peroxidation. Chronic ingestion of heated oil was shown to cause an elevation of blood pressure [49] and necrotic cardiac changes [50]. This would disrupt the endogenous antioxidant defense in our body in order to overcome the overwhelming of oxidative stress. Linoleyl, peroxy, and alkoxy radicals were reported to be produced in heated oils. These radicals act on the fatty acids of the oil producing oxidized products via hemolytic -scission [25]. Dietaries fresh palm and soy oil were shown to attenuate oxidative stress and augment antioxidant enzymes activities in rat models [51-53]. Their findings are in agreement with the present study where a significant reduction in plasma lipid peroxidation was observed in the fresh oil-fed male rats. This positive effect was attributed to the rich antioxidant content of the oils.

222 Lipid Peroxidation

and cottonseed oils [40,42].

appearance of -tocopherolquinones.

several hours [48].

distributed at a wider range of food and vegetable oils such as soybean, corn, sunflower

Once heating did not significantly affect - and -tocopherols contents in the soy oil, but the -tocopherol content was apparently reduced. However in five times heated, all tocopherol isoforms content were significantly decreased. When the percentage difference relative to the fresh soy oil calculated, the most prominent reduction was seen in -tocopherol content after once heating, followed by the -tocopherol and -tocopherol contents. Quite a similar finding was reported by Rennick & Warner [43] that the -tocopherol content of soy oil was significantly decreased when was heated for 5 hours and was further decreased after heated for 10 hours. The loss of the tocopherol content was accompanied by an increasing

From the findings obtained, it seemed that the loss of tocotrienol was more than the same isoform of tocopherol. The vitamin E chemical structure is consisted of a chromanol ring and a 15-carbon phytyl side chain. Tocotrienol differs from tocopherol by the presence of three unsaturated bonds in its phytyl chain [44]. The presence of unsaturations in the tocotrienol phytyl chain may explain the susceptibility of the compound to repeated heating. It was also noted that the isoform of both tocopherol and tocotrienol was the mostly susceptible to oxidative loss in both palm and soy oils. The second sensitive isoform was the isoform (both -tocopherol and tocotrienol), and the least sensitive being the isoform. The isoform has three methylated groups, the isoform has two while the isoform has only one methylated group on the chromanol ring [45]. However Miyagawa et al. [46] had reported that the decomposition rate of -tocopherol was the fastest, followed by - and -tocopherol when a mixture of soy and rapeseed oil was used to deep fry potato. The discrepancy could be due to the different type of oils used in their study. It can be postulated that the degree and position of the methylation on the chromanol ring also determine the susceptibility of the vitamer to oxidative loss in addition to the degree of saturation of the phytyl chain.

The higher vitamin E content in the palm oil than soy oil might contribute to lesser peroxide value in the former oil. Both tocopherols and tocotrienols possess antioxidant property, with the latter having greater antioxidant property [6]. Vitamin E could effectively protect the fatty acids in the oil from oxidation. During oil heating, the vitamin E is consumed by scavenging the lipid free radicals which are derived from the oxidation of unsaturated fatty acids in the oils. -Tocopherol addition to frying oil increased the stability and resistance of polyunsaturated fatty acid against oxidation [47]. Inclusion of antioxidants lemon seed extract and tert-butylhydroquinone was demonstrated to retard lipid oxidation and contribute to the -tocopherol retention in the soy oil heated at high temperatures for

Male rats that were fed heated oils had elevated plasma lipid peroxidation content. The elevation was more prominent with the five times heated oils. The increased peroxide value of the heated oils may be associated with the significant increase in plasma lipid peroxidation. Chronic ingestion of heated oil was shown to cause an elevation of blood pressure [49] and necrotic cardiac changes [50]. This would disrupt the endogenous Heating diminished the vitamin E content and rendered the oils to lose their beneficial effects. Chronic ingestion of heated soy oil diet was shown to induce the increase in hepatic lipid peroxidation in rats [54]. They also observed a reduction in serum and hepatic vitamin E content in rats that were fed heated oil containing diet. An evidence of hepatic damage was also reported with a significant elevation of aspartate and alanine transaminases, the marker enzymes for liver function in rats that were given combination of heated soy and rapeseed oils [55]. The administration of dietary heated soy oil affected the activities of antioxidant enzymes in various rat organs. It increased superoxide dismutase in the liver and brain, while increment in glutathione reductase was seen in the liver and kidneys [26]. Heated palm oil also increased catalase, glutathione peroxidase and glutathione Stransferase in the rat liver [35]. It shows dietary heated oil contained prooxidant substances that would evoke the body primary defenses.

Plasma lipid peroxidation was increased significantly in the rats that were given heated soy oil (once and five times heated) when compared to the heated palm oil-fed groups. This finding was suggestive of the better effect of the palm oil in terms of oxidative stability when exposed to extreme heat. High composition of saturated fats in palm oil confers it to withstand thermal oxidative changes, in addition to its rich content of tocotrienols. The better effect of heated palm oil compared to heated soy oil was reported elsewhere [56].

Ovariectomized female rats were often used as an experimental postmenopausal model. Ovariectomy results in an estrogen deficiency state. The estrogen provides protection to the body against oxidative stress [16]. In the present study, dietaries once heated and five times heated palm and soy oil for four months had significantly increased the plasma lipid peroxidation in ovariectomized female rats. The result is in agreement with other studies which also reported that in ovariectomized-induced estrogen deficiency, an increase in oxidative stress accompanied by a reduction in antioxidant status was seen [57-58]. A study by Sánchez-Rodríguez [59] had recently found that menopause could be one of the main risk factors for oxidative stress. Therefore, this finding suggests that chronic ingestion of repeatedly heated vegetable oils is detrimental to health and could further aggravate the increase in oxidative stress in postmenopausal women. Shuid et al. [56] also had demonstrated that fresh and once heated palm and soy oil diets for six months reduced the risk of osteoporosis in ovariectomized rats, a positive effect that was not seen in rats that were fed five times heated oils.

The patterns of plasma lipid peroxidation increase in both male and ovariectomized female rats were somewhat similar. However, the plasma lipid peroxidation of the fresh oil-fed groups in the ovariectomized female rats that were similar to the control group, different from the one observed in the male rats. The addition of dietary 2% cholesterol and the ovariectomy status of the female rats made them more susceptible to oxidative insult. This may explain why there was no significant reduction in plasma TBARS seen in the female rats fed fresh oils compared to the control. The plasma lipid peroxidation was significantly elevated in the five times heated soy oil group compared to the same heating frequency of palm oil. The difference was also attributed to the higher component of polyunsaturated fatty acids present in the soy oil, which are prone to oxidation.

## **5. Conclusion**

Our findings suggest that it is recommended not to heat cooking oil more than once in view of its possible deleterious effect on health. The use of palm oil possibly has better effect on health due to its stability against oxidative insult.

## **Author details**

Kamsiah Jaarin and Yusof Kamisah *Department of Pharmacology, Faculty of Medicine,Universiti Kebangsaan Malaysia, Malaysia* 

## **Acknowledgement**

The study was funded by grants from Universiti Kebangsaan Malaysia (UKM-GUP-SK-08- 21-299) and Malaysian Ministry of Science, Technology and Innovations (IRPA 06-02-02- 0050-EA242). The authors also would like to thank Prof. Dr Jumat Salimon, Ms Xin-Fang Leong and Mr. Chun-Yi Ng for their technical help as well as Ms Jurika Sharaton Abdul Wahed for editorial assistance.

#### **6. References**


[7] Edem DO (2002) Palm oil: biochemical, physiological, nutritional, hematological, and toxicological aspects: a review. Plant Foods Hum. Nutr. 57(3-4): 319-341.

224 Lipid Peroxidation

**5. Conclusion** 

**Author details** 

**Acknowledgement** 

Wahed for editorial assistance.

Crit. Rev. Food Sci. Nutr. 46(1): 1-22.

profile in rats. Pakistan J. Nutr. 4(2): 89-96

Tocotrienol. Curr. Pharm. Des. 17(21): 2196-2205.

**6. References** 

The patterns of plasma lipid peroxidation increase in both male and ovariectomized female rats were somewhat similar. However, the plasma lipid peroxidation of the fresh oil-fed groups in the ovariectomized female rats that were similar to the control group, different from the one observed in the male rats. The addition of dietary 2% cholesterol and the ovariectomy status of the female rats made them more susceptible to oxidative insult. This may explain why there was no significant reduction in plasma TBARS seen in the female rats fed fresh oils compared to the control. The plasma lipid peroxidation was significantly elevated in the five times heated soy oil group compared to the same heating frequency of palm oil. The difference was also attributed to the higher component of polyunsaturated

Our findings suggest that it is recommended not to heat cooking oil more than once in view of its possible deleterious effect on health. The use of palm oil possibly has better effect on

*Department of Pharmacology, Faculty of Medicine,Universiti Kebangsaan Malaysia, Malaysia* 

The study was funded by grants from Universiti Kebangsaan Malaysia (UKM-GUP-SK-08- 21-299) and Malaysian Ministry of Science, Technology and Innovations (IRPA 06-02-02- 0050-EA242). The authors also would like to thank Prof. Dr Jumat Salimon, Ms Xin-Fang Leong and Mr. Chun-Yi Ng for their technical help as well as Ms Jurika Sharaton Abdul

[1] Rani AKS, Reddy SY, Chetana R (2010) Quality changes in trans and trans free fats/oils

[4] Kamisah Y, Adam A, Wan Ngah WZ, Gapor MT, Azizah O, Marzuki A (2005) Chronic intake of red palm olein and palm olein produce beneficial effects on plasma lipid

[5] Clemente TE, Cahoon EB (2009) Soybean oil: genetic approaches for modification of

[6] Bardhan J, Chakraborty R, Raychaudhuri U (2011). The 21st century form of vitamin E -

[2] Choe E, Min DB (2007) Chemistry of deep-fat frying oils. J. Food Sci. 72(5):R77-R86. [3] Choe, E. & Min, D. B (2006) Chemistry and reactions of reactive oxygen species in foods.

and products during frying. Eur. Food Res. Technol. 230(6): 803–811.

functionality and total content. Plant Physiol. 151(3): 1030-1040.

fatty acids present in the soy oil, which are prone to oxidation.

health due to its stability against oxidative insult.

Kamsiah Jaarin and Yusof Kamisah


[35] Purushothama S, Ramachandran HD, Narasimhamurthy K, Raina PL (2003) Long-term feeding effects of heated and fried oils on hepatic antioxidant enzymes, absorption and excretion of fat in rats. Mol. Cell Biochem. 247(1-2): 95-99.

226 Lipid Peroxidation

211.

(in press)

57(12): 5391-5400.

Chem. 52(14): 4438-4443.

91(5): 927-933.

1999 Sep 13;50(2):269-75.

endothelial cells. PLoS One. 6(12): e29600.

[21] Ledwozyw A, Michalak J, Stepian A, Kadziolka A (1986) A relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during

[22] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with Folin

[23] Matthaus B (2006) Utilization of high-oleic rapeseed oil for deep-fat frying of French fries compared to other commonly used edible oils. Eur. J. Lipid Sci. Tech. 108(3): 200-

[24] Sayon-Orea C, Bes-Rastrollo M, Basterra-Gortari FJ, Beunza JJ, Guallar-Castillon P, de la Fuente-Arrillaga C, Martinez-Gonzalez MA (2012) Consumption of fried foods and weight gain in a Mediterranean cohort: The SUN project. Nutr. Metab. Cardiovasc. Dis.

[25] Picariello G, Paduano A, Sacchi R, Addeo F (2009) Maldi-tof mass spectrometry profiling of polar and nonpolar fractions in heated vegetable oils. J. Agric. Food Chem.

[26] Awney HA (2011) The effects of Bifidobacteria on the lipid profile and oxidative stress biomarkers of male rats fed thermally oxidized soybean oil. Biomarkers. 16(5): 445-452. [27] Leskova E, Kubikova J, Kovacikova E, Kosicka M, Pobruska J, Holcikova K (2006) Vitamin losses: Retention during heat treatment and continual changes expressed by

[28] Velasco J, Marmesat S, Bordeaux O, Márquez-Ruiz G, Dobarganes C (2004) Formation and evolution of monoepoxy fatty acids in thermoxidized olive and sunflower oils and quantitation in used frying oils from restaurants and fried-food outlets. J. Agr. Food

[29] Iwata NG, Pham M, Rizzo NO, Cheng AM, Maloney E, Kim F (2011) Trans fatty acids induce vascular inflammation and reduce vascular nitric oxide production in

[30] Bryk D, Zapolska-Downar D, Malecki M, Hajdukiewicz K, Sitkiewicz D (2011) Trans fatty acids induce a proinflammatory response in endothelial cells through ROS-

[32] Karoui IJ, Dhifi W, Jemia MB, Marzouk B (2011) Thermal stability of corn oil flavoured with Thymus capitatus under heating and deep-frying conditions. J. Sci. Food Agr.

[33] Kochhar SP, Henry CJ (2009) Oxidative stability and shelf-life evaluation of selected

[34] Moya Moreno MC, Mendoza Olivares D, Amézquita López FJ, Gimeno Adelantado JV, Bosch Reig F. Analytical evaluation of polyunsaturated fatty acids degradation during thermal oxidation of edible oils by Fourier transform infrared spectroscopy. Talanta.

dependent nuclear factor-κB activation. J. Physiol. Pharmacol. 62(2): 229-238. [31] Saka S, Aouacheri W, Abdennour C (2002) The capacity of glutathione reductase in cell

protection from the toxic effect of heated oils. Biochimie 84(7): 661-665.

culinary oils. Inter. J. Food Sci. Nutr. 60(7 Suppl): 289-296.

human atherosclerosis. Clin. Chim. Acta. 155: 275-285.

mathematical models. J. Food Comp. Anal. 19: 252–276.

Phenol reagent. J. Biol. Chem. 193: 265-275.


## **Effects of Hydroperoxide in Lipid Peroxidation on Dough Fermentation**

Toshiyuki Toyosaki

228 Lipid Peroxidation

Res. 39: 567-572.

1297-1303.

19(3): 361-367.

[50] Leong XF, Aishah A, Aini UN, Das S, Jaarin K (2008) Heated palm oil causes rise in blood pressure and cardiac changes in heart muscle in experimental rats. Arch. Med.

[51] Bayorh MA, Abukhalaf IK, Ganafa AA (2005) Effect of palm oil on blood pressure, endothelial function and oxidative stress. Asia Pac. J. Clin. Nutr. 14(4): 325-339. [52] Narang D, Sood S, Thomas M, Dinda AK, Maulik SK (2005) Dietary palm olein oil augments cardiac antioxidant enzymes and protects against isoproterenol-induced

[53] Hassan HA, Abdel-Wahhab MA (2012) Effect of soybean oil on atherogenic metabolic risks associated with estrogen deficiency in ovariectomized rats : Dietary soybean oil modulate atherogenic risks in overiectomized rats. J. Physiol. Biochem. (in press). [54] Corcos-Benedetti P, Di Felice M, Gentili V, Tagliamonte B, Tomassi G (1990) Influence of dietary thermally oxidized soybean oil on the oxidative status of rats of different

[55] Totani N, Burenjargal M, Yawata M, Ojiri Y (2008) Chemical properties and cytotoxicity

[56] Shuid AN, Chuan LH, Mohamed N, Jaarin K, Fong YS, Soelaiman IN (2007) Recycled palm oil is better than soy oil in maintaining bone properties in a menopausal

[57] Huang YH, Zhang QH (2010) Genistein reduced the neural apoptosis in the brain of ovariectomised rats by modulating mitochondrial oxidative stress. Br. J. Nutr. 104(9):

[58] Topçuoglu A, Uzun H, Balci H, Karakus M, Coban I, Altug T, Aydin S, Topçuoglu D, Cakatay U (2009) Effects of estrogens on oxidative protein damage in plasma and

[59] Sánchez-Rodríguez MA, Zacarías-Flores M, Arronte-Rosales A, Correa-Muñoz E, Mendoza-Núñez VM (2012) Menopause as risk factor for oxidative stress. Menopause.

syndrome model of ovariectomized rat. Asia Pac J Clin Nutr. 16(3): 393-402.

tissues in ovariectomised rats. Clin. Invest. Med. 32(2): E133-143.

myocardial necrosis in rats. J. Pharm. Pharmacol. 57(11): 1445-1451.

ages. Ann. Nutr. Metab. 34(4): 221-231.

of thermally oxidized oil. J. Oleo. Sci. 57(3): 153-160.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/39187

## **1. Introduction**

The oxidation of lipids in foods is responsible for the formation of off-flavors and chemical compounds that may be detrimental to health; it is a well known problem in the food chemistry and biochemistry fields (Ames et al., 1994; Gardnaer, 1996; Grosch, 1987; Pokorny, 1999; Shewfelt & Del Rosario, 2000; Mercier & Gelinas, 2001; Toyosaki & Sakane, 2002; Toyosaki & Koketsu, 2004). Currently, the various lipid peroxides produced by such lipid peroxidation are treated only as a nuisance. However, among longstanding traditional foods there are foods with fine flavors that are brought out by inducing lipid peroxidation; such foods include fine, thin noodles and certain dried foods. Thus, lipid peroxides produced by lipid peroxidation can also be advantageous. The properties of foods can be improved by better use of the properties of lipid peroxides. The current work provided an interesting finding: when lipoxygenase was added during the fermentation of bread dough, the fermentation of dough was promoted. During this event, hydroperoxides produced by lipid peroxidation triggered the promotion of fermentation and promoted the fermentation of dough. The phenomenon by which hydroperoxides are produced by lipid peroxidation and promote the fermentation of bread dough is decidedly not beneficial when assessed from a nutritional standpoint, but this phenomenon is extremely desirable when assessed from a food science standpoint. The objective of the current study was to investigate the bread dough fermentation-promoting action of hydroperoxides produced by lipid peroxidation in bread dough and the mechanism that is involved.

## **2. Methods**

#### **Preparation of dough**

The wheat flour (strong flour) that was used to make adjustments in the bread dough was the type that is readily commercially available. The lipid added was linoleic acid (more than

© 2012 Toyosaki, 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. © 2012 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.

95% pure), which was added at a 3% level. Other ingredients used to make the bread were all commercially available. Lipoxygenase was added at the end of bread dough adjustment and underwent primary fermentation in an incubator at 37°C with 75-80% humidity. After fermentation, gas was released; after a bench time of 10 min, the dough underwent final fermentation for 90 min and was then baked for 12 min at 200°C.

#### **Preparation of the model system of gluten and linoleic acid**

By mixing a fixed amount of commercially available gluten and linoleic acid (more than 95% pure) of 3% level, which served as the test sample, a model system was created. This sample underwent fermentation by lipoxygenase induction.

#### **Measurement of the rate of dough expansion**

To determine the rate of dough expansion with fermentation, a fixed amount of dough was placed in a graduated cylinder and fermented in an incubator (temperature 30°C, humidity 75%). The rate of expansion over a fixed period of time was then measured.

#### **Measurement of hydroperoxide**

Hydroperoxide concentration was calculated in terms of 2',7'-dichlorofluorescein (DCF). To de-emulsify, the 5.0 ml samples were centrifuged (10,000 x g, 30 min). The linoleic acid of the supernatant was then measured to determine the hydroperoxide level using the method of Cathcart et al. (1984). First, 1.0 ml of a 1.0 mM solution of DCF in ethanol and 2.0 ml of 0.01N NaOH were mixed and stirred for 30 min before being neutralized with 10 ml of 25 mM phosphate buffer (pH 7.2). Then 2.0 ml of the neutralized DCF solution were added to a solution of hematin (10 mM) in 25 mM phosphate buffer (pH 7.2; 0.01 mg DCF/ml); subsequently, 2.0 ml of this hematin-DCF solution and 10 ml of the linoleic acid sample were mixed and left at 50°C for 50 min, before fluorometry treatment (excitation. 400 nm; emission. 470 nm) to measure DCF. This method measures hydroperoxide with more sensitivity than the iron rhodanide method that is usually used.

#### **Diethylaminoethyl (DEAE) column chromatography**

The extracted dough was separated by Tris-HCl buffer (pH 8.0). The extracted sample fractions were separated by DEAE-cellulose (DE52, Whatman, Ltd., Tokyo, Japan) column chromatography as follows. A DEAE-cellulose column (3.8 x 54 cm) was equilibrated with 50 mM Tris-HCl buffer (pH 8.0), washed with the same buffer, and developed in a linear gradient made with 300 ml of this buffer and 300 ml of the same buffer containing 0.6 M NaCl. The flow rate was 30 ml/hr, and 3.0 ml fractions were collected.

#### **Measurement of the amount of protein**

The amount of protein was measured using the Lowry method(1951).

#### **SDS-polyacrylamide gel electrophoresis (PAGE)**

The measurement was done according to the method of Laemmli (1979). Electrophoresis was performed using the Mini-Protean II Electrophoresis Cell (Bio-Rad Laboratories, Inc., Tokyo, Japan) at 18 mA/gel with Ready Gel J of differing gel concentrations. After electrophoresis, the gels were stained using Coomassie brilliant blue-R250. In addition, automated electrophoresis (Phast System; Pharmacia LKB, Biotechnology AB, Uppsala, Sweden) equipment was used.

#### **Statistical analysis**

230 Lipid Peroxidation

95% pure), which was added at a 3% level. Other ingredients used to make the bread were all commercially available. Lipoxygenase was added at the end of bread dough adjustment and underwent primary fermentation in an incubator at 37°C with 75-80% humidity. After fermentation, gas was released; after a bench time of 10 min, the dough underwent final

By mixing a fixed amount of commercially available gluten and linoleic acid (more than 95% pure) of 3% level, which served as the test sample, a model system was created. This sample

To determine the rate of dough expansion with fermentation, a fixed amount of dough was placed in a graduated cylinder and fermented in an incubator (temperature 30°C, humidity

Hydroperoxide concentration was calculated in terms of 2',7'-dichlorofluorescein (DCF). To de-emulsify, the 5.0 ml samples were centrifuged (10,000 x g, 30 min). The linoleic acid of the supernatant was then measured to determine the hydroperoxide level using the method of Cathcart et al. (1984). First, 1.0 ml of a 1.0 mM solution of DCF in ethanol and 2.0 ml of 0.01N NaOH were mixed and stirred for 30 min before being neutralized with 10 ml of 25 mM phosphate buffer (pH 7.2). Then 2.0 ml of the neutralized DCF solution were added to a solution of hematin (10 mM) in 25 mM phosphate buffer (pH 7.2; 0.01 mg DCF/ml); subsequently, 2.0 ml of this hematin-DCF solution and 10 ml of the linoleic acid sample were mixed and left at 50°C for 50 min, before fluorometry treatment (excitation. 400 nm; emission. 470 nm) to measure DCF. This method measures hydroperoxide with more

The extracted dough was separated by Tris-HCl buffer (pH 8.0). The extracted sample fractions were separated by DEAE-cellulose (DE52, Whatman, Ltd., Tokyo, Japan) column chromatography as follows. A DEAE-cellulose column (3.8 x 54 cm) was equilibrated with 50 mM Tris-HCl buffer (pH 8.0), washed with the same buffer, and developed in a linear gradient made with 300 ml of this buffer and 300 ml of the same buffer containing 0.6 M

The measurement was done according to the method of Laemmli (1979). Electrophoresis was performed using the Mini-Protean II Electrophoresis Cell (Bio-Rad Laboratories, Inc.,

75%). The rate of expansion over a fixed period of time was then measured.

sensitivity than the iron rhodanide method that is usually used.

NaCl. The flow rate was 30 ml/hr, and 3.0 ml fractions were collected.

The amount of protein was measured using the Lowry method(1951).

**Diethylaminoethyl (DEAE) column chromatography** 

**Measurement of the amount of protein** 

**SDS-polyacrylamide gel electrophoresis (PAGE)** 

fermentation for 90 min and was then baked for 12 min at 200°C.

**Preparation of the model system of gluten and linoleic acid** 

underwent fermentation by lipoxygenase induction.

**Measurement of the rate of dough expansion** 

**Measurement of hydroperoxide** 

Analysis of variance (ANOVA) was performed and mean comparisons were obtained by Duncan's multiple range test (Steel & Torrie, 1980). Significance was established at *P*<0.05.

#### **3. Results and discussion**

#### **Lipid peroxidation during fermentation and accompanying changes in the rate of expansion**

After 3% linoleic acid was added to the other bread ingredients, the doughs with and without lipoxygenase were individually mixed with a mixer for a fixed time. Next, the dough underwent primary fermentation in an incubator for 90 min, and the amount of hydroperoxide produced over this time period was measured. These results are shown in Fig. 1. Lipid peroxidation by lipoxygenase induction increased as the fermentation time progressed. However, the overall amount of hydroperoxide produced in the lipoxygenasefree dough tended not to increase. The rate of expansion during this time is shown in Fig. 2.

**Figure 1.** Changes in the amount of hydroperoxide produced with the fermentation of dough. Each value represents the mean standard error in triplicate.

For up to 40 min, dough with lipoxygenase expanded rapidly after the start of fermentation, but after this time the expansion tended to decrease abruptly. In contrast, lipoxygenase-free dough reached its maximum rate of expansion in 30 min from the start of fermentation, and this tended to decrease gradually afterwards. Further, in the dough without lipoxygenase, changes in the rate of expansion per unit time were smaller than in the dough with lipoxygenase. Changes brought about by this phenomenon are quite likely due to the effect that hydroperoxide, which is produced by lipid peroxidation, has on the fermentation stage.

**Figure 2.** Changes in the rate of expansion with the fermentation of dough. Each value represents the mean standard error in triplicate.

#### **Relationship between the rate of expansion and hydroperoxide**

A model system of gluten and linoleic acid was created, and the involvement of the hydroperoxide that was produced in the fermentation of dough was examined. These results are shown in Fig. 3. The rate of dough expansion was affected by hydroperoxide concentration, and in this experiment the rate of expansion reached its maximum at a hydroperoxide concentration of 30-40 mM. Based on these results, the fermentation of dough was influenced by the concentrations of hydroperoxide that were produced.

**Figure 3.** Changes in the rate of dough expansion with hydroperoxides. Each value represents the mean standard error in triplicate.

#### **Effects of yeast and gluten during fermentation**

232 Lipid Peroxidation

stage.

mean standard error in triplicate.

produced.

For up to 40 min, dough with lipoxygenase expanded rapidly after the start of fermentation, but after this time the expansion tended to decrease abruptly. In contrast, lipoxygenase-free dough reached its maximum rate of expansion in 30 min from the start of fermentation, and this tended to decrease gradually afterwards. Further, in the dough without lipoxygenase, changes in the rate of expansion per unit time were smaller than in the dough with lipoxygenase. Changes brought about by this phenomenon are quite likely due to the effect that hydroperoxide, which is produced by lipid peroxidation, has on the fermentation

**Figure 2.** Changes in the rate of expansion with the fermentation of dough. Each value represents the

A model system of gluten and linoleic acid was created, and the involvement of the hydroperoxide that was produced in the fermentation of dough was examined. These results are shown in Fig. 3. The rate of dough expansion was affected by hydroperoxide concentration, and in this experiment the rate of expansion reached its maximum at a hydroperoxide concentration of 30-40 mM. Based on these results, the fermentation of dough was influenced by the concentrations of hydroperoxide that were

**Relationship between the rate of expansion and hydroperoxide** 

The effects of the yeast on dough fermentation were also studied. The comparison of the dough with and without lipoxygenase is shown in Fig. 4. Both doughs with ≤ 2.0% yeast had similar rates of expansion that tended to increase with fermentation time. There were almost no changes in the rate of expansion with yeast concentrations of ≥ 2.5%; in fact, the rate of expansion tended to decrease. However, the rate of expansion of dough with lipoxygenase tended to increase more than the rate of the dough without lipoxygenase. Since a detailed study of the relationship between yeast and hydroperoxide produced was not done in this experiment, further study is needed.

Next, changes in the amount of hydroperoxide were studied; these results are shown in Fig. 5. For the dough with lipoxygenase, the amount of hydroperoxide reached its maximum when the yeast concentration was 1%; as the yeast concentration increased, the amount of hydroperoxide that was produced tended to decrease. Comparing these results with those in Fig. 4 indicates that there is a relationship between the amount of hydroperoxide produced and the yeast concentration; the specifics of this relationship need to be further investigated. Lipoxygenase-free dough produced almost no hydroperoxide. However, based on the results in Fig. 4, the production of hydroperoxide may not be the sole factor involved in the fermentation of dough. Thus, the hydroperoxide that is produced may be synergistically involved in the mechanism of yeast fermentation.

**Figure 4.** Effect of yeast contents on the rate of dough expansion. Each value represents the mean standard error in triplicate.

Next, the effect of differences in gluten content on dough fermentation was studied. The results are shown in Fig. 6. The expansion of dough began at a gluten content of 40%, and the rate of expansion reached its maximum at a gluten content of 60%. Beyond this concentration, the rate of expansion tended to gradually decrease. Dough with lipoxygenase had a rate of expansion of about 35% at a gluten content of 60%, while lipoxygenase-free dough had a rate of expansion of 15%. Thus, the presence of hydroperoxide had an effect on the rate of expansion; the hydroperoxide that was produced promoted fermentation.

#### **The relationship between gluten and hydroperoxide**

Hydroperoxide is involved in the fermentation of dough in a facilitatory manner, and, as a result, the rate of dough expansion is increased. Consequently, this phenomenon has a positive effect on dough. To study the effect of the hydroperoxide that is produced during dough fermentation on gluten, the gluten was separated and purified after the completion of fermentation using affinity chromatography, so that, ultimately, the gluten fraction was obtained. This gluten fraction was subjected to SDS–gel electrophoresis, and the relationship between gluten and hydroperoxide in the fermentation stage was studied; the results are shown in Fig. 7. In the dough without lipoxygenase, there were almost no changes in the molecular weight of gluten during 100 min of fermentation time. In contrast, in the dough with lipoxygenase, changes in the molecular weight of gluten were seen with fermentation, and formation of gluten polymers was noted with fermentation. This phenomenon was caused by hydroperoxide that was produced, which acted on the gluten and may have induced denaturation. A comparison of the results shown in Figure 4 and 5 shows that the gluten network was tightened, because the hydroperoxide that was produced by the addition of lipoxygenase denatured the gluten and, subsequently, increased dough expansion.

234 Lipid Peroxidation

standard error in triplicate.

**Figure 4.** Effect of yeast contents on the rate of dough expansion. Each value represents the mean

the rate of expansion; the hydroperoxide that was produced promoted fermentation.

**The relationship between gluten and hydroperoxide** 

Next, the effect of differences in gluten content on dough fermentation was studied. The results are shown in Fig. 6. The expansion of dough began at a gluten content of 40%, and the rate of expansion reached its maximum at a gluten content of 60%. Beyond this concentration, the rate of expansion tended to gradually decrease. Dough with lipoxygenase had a rate of expansion of about 35% at a gluten content of 60%, while lipoxygenase-free dough had a rate of expansion of 15%. Thus, the presence of hydroperoxide had an effect on

Hydroperoxide is involved in the fermentation of dough in a facilitatory manner, and, as a result, the rate of dough expansion is increased. Consequently, this phenomenon has a positive effect on dough. To study the effect of the hydroperoxide that is produced during dough fermentation on gluten, the gluten was separated and purified after the completion of fermentation using affinity chromatography, so that, ultimately, the gluten fraction was

**Figure 5.** Effect of yeast contents on the amount of hydroperoxide produced in dough. Each value represents the mean standard error in triplicate.

**Figure 6.** Effect of gluten content on the rate of dough expansion. Each value represents the mean standard error in triplicate.

**Figure 7.** Changes in the molecular weight of gluten when dough was fermented for 100 min.

#### **The mechanism by which hydroperoxide acts to promote fermentation**

The various experimental results that were obtained were comprehensively analyzed to determine the mechanism of action by which hydroperoxide acts to promote fermentation; this is shown in Fig. 8. During gluten formation, gluten is formed when gliadin and glutenin form a network structure. When gluten is crosslinked in the presence of hydroperoxide, the molecules themselves form macromolecules. As a result, expansion is promoted by the uptake of large amounts of carbon dioxide gas produced during dough fermentation. This phenomenon is ultimately advantageous when baking dough, and it improves the bread's texture. When very little lipid peroxidation is induced, the unoxidized linoleic acid has no interaction with gluten, and, as a result, gluten crosslinking does not occur, which results in baked bread with a poor texture.

**Figure 8.** The mechanism by which hydroperoxides accelerate fermentation.

### **4. Conclusions**

236 Lipid Peroxidation

standard error in triplicate.

**Figure 6.** Effect of gluten content on the rate of dough expansion. Each value represents the mean

**Figure 7.** Changes in the molecular weight of gluten when dough was fermented for 100 min.

The current research demonstrated that well-fermented dough can be produced by the induction of lipid peroxidation when fermenting dough. The induction of lipid peroxidation was achieved in the current study by using lipoxygenase induction, but a similar phenomenon should also occur with lipid peroxidation induced by other methods. This phenomenon is advantageous when baking bread and can be used to enhance the quality of

baked bread. Based on the results of these tests of physical properties, further detailed study is needed of the effect of lipid peroxidation on the flavor of baked bread.

#### **Author details**

Toshiyuki Toyosaki *Department of Foods and Nutrition, Koran Women's Junior College, Fukuoka, Japan* 

#### **5. References**

