**2. Formation of OxPLs**

OxPLs are generated by the oxidation of polyunsaturated fatty acid residues, which are usually present in the phospholipids at the *sn*-2 position. Oxidation of phospholipids is initiated either enzymatically by lipoxygenases or by reactive oxygen species and propagates *via* the classical mechanism of lipid peroxidation chain reaction. This implies that the production of OxPLs cannot be regulated by adjusting the amount or activity of enzymes. Hence there is a probability of the uncontrolled generation of OxPLs during oxidative stress. Several evidences suggest that OxPLs are formed from Poly Unsaturated Fatty Acids (PUFAs) at the sn-2 position (Bochkov et al 2007, Podrez et al 2002). Bioactive oxidized phospholipids may contain fragmentation products of PUFA, such as 1-palmitoyl-2-oxovaleroyl-*sn*-glycero-3-phosphorylcholine and 9-keto-10-dodecendioic acid ester of 2 lyso-phosphatidyl choline (KOdiA-PC); prostaglandins, such as 15 deoxy-delta 12, 14 prostaglandin I2 (PGI2) and 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-*sn*-glycero-3 phosphoryl choline (PEIPC); and levuglandins. These molecules exhibit different biological activities. Chromatographic separation of many products formed by oxidation of 1 palmitoyl-2-arachidonoyl-*sn*-glycero-3-phosphorylcholine (PAPC) led to the identification of 1-palmitoyl-2-(5-oxovaleroyl)-*sn*-glycero-3-phosphatidylcholine (POVPC), 1-palmitoyl-2 glutaroyl-*sn*-glycero-3-phosphatidylcholine (PGPC) and 1-palmitoyl-2-(5,6-epoxyisopropane E2)-*sn*-glycero-3-phosphatidylcholine (PEIPC) as potent lipid mediators of inflammation. High structural variation may explain why OxPLs demonstrate a remarkable variety of biological activities (FIGURE-1).

Enzymatic and non-enzymatic reactions, free-radical, and radical-free processes are capable of initiating wide spectrum of reactions causing oxidation of PUFAs. Majority of these reactions produce identical primary oxidation products (i.e., peroxyl radicals and hydroperoxides). Subsequent oxidation of OxPLs is an enzyme-independent stochastic process producing a wide spectrum of OxPLs. Peroxidation products thus generated proceeds according to several mechanisms such as oxidation of PUFA residue, cyclization of peroxyl radical or oxidative fragmentation of esterified PUFAs generating either fulllength residues incorporating several oxygen atoms, or shortened fatty acid residues. Introduction of additional oxygen atoms into PUFAs is a common mechanism that increases complexity of OxPLs mixtures however biological activities of poly-oxygenated PLs are still not characterized. On the other hand, cyclization of peroxyl radical produces cyclic peroxide, which undergoes re-arrangements yielding bicyclic endoperoxide, or oxidation introducing additional non-cyclic or cyclic peroxide group. Cyclization of peroxyl radical is only possible for FAs having three or more double bonds (Salomon et al 2005).

**2. Formation of OxPLs** 

biological activities (FIGURE-1).

2005).

(Tsimikas et al 2005).

LDL. Increased levels of OxPLs/apoB are implicated in coronary artery disease, progression of carotid and femoral atherosclerosis and the prediction of cardiovascular events

OxPLs are generated by the oxidation of polyunsaturated fatty acid residues, which are usually present in the phospholipids at the *sn*-2 position. Oxidation of phospholipids is initiated either enzymatically by lipoxygenases or by reactive oxygen species and propagates *via* the classical mechanism of lipid peroxidation chain reaction. This implies that the production of OxPLs cannot be regulated by adjusting the amount or activity of enzymes. Hence there is a probability of the uncontrolled generation of OxPLs during oxidative stress. Several evidences suggest that OxPLs are formed from Poly Unsaturated Fatty Acids (PUFAs) at the sn-2 position (Bochkov et al 2007, Podrez et al 2002). Bioactive oxidized phospholipids may contain fragmentation products of PUFA, such as 1-palmitoyl-2-oxovaleroyl-*sn*-glycero-3-phosphorylcholine and 9-keto-10-dodecendioic acid ester of 2 lyso-phosphatidyl choline (KOdiA-PC); prostaglandins, such as 15 deoxy-delta 12, 14 prostaglandin I2 (PGI2) and 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-*sn*-glycero-3 phosphoryl choline (PEIPC); and levuglandins. These molecules exhibit different biological activities. Chromatographic separation of many products formed by oxidation of 1 palmitoyl-2-arachidonoyl-*sn*-glycero-3-phosphorylcholine (PAPC) led to the identification of 1-palmitoyl-2-(5-oxovaleroyl)-*sn*-glycero-3-phosphatidylcholine (POVPC), 1-palmitoyl-2 glutaroyl-*sn*-glycero-3-phosphatidylcholine (PGPC) and 1-palmitoyl-2-(5,6-epoxyisopropane E2)-*sn*-glycero-3-phosphatidylcholine (PEIPC) as potent lipid mediators of inflammation. High structural variation may explain why OxPLs demonstrate a remarkable variety of

Enzymatic and non-enzymatic reactions, free-radical, and radical-free processes are capable of initiating wide spectrum of reactions causing oxidation of PUFAs. Majority of these reactions produce identical primary oxidation products (i.e., peroxyl radicals and hydroperoxides). Subsequent oxidation of OxPLs is an enzyme-independent stochastic process producing a wide spectrum of OxPLs. Peroxidation products thus generated proceeds according to several mechanisms such as oxidation of PUFA residue, cyclization of peroxyl radical or oxidative fragmentation of esterified PUFAs generating either fulllength residues incorporating several oxygen atoms, or shortened fatty acid residues. Introduction of additional oxygen atoms into PUFAs is a common mechanism that increases complexity of OxPLs mixtures however biological activities of poly-oxygenated PLs are still not characterized. On the other hand, cyclization of peroxyl radical produces cyclic peroxide, which undergoes re-arrangements yielding bicyclic endoperoxide, or oxidation introducing additional non-cyclic or cyclic peroxide group. Cyclization of peroxyl radical is only possible for FAs having three or more double bonds (Salomon et al

**Figure 1.** Representative chemical structures of oxidized phospholipids formed during oxidation of PAPC.

#### **2.1. Oxidative cleavage and generation of fragmented OxPLs species**

Peroxides/ peroxyls are transformed into advanced oxidation products by fragmentation of hydroperoxides. γ-Hydroxy (or oxo) a,b-unsaturated PLs with terminal aldehyde groups are produced from hydroperoxides via oxidation/fragmentation or polymerization/cleavage. Oxidative fragmentation of hydroperoxides occurs via several mechanisms including bscission, Hock rearrangement, or cyclization of alkoxy radical produced from hydroperoxide (Gugiu et al 2006). γ-Hydroxy (or oxo)-α,β-unsaturated aldehyde PLs are highly reactive compounds, that are able to covalently link to amino groups of proteins, as well as thiol groups of biomolecules (Hoff et al 2003). On the other hand, peroxyl radical can cross-react with double bonds present in hydroperoxides yielding peroxydimers, these are unstable products and spontaneously break down forming either new radicals or α,βunsaturated aldehydes (Schneider et al 2008). In addition to these products, saturated fragmented species containing terminal carbonyl groups are produced by oxidative fragmentation of PUFA-PLs, most common amongst which are oxononanoate and azelaoate

formed from linoleic acid, oxovaleroate, and glutaroate generated from arachidonic acid, or oxobutyrate and succinate produced from docosahexaenoic acid (Gu et al 2003, Podrez et al 2002). Saturated fragmented OxPLs can be formed by further oxidation of γ-hydroxy (or oxo)-α, β-unsaturated PLs in addition to direct formation from hydroperoxides, (Podrez et al 2002). Saturated fragmented OxPLs l lack double bonds and hence they are resistant to further oxidation as the absence of double bonds within fragmented chains results in reduced reactivity of aldehyde containing saturated OxPLs as compared to α,β-unsaturated fragmented OxPLs.

#### **2.2. Non-enzymatic oxidation of PL-PUFAs**

This process is initiated by free radicals or non-radical reactive oxygen species (ROS). Free radical-mediated chain reaction is initiated by the formation of carbon-centered radicals and/or hydroperoxides of PUFAs (peroxidation of PUFAs). Due to the presence of methylene groups located between double bonds (bisallylic methylene groups), PUFAs are more susceptible to oxidation as compared to saturated FAs. As a result they are characterized by weakened hydrogen-carbon bonds. Free radicals can abstract hydrogen from bisallylic methylene leading to the formation of carbon-centered radicals within PUFAs. Now occurs the initiation step of lipid peroxidation, Carbon-centered radicals rapidly react with molecular oxygen, producing peroxyl radicals. These Peroxyl radicals react with bisallylic methylene groups in other PUFA molecules, leading to the transformation of peroxyl radicals to hydroperoxides and generation of new carboncentered radicals. Thus, additional cycles of peroxidation are initiated. PUFA hydroperoxides in turn produce reactive alkoxyl and hydroxyl radicals via iron or coppercatalyzed Fenton-like reactions, further propagating the chain reaction (Bochkov et al 2010).

#### **2.3. Enzymatic oxidation of PL-PUFAs**

1, 4-pentadiene motifs are recognized within unsaturated fatty acids by lipoxygenases (LOXs) and molecular oxygen with high stereoselectivity is introduced. The majority of lipoxygenases oxidize only unesterified PUFAs. Only one group (12/15-LOX) amongst all known LOXs is capable of oxidizing PL-esterified fatty acids. This class of enzymes is present in different biological species and includes mouse, rat, rabbit, bovine, and porcine leukocytetype 12- LOX, rabbit and human reticulocyte-type 15-LOX, and soybean LOX (Huang et al 2008, Wittwer et al 2007). Switching of activity of electron transport in mitochondria to peroxidation by cytochrome c (cyt c) has been suggested by Kagan et al (2005). This transformation begins when cyt c binds to negatively charged cardiolipin (CL), leading to conformational changes and subsequent release of PL-protein complex from mitochondria into cytosol. The complex of cyt c with CL activated by traces of PUFA-OOH or H2O2 acquires the ability to oxidize CL, PS, or PI, with formation of PL-OOH (Kagan et al 2009).

Alternatively, OxPLs are also generated by re-esterification of free oxidized PUFAs into lyso-PLs. Several types of OxPLs have been found to be generated by this mechanism both *in vivo* and *in vitro* (Arai et al 1997, Birkle et al 1984).

#### **2.4. Detoxification of reactive OxPLs**

412 Lipoproteins – Role in Health and Diseases

fragmented OxPLs.

**2.2. Non-enzymatic oxidation of PL-PUFAs** 

**2.3. Enzymatic oxidation of PL-PUFAs** 

*in vivo* and *in vitro* (Arai et al 1997, Birkle et al 1984).

formed from linoleic acid, oxovaleroate, and glutaroate generated from arachidonic acid, or oxobutyrate and succinate produced from docosahexaenoic acid (Gu et al 2003, Podrez et al 2002). Saturated fragmented OxPLs can be formed by further oxidation of γ-hydroxy (or oxo)-α, β-unsaturated PLs in addition to direct formation from hydroperoxides, (Podrez et al 2002). Saturated fragmented OxPLs l lack double bonds and hence they are resistant to further oxidation as the absence of double bonds within fragmented chains results in reduced reactivity of aldehyde containing saturated OxPLs as compared to α,β-unsaturated

This process is initiated by free radicals or non-radical reactive oxygen species (ROS). Free radical-mediated chain reaction is initiated by the formation of carbon-centered radicals and/or hydroperoxides of PUFAs (peroxidation of PUFAs). Due to the presence of methylene groups located between double bonds (bisallylic methylene groups), PUFAs are more susceptible to oxidation as compared to saturated FAs. As a result they are characterized by weakened hydrogen-carbon bonds. Free radicals can abstract hydrogen from bisallylic methylene leading to the formation of carbon-centered radicals within PUFAs. Now occurs the initiation step of lipid peroxidation, Carbon-centered radicals rapidly react with molecular oxygen, producing peroxyl radicals. These Peroxyl radicals react with bisallylic methylene groups in other PUFA molecules, leading to the transformation of peroxyl radicals to hydroperoxides and generation of new carboncentered radicals. Thus, additional cycles of peroxidation are initiated. PUFA hydroperoxides in turn produce reactive alkoxyl and hydroxyl radicals via iron or coppercatalyzed Fenton-like reactions, further propagating the chain reaction (Bochkov et al 2010).

1, 4-pentadiene motifs are recognized within unsaturated fatty acids by lipoxygenases (LOXs) and molecular oxygen with high stereoselectivity is introduced. The majority of lipoxygenases oxidize only unesterified PUFAs. Only one group (12/15-LOX) amongst all known LOXs is capable of oxidizing PL-esterified fatty acids. This class of enzymes is present in different biological species and includes mouse, rat, rabbit, bovine, and porcine leukocytetype 12- LOX, rabbit and human reticulocyte-type 15-LOX, and soybean LOX (Huang et al 2008, Wittwer et al 2007). Switching of activity of electron transport in mitochondria to peroxidation by cytochrome c (cyt c) has been suggested by Kagan et al (2005). This transformation begins when cyt c binds to negatively charged cardiolipin (CL), leading to conformational changes and subsequent release of PL-protein complex from mitochondria into cytosol. The complex of cyt c with CL activated by traces of PUFA-OOH or H2O2 acquires the ability to oxidize CL, PS, or PI, with formation of PL-OOH (Kagan et al 2009).

Alternatively, OxPLs are also generated by re-esterification of free oxidized PUFAs into lyso-PLs. Several types of OxPLs have been found to be generated by this mechanism both Detoxification of OxPLs comprises the mechanisms that terminate peroxidation chain reaction and inactivate chemically reactive toxic groups produced by oxidation. Hydroxides are characterized by significantly lower chemical reactivity and therefore are considered to be stable and non-toxic compared to hydroperoxides (Spiteller et al 1997). Most commonly, the enzyme catalyzing the reduction of hydroperoxides to hydroxides is glutathione peroxidase (GPx). Lipid hydroperoxides are reduced in a reaction that involve selenocysteine residue of GPx and glutathione thus generating lipid hydroxide and oxidized glutathione. With respect to membrane-bound hydroperoxides of PL esterified PUFAs, PL glutathione peroxidase (GPx4) has the highest activity amongst GPx enzymes (Savaskan et al 2007).

A variety of products containing aldehyde and keto functional groups are formed upon oxidation of OxPLs which are further reduced by aldo-keto reductases to respective hydroxyl groups. Apart from playing physiological role in metabolism of sugar aldehydes, aldo-keto reductases also play a role in detoxification of toxic phospholipid aldehydes (Jin et al 2007).

Another aspect of detoxification is OxPLs cleavage. Platelet activating factor acetylhydrolase (PAF-AH) has been recognized for its ability to cleave and thus inactivate PAF (McIntyre et al 2009).The enzyme was shown to hydrolyze fragmented saturated OxPLs (Stremler et al 1991), as well as long-chain OxPLs, including esterified F2-isoprostanes, PC-hydroperoxides and PEIPC (Kriska et al 2007, Davis et al 2008).
