Critical Review. Reactivity of Peroxynitrite and Nitric Oxide with LDL

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1 IUBMB Life, 57(6): , June 2005 Critical Review Reactivity of Peroxynitrite and Nitric Oxide with LDL Horacio Botti, Andre s Trostchansky, Carlos Batthya ny and Homero Rubbo Departamento de Bioquı mica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la Repu blica, Montevideo, Uruguay. Summary Low density lipoprotein (LDL) oxidation by peroxynitrite is a complex process, finely modulated by control of peroxynitrite formation, LDL availability and free-radical scavenging by nitric oxide ( NO), ascorbate and a-tocopherol (a-toh). In the presence of CO 2, lipid targets are spared at the expense of surface constituents. Since surface damage may lead to oxidation-induced LDL aggregation and particle recognition by scavenger receptors, CO 2 cannot be considered an inhibitor of peroxynitrite-dependent LDL modifications. Chromanols, urate and ascorbate cannot scavenge peroxynitrite in the vasculature, although intermediates of urate oxidation and high ascorbate concentrations may do so in vitro. Most if not all of the protection against peroxynitrite-induced LDL oxidation afforded by urate, ascorbate, chromanols and also NO should be considered to depend on their free radical scavenging abilities, including inactivation of lipid peroxyl radicals (LOO), NO 2, and CO 3 - ; as well as their capacity to reduce high oxidation states of metal centers. Peroxynitrite direct interception by reduced manganese (II) porphyrins is possibly the most powerful although unspecific strategy to inhibit peroxynitrite reactions. In light of the recent demonstration of nitrated bioactive lipids in vivo, renewed interest in the mechanisms of peroxynitrite- and nitric oxidemediated lipid nitration and nitrosation is guaranteed. IUBMB Life, 57: , 2005 Keywords Low density lipoprotein; peroxynitrite; nitric oxide. INTRODUCTION Low density lipoprotein (LDL) oxidation has been extensively studied since the inspiring reports from Steinberg and Chisolm that related LDL oxidative modifications with increased particle atherogenicity (1). The number of annual publications on LDL oxidation has already reached saturation, indicating the need for data consolidation and the Received 1 February 2005; accepted 28 February 2005 Address correspondence to: Homero Rubbo, Departamento de Bioquı mica, Facultad de Medicina, General Flores 2125, Montevideo, Uruguay Tel: Fax: hrubbo@fmed.edu.uy emergence of new questions and approaches. Among other questions, authors have asked about: (i) the identity and sources of oxidants; (ii) the role of LDL endogenous and water-soluble reductants; (iii) the relevance of diffusion and compartmentalization; and (iv) the kinetic and chemical mechanism of reaction. Beckman and colleagues (2) were the first to note that peroxynitrite, 1 the product of the reaction between nitric oxide ( NO) and superoxide anion (O 2 - ) radicals, can be an important mediator of vascular oxidative damage. In fact, peroxynitrite is a powerful oxidant for LDL that renders modified LDL with increased atherogenic potential (3, 4). This review focuses on peroxynitrite and NO interactions in LDL oxidation. In addition, comparisons with related biochemical oxidants and possibly in vivo relevant modulatory mechanisms will also be discussed. PEROXYNITRITE FORMATION: A LIKELY STEP IN LDL OXIDATION Superoxide anion is almost certainly involved as a starting point of complex metabolic and signaling networks leading to LDL oxidation in vivo. While resting endothelium is a poor source of O 2 -, increased LDL oxidation at the arterial intima may result from different processes, including vascular cell and leukocyte activation leading to NAD(P)H oxidases assembly, increased xanthine oxidase activity, mitochondrial dysfunction and/or endothelial NOS uncoupling (5). Superoxide anion is itself a selective oxidant, and LDL constituents are not chemically susceptible to oxidation by O 2 -. In addition, due to its hydrophobic character and low permeability to ions, most LDL components are physically not available for O 2 -. Therefore, O 2 - is not reactive enough toward LDL and needs to be activated before LDL oxidation takes place. Superoxide anion-dependent LDL oxidation can be brought about in different ways, but more likely by near diffusion-limited recombination of O 2 - with NO to yield peroxynitrite, or by its one-electron reduction through uncatalyzed or superoxide dismutase (SOD)-catalyzed pathways leading to hydrogen ISSN print/issn online ª 2005 IUBMB DOI: /

2 408 BOTTI ET AL. peroxide (H 2 O 2 ) formation. An increase of protein 3-nitrotyrosine has been found in atherosclerotic tissues and has been previously considered as a specific footprint of peroxynitrite formation and reactivity during atherogenesis (6). However, peroxynitrite formation is theoretically unnecessary to explain biological nitration because this reaction can also be performed in vitro by H 2 O 2 /nitrite anion/peroxidase systems. The colocalization of nitrated proteins with peroxidasespecific epitopes and protein modifications (like 3-chlorotyrisine) does not discard peroxynitrite participation in protein nitration in vivo as peroxynitrite can induce peroxidasemediated oxidations. Moreover, peroxynitrite remains the most likely player in 3-nitro-tyrosine formation in the absence of active peroxidases (7). Clearly these criteria do not suffice to assert or reject peroxynitrite involvement in any biological process, and so at this moment it must be considered that a rich milieu of oxidizing, nitrating and chlorinating species is present in inflammatory cardiovascular diseases. MULTIPLE TARGETS AND PATHWAYS OF PEROXYNITRITE-INDUCED LDL OXIDATION LDL is composed of lipids (78% of mass) and a single polypeptide chain of 4536 residues, namely apoprotein B-100 (apo B-100). Most of the protein (8) and a monolayer of amphipathic lipids (mainly phosphatidyl cholines and cholesterol, *33 and *9% of lipid mass, respectively) are arranged in the particle surface. A hydrophobic particle core (*50% of particle volume) structured on triacylglicerides (TAG) and cholesterol esters has been classically described, although this view has been recently challenged (9). The concentration of unsaturated lipid moieties in LDL is *0.9 M. LDL carries lipophilic reductants as minor components, in particular a- tocopherol (a-toh), the most abundant (*1 mm). It has been proposed that in contrast to bulk LDL constituents, chromanols and carotenoids are selectively oxidized by peroxynitrite (10, 11). In contrast, we have found evidence supporting the fact that lipid peroxidation mediates the consumption of these reductants during peroxynitrite-induced LDL oxidation, briefly: (i) Nitric oxide reacts very slowly with peroxynitrite, but potently inhibits peroxynitrite flux-dependent a-toh and lipid oxidation in LDL; in fact kinetic analysis by computer-assisted simulations support the fact that the main mechanism of NO inhibition of a-toh oxidation in LDL is its reaction with lipid peroxyl radicals; (ii) low NO fluxes and ascorbate concentrations additively inhibit a-toh oxidation in agreement with one-electron sequential oxidation of a-toh to a-tocopheryl quinone in LDL; (iii) the yield of oxidation of a-toh in LDL is only fractional with respect to peroxynitrite concentration and its dependence on LDL concentration support that peroxynitriteinduced a-toh in LDL is indirect and therefore free radical mediated; (iv) Trolox C, a water soluble chromanol, does not react directly with peroxynitrite; moreover, to our knowledge there is no well-evidenced direct one electron oxidation of any organic molecule by peroxynitrite (12, 13). Peroxynitrite chemistry involves a large array of reactive species and pathways. Peroxynitrous acid (ONOOH) is a strong electrophile that can directly react with suitable protein groups through bimolecular processes. These reactions are in most cases concerted (two electron) oxidations. Reduced cysteinyl, methionyl and tryptophanyl residues represent the most likely direct targets of peroxynitrite in apoproteins (14), including apo B-100. Peroxynitrite can induce lipid oxidation (15), but kinetics and mechanism of this process are only partially defined. Radicals formed after peroxynitrous acid (ONOOH) homolysis (nitrogen dioxide, NO 2 ; and hydroxyl radical, OH) can certainly be involved in lipid oxidation initiation in vitro, nevertheless, the homolytic step of the reaction is possibly too slow to be relevant in vivo (k = 0.9s 71, ph 7.4, 378C). Contrarily, peroxynitrite anion (ONOO - ) rapidly reacts with CO 2 (k * M 71 s 71 ) to give NO 2 and carbonate radical anion (CO - 3 ) in 35 40% yield. Alternatively, it has been shown that ONOOH rapidly (k * 2x10 7 M 71 s 71 (16)) oxidizes resting (ferric) myeloperoxidase (MPO) to apparently yield MPO compound II and NO 2 (Figure 1). The question concerning which of these oxidative pathways (direct, CO 2 - and metalloprotein-dependent) may be more relevant for peroxynitrite-mediated LDL oxidation in vivo is presently unresolved. The calculated rate constant for the direct reaction of peroxynitrite with LDL on the basis of apo B-100 amino acid composition gives *5 x10 4 M 71 s 71 at ph 7.4 and 378C which represents a lower estimate of the second order rate constant of peroxynitrite reaction with LDL (k LDL ). Peroxynitrite-mediated a-toh oxidation depends on free radical generation from peroxynitrous acid (discussed later on) and the apparent yield diminishes in vitro if LDL concentration is increased beyond 2 mm, halving at 8 mm LDL at ph 7.4 and 258C (12), then assuming simple competition between peroxynitrite decomposition (leading to a-toh oxidation) and direct reaction with LDL, we conclude that k LDL is *1 x10 5 M 71 s 71 at ph 7.4 and 378C, only twice the calculated one, suggesting that apo B-100 may be the preferred direct target for peroxynitrite in LDL. Although, peroxynitrite reaction with CO 2 will be preferred in vivo (k app *1.3 mm x 4.6 x 10 4 M 71 s 71 =60 s -1 ), it is not clear if this pathway will enhance or inhibit apo B-100 oxidation in complex biological media like plasma or the arterial intima. Finally, MPO can be present in high local concentrations in the inflamed arterial wall, and may be able to catalyze peroxynitrite-mediated oxidations. MECHANISMS OF PEROXYNITRITE-INDUCED OXIDATION OF a-tocopherol AND UNSATURATED FATTY ACIDS IN LDL The kinetics of lipid oxidation in homogeneous phases differs from those of lipid oxidation in LDL. Penetration of

3 NITRIC OXIDE, PEROXYNITRITE AND LDL INTERACTIONS 409 Figure 1. Overview of peroxynitrite and nitric oxide reactivity in LDL. Nitric oxide and O 2 - combine to form peroxynitrite anion which is in equilibrium with its conjugated acid and rapidly reacts with CO 2 to yield NO 2 and CO 3 - free radicals, which can lead to apo B-100 tyrosine nitration (pathway A). Peroxynitrous acid may engage in MPO-mediated LDL oxidation, which may primarily result in lipid peroxidation and its associated secondary damage to LDL lipid and protein reductants (pathway B). Peroxynitrous acid can also directly react with LDL with a moderately high apparent overall rate constant at high pathological LDL concentrations, particularly with apo B-100 nucleophiles i.e., thiols (pathway C). Although ONOOH can homolyze to NO 2 and OH radicals, this reaction is possibly too slow to be relevant in vivo. Nitric oxide and possibly NO 2 can rapidly diffuse and equilibrate between compartments. Lipid peroxidation is exquisitely sensitive to inhibition by NO, which mainly depends on NO reaction with LOO to form unstable organic peroxynitrites. Manganese (II) porphyrins can act as catalytic antioxidants against peroxynitrite, O 2 - and CO 3 -. the particle is a key step for LDL lipid oxidation initiation but not for lipid oxidation in solution (17). Knowledge of the kinetic and chemical reaction mechanisms by which endogenous reductants are oxidized by peroxynitrite and its derived reactive species, is critical to understanding their role in LDL oxidation. In this direction, an important task is to establish if any of them can directly and efficiently scavenge peroxynitrite in vitro and in vivo. The mechanism of reaction of peroxynitrite with chromanols (a-toh, g-toh and Trolox C, a water soluble analogue of a-toh) oxidation has been controversial. Initial work showed near quantitative formation of the quinone forms and low yields of chromanoxyl radicals from the reaction of peroxynitrite with a-toh and Trolox C, suggesting a predominant bimolecular concerted mechanism of oxidation (18). One report supported a rapid one-electron direct oxidation of Trolox C and Trolox C radical by peroxynitrite (19). Therefore, we developed a detailed kinetic study of the reaction, and showed that Trolox C oxidation is entirely dependent on peroxynitrite peroxo bond homolysis, either in the absence or presence of CO 2, implying that chromanols are not direct peroxynitrite scavengers (13). Unlike g-toh oxidation, two-electron sequential oxidation of fully substituted chromanols may not involve chromanoxyl radical reaction with NO 2, since no ring nitration product is observed and the reaction is at least partially reversible (13). Peroxynitrite is a weak electrolyte (pk a = 6.8), and its protonated form can transverse biomembranes by simple diffusion (14). Peroxynitrite reaction with most biomolecules are activation-controlled, having k M 71 s 71. Peroxynitrous acid partitioning into a lipid milieu has not been studied, but may be low and comparable to that of water. Therefore, we anticipate that first (i.e., peroxo bond homolysis) and second order reactions of ONOOH in highly hydrophobic environments will be disfavored and that water borne peroxynitrite-derived oxidants may be responsible for lipid oxidation initiation instead (12). Due to its high reactivity, it is expected that peroxynitritederived OH-mediated oxidation will be in many aspects similar to ionizing radiation-induced damage, that is: OH (the primary radical) generates random surface damage and

4 410 BOTTI ET AL. associated secondary radicals, which then causes more selective secondary damage and third generation radicals. In fact, OH deriving from peroxynitrite may cause extensive polypeptide scission which is enhanced by lipid peroxidation chain reactions (12). Alternatively, it is expected that NO 2 and CO - 3 will inflict more selective and distinctive damage than OH. Nitrogen dioxide is a small-uncharged radical, which can diffuse into LDL and cause lipid oxidation. Carbonate anion radical is a strong acid and negatively charged molecule restricted to aqueous environments. In this regard, it has been shown that while inhibiting LDL lipid oxidation, CO 2 enhances LDL tyrosyl residue nitration (12, 20), meaning that in the presence of CO 2 oxidative damage is redirected to oxidation and nitration of susceptible surface targets. PREVENTION OF PEROXYNITRITE-INDUCED LDL OXIDATION Prevention of peroxynitrite formation Natural defenses against peroxynitrite-mediated oxidative damage are arranged in many levels, including prevention, direct scavenging and repair. Efficient inhibition of peroxynitrite formation may be achieved by control of - NO and O 2 formation. It is widely accepted that SOD inhibits peroxynitrite formation in vivo. At a given flux of O - 2, high NO and low SOD local concentrations will favor peroxynitrite formation and vice versa. However, SOD may not inhibit peroxynitrite formation if constant fluxes of - NO and O 2 are generated in vitro in the absence of efficient NOconsuming first order processes (21), meaning that lowering of NO half-life complement SOD actions in vivo. Prevention of peroxynitrite reaction with LDL targets (a) Urate. Prevention of LDL oxidation can be achieved by peroxynitrite scavenging by natural as well as synthetic antioxidants. Urate, present in plasma at relatively high concentrations ( mm) has been previously proposed as a peroxynitrite scavenger (22). Nevertheless, reports have been contrasting. A low phindependent overall reaction rate constant between peroxynitrite and urate has been reported, with k = M -1 s 71, giving an apparent first order rate constant of reaction in plasma of 0.05 s 71, just too low to be biologically significant (23, 24). Recently, it has been suggested that urate-dependent acceleration of peroxynitrite decay might be due to peroxynitrite reaction with dehydrourate, which in turn results from the two-electron sequential free radical oxidation of urate (25). This evidence supports that urate is not a direct, but rather a peroxynitrite, derived radical scavenger. (b) Ascorbate. Two independent reports on the mechanism of peroxynitrite-mediated ascorbate oxidation demonstrated that peroxynitrite directly reacts with ascorbate, although a rather low bimolecular rate constant was reported (k = 235 M 71 s 71, 258C, ph 7.4) (26, 27). Nevertheless, the kinetic reaction mechanism has been recently reinvestigated (28). It has been reported that ascorbate rapidly reacts with ONOOH at high ascorbate concentrations through a multiple step mechanism that involves the initial formation of an unstable complex (k a = 1.5 x 10 6 M 71 s 71 and k d =1 x 10 3 s 71 ) and its subsequent reaction with excess ascorbate, yielding ascorbate, dehydroascorbate and NO - 2 (28). At low ascorbate concentrations, a slow isomerization of peroxynitrite to nitrate (NO - 3 ) is proposed to take place via this intermediate (28). As previously discussed (27), ascorbate cannot be considered an efficient peroxynitrite scavenger but is an efficient electro reductant in vascular extracellular fluids. (c) Manganese porphyrins. These compounds represent - good O 2 and excellent peroxynitrite scavengers in vitro. It has been reported that the limiting step for these compounds to act as catalytic antioxidants is the regeneration of manganic (Mn 3+ ) and manganous (Mn 2+ ) forms from higher oxidation states of the complex, therefore suitable reductants like urate in plasma are needed (29). Efficient scavenging of O - 2 by Mn-porphyrins is difficult if SOD is already present. However, by attenuating peroxynitrite formation, SOD may facilitate the catalytic Mn-porphyrin-mediated peroxynitrite scavenging. The ubiquitous presence in biological media of highly concentrated CO 2 competes with peroxynitrite interception by Mn-porphyrins and any other possible scavenger. Importantly, it has been recently demonstrated that some Mn-porphyrins in - their manganous state can also rapidly scavenge CO 3 (k*1 x10 9 M -1 s -1 )(30), and so, like natural antioxidant defenses, Mn-porphyrins can act at many levels to prevent peroxynitrite-mediated oxidative damage. Possible limitations of peroxynitrite interception by Mnporphyrins may be the formation of NO 2 and high oxidation states of the Mn-porphyrins, which may lead to increased LDL oxidation and nitration in the absence of coantioxidants (31). In addition, since MPO rapidly reacts with peroxynitrite and specifically associates with anionic moieties in proteins its presence may preclude Mn-porphyrin-dependent protection against peroxynitrite-mediated oxidation and nitration. (d) a-toh and NO. a-tocopherol plus ascorbate and a- TOH plus NO pairs have synergistic protective actions against lipid oxidation (32, 33). In addition NO and ascorbate exert additive protection towards peroxynitrite-induced a-toh oxidation in LDL (12). Despite these interactions, the role of a-toh in peroxynitriteinduced LDL oxidation in the absence of ascorbate has been debated. Since a-toh oxidation depends on peroxynitrite homolysis (12, 13), and not on direct

5 NITRIC OXIDE, PEROXYNITRITE AND LDL INTERACTIONS 411 oxidation by peroxynitrite, chromanols should not be considered peroxynitrite scavengers. Since peroxynitrite-induced a-toh oxidation is a free radical process, the proposed peroxynitrite-dependent tocopherolmediated peroxidation (TMP) of LDL (34) is at least chemically possible. Tocopherol-mediated peroxidation of LDL lipids has been proposed on the basis of an enhanced lipid hydro(pero)xide (LO(O)H) accumulation in LDL particles with increased a-toh content after peroxynitrite treatment (34). However, lipid oxidation cannot be thoroughly analyzed using any single marker. In this particular case, measured oxidation products accounted for less than 20% of a-toh consumption (34). These results can be reinterpreted on the basis of a competition between LOO reaction with a-toh and tocopheroxyl radical to yield LOOH and LOH versus LOO radical termination reactions with a second lipid radical or peroxynitrite-derived NO 2. Therefore, we expect that in LDL (*1mM) exposed to bolus addition of peroxynitrite ( 5 200mM), an increase in a-toh concentration will result in higher yields of LO(O)H as well as lower yields of other lipid oxidation products. The chain breaking antioxidant action of a- TOH requires low rates of LOO radical formation, which minimize LOO recombination, as obtained with exposure to low fluxes (0.5 5 mmmin -1 ) of peroxynitrite. Moreover, it has been shown that TMP strongly depends on diffusion properties of initiator radicals in lipid milieus (17). In this respect, we estimate that the diffusion coefficient of NO 2 and OH in LDL will be orders of magnitude greater than that of charged and bulky azo compound-derived radicals. The scarce LDL lipid oxidation observed in the presence of physiological concentrations of CO 2 (in the absence of ascorbate and NO) may implicate CO 3 initiated TMP, although this remains to be demonstrated. Peroxynitrite can induce initiation and sustain propagation of lipid oxidation (35). On the contrary, NO is a powerful antioxidant through it s almost diffusion limited termination reactions with lipid peroxyl radicals (LOO ) (36), yielding nitrogenated lipids (37). The small volume and low polarity of NO minimizes solvatation and favors its diffusion and solubility in hydrophobic environments. The partition coefficient (K P, relative solubility between lipid phase and water) of NO in LDL and liposomes has been recently determined (K P * 4) (38). If average steady state concentrations of NO (*100 nm) and LDL (2 mm) in the vascular system as well as LDL volume (4 x L) are considered, it can be calculated that one particle over 1000 will contain one molecule of NO at any time and that only *2% of NO will be directly available inside LDL particles. This implies that in contrary to a-toh, NO is not compartmentalized into LDL. Therefore, NO antioxidant efficiency in LDL can only be explained on the basis of exceptional transport properties of the system. In fact, it has been determined that diffusion of NO is slightly hindered in biomembranes but to a minimal extent in LDL, with a ratio of observed diffusion constants in LDL and in buffer D LDL /D B of * 0.5 (39). The reported facile diffusion of NO in LDL surface will certainly make LDL core lipids readily available for NO (39). Nitric oxide can spare a-toh and ascorbate exposed to peroxynitrite fluxes in vitro (12). This is explained by the rather low reaction rate constants of LOO and NO 2 with LDL targets like a-toh and unsaturated lipids (k a-toh = M 71 s 71, and ascorbate, respectively, in comparison with NO-dependent LOO and NO 2 trapping (12, 33). In LDL, the primary action of NO is to react with lipid free radicals (12). Nevertheless, NO reactions with oxidizing lipids are complex. In fact, it has been reported that the organic alkyl peroxynitrites formed from NO reaction with LOO are unstable and that 2 3 NO molecules may be needed to inactivate one LOO (40). In addition, NO 2 scavenging by NO in aqueous and lipid environments can also take place during peroxynitrite-induced LDL oxidation. This pathway may be relevant only at high and possibly pathological NO concentrations. In addition, nitrated lipids are now demonstrated to occur in vivo (41) but the role that NO and/or NO-derived reactive species play in its formation remains to be defined. SOME PRACTICAL ISSUES CONCERNING EXPERIMENTATION WITH LDL AND PEROXYNITRITE Synthetic peroxynitrite can be handled as an external reactant to enhance controlled experimentation. This approach allows avoidance of the complexity of independently generating NO and O 2 - fluxes, the possible interferences of enzymes and substrates and the uncertainties inherent to the use of SIN-1 (3-morpholinosyndnomine) (12). The approaches used for introducing peroxynitrite into reaction systems is critical for modeling biological conditions, since free radical termination reactions involving peroxynitrite-derived radicals as well as LDL secondary radicals may predominate if peroxynitrite is added as a bolus, but continued infusion of peroxynitrite at low rates allows free radical chain reactions to occur (35). It is also of paramount importance to study a wide range of target concentrations, since at low target concentrations it may be difficult to distinguish direct oxidations from peroxynitrite homolysis-dependent processes and, on the contrary high target concentrations may out-compete homolysis (12). ACKNOWLEDGEMENTS This work was supported by grants from Programa Desarrollo Tecnologico, Dinacyt and PEDECIBA, Uruguay, FOGARTY-NIH, Wellcome Trust and the Guggenheim Foundation.

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