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• W2076241437 abstract "A key early event in the development of atherosclerosis is the oxidation of low density lipoprotein (LDL) via different mechanisms including free radical reactions with both protein and lipid components. Nitric oxide (⋅NO) is capable of inhibiting LDL oxidation by scavenging radical species involved in oxidative chain propagation reactions. Herein, the diffusion of⋅NO into LDL is studied by fluorescence quenching of pyrene derivatives. Selected probes 1-(pyrenyl)methyltrimethylammonium (PMTMA) and 1-(pyrenyl)-methyl-3-(9-octadecenoyloxy)-22,23-bisnor-5-cholenate (PMChO) were chosen so that they could be incorporated at different depths of the LDL particle. Indeed, PMTMA and PMChO were located in the surface and core of LDL, respectively, as indicated by changes in fluorescence spectra, fluorescence quenching studies with water-soluble quenchers and the lifetime values (τo) of the excited probes. The apparent second order rate quenching constants of ⋅NO (kNO) for both probes were 2.6–3.8 × 1010m−1 s−1 and 1.2 × 1010m−1s−1 in solution and native LDL, respectively, indicating that there is no significant barrier to the diffusion of ⋅NO to the surface and core of LDL. Nitric oxide was also capable of diffusing through oxidized LDL. Considering the preferential partitioning of⋅NO in apolar milieu (6–8 for n-octanol:water) and therefore a larger ⋅NO concentration in LDL with respect to the aqueous phase, a corrected kNO value of ∼0.2 × 1010m−1s−1 can be determined, which still is sufficiently large and consistent with a facile diffusion of ⋅NO through LDL. Applying the Einstein-Smoluchowsky treatment, the apparent diffusion coefficient (D′NO) of⋅NO in native LDL is on average 2 × 10−5cm2 s−1, six times larger than that previously reported for erythrocyte plasma membrane. Thus, our observations support that ⋅NO readily traverses the LDL surface accessing the hydrophobic lipid core of the particle and affirm a role for ⋅NO as a major lipophilic antioxidant in LDL. A key early event in the development of atherosclerosis is the oxidation of low density lipoprotein (LDL) via different mechanisms including free radical reactions with both protein and lipid components. Nitric oxide (⋅NO) is capable of inhibiting LDL oxidation by scavenging radical species involved in oxidative chain propagation reactions. Herein, the diffusion of⋅NO into LDL is studied by fluorescence quenching of pyrene derivatives. Selected probes 1-(pyrenyl)methyltrimethylammonium (PMTMA) and 1-(pyrenyl)-methyl-3-(9-octadecenoyloxy)-22,23-bisnor-5-cholenate (PMChO) were chosen so that they could be incorporated at different depths of the LDL particle. Indeed, PMTMA and PMChO were located in the surface and core of LDL, respectively, as indicated by changes in fluorescence spectra, fluorescence quenching studies with water-soluble quenchers and the lifetime values (τo) of the excited probes. The apparent second order rate quenching constants of ⋅NO (kNO) for both probes were 2.6–3.8 × 1010m−1 s−1 and 1.2 × 1010m−1s−1 in solution and native LDL, respectively, indicating that there is no significant barrier to the diffusion of ⋅NO to the surface and core of LDL. Nitric oxide was also capable of diffusing through oxidized LDL. Considering the preferential partitioning of⋅NO in apolar milieu (6–8 for n-octanol:water) and therefore a larger ⋅NO concentration in LDL with respect to the aqueous phase, a corrected kNO value of ∼0.2 × 1010m−1s−1 can be determined, which still is sufficiently large and consistent with a facile diffusion of ⋅NO through LDL. Applying the Einstein-Smoluchowsky treatment, the apparent diffusion coefficient (D′NO) of⋅NO in native LDL is on average 2 × 10−5cm2 s−1, six times larger than that previously reported for erythrocyte plasma membrane. Thus, our observations support that ⋅NO readily traverses the LDL surface accessing the hydrophobic lipid core of the particle and affirm a role for ⋅NO as a major lipophilic antioxidant in LDL. low density lipoprotein 1-(pyrenyl)methyltrimethylammonium 1-(pyrenyl)-methyl-3-(9-octadecenoyloxy)-22,23-bisnor-5-cholenate 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine native low density lipoprotein oxidized low density lipoprotein Lipid accumulation in the vascular wall is a characteristic feature of the early pathogenesis of atherosclerosis, with associated lipoprotein oxidation representing a critical component of endothelial dysfunction and foam cell formation (1Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. N. Engl. J. Med. 1989; 320: 915-924Google Scholar, 2Chisolm G.M. Steinberg D. Free Radic. Biol. Med. 2000; 28: 1815-1826Google Scholar). Low density lipoprotein (LDL)1 is the major vehicle for cholesterol transport in human plasma and, in a modified-oxidized form, serves as the major source of cholesteryl ester deposited in atheroma (3Goldstein J.L. Hazzard W.R. Schrott H.G. Bierman E.L. Motulsky A.G. J. Clin. Invest. 1973; 52: 1533-1543Google Scholar, 4Steinberg D. J. Biol. Chem. 1997; 272: 20963-20966Google Scholar). Indeed, oxidized LDL becomes suitable for uptake by macrophages through the scavenger-receptor pathway, promoting the formation of cholesteryl ester-containing foam cells, one of the initial events in atheroma formation (5Goldstein J.L. Ho Y.K. Brown M.S. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1980; 255: 1839-1848Google Scholar, 6Brown M.S. Ho Y.K. Goldstein J.L. J. Biol. Chem. 1980; 255: 9344-9352Google Scholar, 7Steinbrecher U.P. Lougheed M. Kwan W.C. Dirks M. J. Biol. Chem. 1989; 264: 15216-15223Google Scholar, 8Guy R.A. Maguire G.F. Crandall I. Connelly P.W. Kain K.C. Atherosclerosis. 2001; 155: 19-28Google Scholar, 9Boullier A. Gillotte K.L. Horkko S. Green S.R. Friedman P. Dennis E.A. Witztum J.L. Steinberg D. Quehenberger O. J. Biol. Chem. 2000; 275: 9163-9169Google Scholar).The LDL particle (∼2.5 MDa) consists of an apolar core of cholesteryl esters and triglycerides, surrounded by a monolayer of phospholipids, unesterified cholesterol, and one molecule of apolipoprotein B-100 (4,536 amino acids, 550 kDa) (10Schuster B. Prassl R. Nigon F. Chapman M.J. Laggner P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2509-2513Google Scholar, 11Orlova E.V. Sherman M.B. Chiu W. Mowri H. Smith L.C. Gotto Jr., A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8420-8425Google Scholar). LDL is ∼22 nm in diameter and contains 42% cholesteryl ester, 6% triglyceride, 8% cholesterol, 22% phospholipid, and 22% apoB-100 by weight. Thus, each LDL particle would contain about 1600 molecules of cholesteryl ester, 170 of triglyceride, 700 of phospholipid, and 600 molecules of free cholesterol (12Esterbauer H. Gebicki J. Puhl H. Jurgens G. Free Radic. Biol. Med. 1992; 13: 341-390Google Scholar). About 50% of esterified fatty acids in the different lipid classes of LDL are polyunsaturated (12Esterbauer H. Gebicki J. Puhl H. Jurgens G. Free Radic. Biol. Med. 1992; 13: 341-390Google Scholar), an important attribute considering the sensitivity of these species to oxidative reactions in the lipoprotein particle. Cholesteryl esters are the most abundant lipid class in LDL, with cholesteryl linoleate being the principal oxidizable lipid in the hydrophobic core of LDL (12Esterbauer H. Gebicki J. Puhl H. Jurgens G. Free Radic. Biol. Med. 1992; 13: 341-390Google Scholar).Human LDL contains a number of antioxidants that inhibit lipid oxidation, with α-tocopherol the most abundant (∼6 α-tocopherol molecules per LDL particle), and other antioxidants (e.g.carotenoids, ubiquinol-10) present in much lower abundance (12Esterbauer H. Gebicki J. Puhl H. Jurgens G. Free Radic. Biol. Med. 1992; 13: 341-390Google Scholar). α-Tocopherol, localized at the surface of the LDL particle, provides minimal protection to lipid components in the hydrophobic core of LDL. Indeed, the principal oxidizable lipid, cholesteryl linoleate, is localized in the core of the lipoprotein, away from the more polar tocopherols (13Fielding C.J. J. Lipid Res. 1984; 25: 1624-1628Google Scholar, 14Thomas J.P. Kalyanaraman B. Girotti A.W. Arch Biochem. Biophys. 1994; 315: 244-254Google Scholar, 15Kenar J.A. Havrilla C.M. Porter N.A. Guyton J.R. Brown S.A. Klemp K.F. Selinger E. Chem. Res. Toxicol. 1996; 9: 737-744Google Scholar, 16Handelman G.J. Frankel E.N. Fenz R. German J.B. Biochem. Mol. Biol. Int. 1993; 31: 777-788Google Scholar).Low density lipoprotein oxidation is inhibited by both chemically and cell-derived nitric oxide (⋅NO) (17Rubbo H. Parthasarathy S. Barnes S. Kirk M. Kalyanaraman B. Freeman B.A. Arch. Biochem. Biophys. 1995; 324: 15-25Google Scholar, 18Hogg N. Kalyanaraman B. Joseph J. Struck A. Parthasarathy S. FEBS Lett. 1993; 334: 170-174Google Scholar, 19Hogg N. Struck A. Goss S.P. Santanam N. Joseph J. Parthasarathy S. Kalyanaraman B. J. Lipid Res. 1995; 36: 1756-1762Google Scholar, 20Goss S.P. Hogg N. Kalyanaraman B. J. Biol. Chem. 1997; 272: 21647-21653Google Scholar, 21Carr A.C. Frei B. J. Biol. Chem. 2001; 276: 1822-1828Google Scholar). Nitric oxide has multiple physicochemical qualities that make it a potentially more effective lipid antioxidant than α-tocopherol. Nitric oxide (a) readily crosses cell membranes and concentrates in lipophilic milieu by virtue of its uncharged character, low molecular mass, and relatively high lipid/water partition coefficient (n-octanol:water partition coefficient of 6–8:1) (22Malinski T. Taha Z. Grunfeld S. Patton S. Kapturczak M. Tomboulian P. Biochem. Biophys. Res. Commun. 1993; 193: 1076-1082Google Scholar, 23Denicola A. Souza J.M. Radi R. Lissi E. Arch. Biochem. Biophys. 1996; 328: 208-212Google Scholar) and (b) reacts to terminate propagation reactions catalyzed by lipid alkoxyl and peroxyl radical species (24Rubbo H. Radi R. Trujillo M. Telleri R. Kalyanaraman B. Barnes S. Kirk M. Freeman B.A. J. Biol. Chem. 1994; 269: 26066-26075Google Scholar, 25Padmaja S. Huie R.E. Biochem. Biophys. Res. Commun. 1993; 195: 539-544Google Scholar, 26O'Donnell V.B. Chumley P.H. Hogg N. Bloodsworth A. Darley-Usmar V.M. Freeman B.A. Biochemistry. 1997; 36: 15216-15223Google Scholar). Thus, by virtue of its high reactivity with lipid radical species, ⋅NO can spare lipophilic antioxidants (e.g. α-tocopherol) from oxidation (27Rubbo H. Radi R. Anselmi D. Kirk M. Barnes S. Butler J. Eiserich J.P. Freeman B.A. J. Biol. Chem. 2000; 275: 10812-10818Google Scholar).Nitric oxide production by vascular endothelium (28Furchgott R.F. Zawadzki J.V. Nature. 1980; 288: 373-376Google Scholar) and its diffusion into LDL can represent a key antioxidant mechanism, by acting in the hydrophobic core of LDL where the ratio of oxidizable lipids to endogenous antioxidants is much greater than at the LDL particle surface. The present work supports these concepts by revealing the diffusion of ⋅NO into the surface and the core of LDL via fluorescence quenching of pyrene derivatives incorporated at different depths of the lipoprotein.RESULTS AND DISCUSSIONThe structures of the hydrophilic and hydrophobic pyrene derivatives are shown in Fig. 1. The characteristic fine structure of the emission spectra of these pyrene derivatives in solution (Fig.2A) is less defined once they are incorporated into the lipid environment of the LDL and an increase in the emission at 396 nm is observed (Fig. 2B). The fluorescence spectra of PMTMA and PMChO in oxidized LDL were the same as in native LDL (not shown). The selected pyrene probes were chosen so that they could be incorporated at different depths of the LDL particle, with the pyrene moiety responsible for fluorescence emission being effectively quenched by ⋅NO. Pyrene is readily solubilized by membranes and located in the hydrocarbon core of the lipid bilayer (32Sepulveda P. Gallardo S. Lissi E.A. J. Colloid Interface Sci. 1992; 152: 104-113Google Scholar). A strongly cationic derivative like PMTMA is expected to be adsorbed only at the surface of LDL, a microenvironment rich in negatively charged phospholipids. The PMChO derivative is expected to penetrate to the hydrophobic core of LDL due to a structure analogous to esterified cholesterol. The degree of penetration of different pyrene derivatives into membranes has been characterized by proton and carbon NMR (35Gratzel M. Kalyanasundaram K. Thomas J.K. J. Am. Chem. Soc. 1974; 967869Google Scholar) and susceptibility to quenching by highly hydrophilic quenchers (36Lissi E.A. Caceres T. J. Bioenerg. Biomembr. 1989; 21: 375-385Google Scholar). Herein we observed that both probes incorporated into LDL were significantly protected from deactivation by iodide and tryptophan (water soluble quenchers), and that the resistance of PMChO to quenching by water-soluble species is at least 10-fold greater than for PMTMA (Table I, n-LDL column). If theKSV values of Table I are converted tokNO (Equation 3) using τo reported in Table II, the relative tendencies of hydrophilic and hydrophobic pyrene probe deactivation have even greater differences. These results indicate a differential distribution of the two probes in LDL and confirm that PMChO is located deeper into the LDL structure.Figure 2Emission spectra of PMTMA. A, emission spectrum of PMTMA (4 μm) in 50 mmphosphate buffer, pH 7.4. B, emission spectrum of PMTMA once incorporated into LDL (0.1 mg/ml in buffer). The excitation wavelength used was 337 nm. Similar spectra were obtained for PMChO in ethanol and LDL, respectively.View Large Image Figure ViewerDownload (PPT)Table IDeactivation of fluorescent probes by iodide and tryptophanProbeQuencherKSV(m−1)BufferEthanoln-LDLox-LDLPMTMAIodide35040816130PMTMATryptophan2503652190PMChO1-aInsoluble in buffer.Iodide230<250PMChO1-aInsoluble in buffer.Tryptophan265<2951-a Insoluble in buffer. Open table in a new tab Table IILifetimes (τO), Stern-Volmer slopes (KSV) and quenching rate constants by ⋅NO (kNO)PMTMAPMChOτ0KSVkNOτ0KSVkNOnsmm−11010m−1s−1nsmm−11010m−1 s−1Buffer501.32.6Ethanol542.03.71435.53.8EPM2-aEPM, erythrocyte plasma membrane; data taken from Ref. 23.1360.30.2ND2-bND, not determined.NDNDn-LDL801.01.22002.41.2Ox-LDL1002.52.52405.02.12-a EPM, erythrocyte plasma membrane; data taken from Ref. 23Denicola A. Souza J.M. Radi R. Lissi E. Arch. Biochem. Biophys. 1996; 328: 208-212Google Scholar.2-b ND, not determined. Open table in a new tab The half-lives obtained from the exponential fluorescence decay of the excited pyrene derivatives in different environments including native (n-LDL) and oxidized LDL (ox-LDL), are summarized on Table II. The fluorescence lifetime of PMChO in both native and oxidized LDL was greater than for the corresponding τ0 for PMTMA (2.5-fold). This agrees with previous results showing that the undecyl derivative analog (PUTMA) had a 2-fold greater half-time than PMTMA in biomembranes (23Denicola A. Souza J.M. Radi R. Lissi E. Arch. Biochem. Biophys. 1996; 328: 208-212Google Scholar). Since the lifetime of the probe is sensitive to the polarity changes of its surroundings, this result also supports a differential location of the two pyrene derivatives, with PMChO being situated in a more hydrophobic environment than PMTMA.Fig. 3 shows Stern-Volmer plots for PMTMA and PMChO fluorescence quenching by ⋅NO following probe incorporation into native and oxidized LDL. From the slope of these lines (KSV) the kNOvalues were calculated and are collected in Table II. Also, data taken from our previous work using PMTMA incorporated into erythrocyte plasma membranes is included for comparative purposes (23Denicola A. Souza J.M. Radi R. Lissi E. Arch. Biochem. Biophys. 1996; 328: 208-212Google Scholar).Figure 3Stern-Volmer plots for the fluorescence quenching of pyrene derivatives by ⋅NO. PMTMA (circles) or PMChO (triangles) fluorescence in LDL was quenched by increasing concentrations of⋅NO. The LDL suspensions contain 0.03 mg of protein/ml.Solid symbols represent LDL in the native conformation and open symbols represent oxidized LDL. Data shown are mean values ± S.D. of six independent experiments. The slopes of these plots are summarized in Table II asKSV.View Large Image Figure ViewerDownload (PPT)These results (Table II, Fig. 3) reveal critical features of the interaction of ⋅NO with the LDL particle. First, ⋅NO is capable of quenching fluorescence of pyrene derivatives located both at the hydrophilic surface and the hydrophobic core of LDL, indicating that ⋅NO can diffuse through and into the LDL particle. Second, we found that ⋅NO diffusion readily occurs in both native and oxidized LDL.To further investigate the diffusion of ⋅NO into LDL, PMChO-containing LDL was exposed to a ⋅NO flux generated from the decomposition of the NOC-7 (t½∼30 min under our experimental conditions). There was a concentration and time-dependent quenching of PMChO fluorescence from NOC-7 (Fig. 4), in agreement with the access of increasing concentrations of ⋅NO into the hydrophobic core of LDL.Figure 4Time course of pyrene fluorescence quenching in LDL exposed to a flux of ⋅NO. PMChO-containing LDL (0.03 mg/ml) was exposed to 1.9 or 3.8 mm NOC-7 (corresponding to a ⋅NO flux of 1.5 and 2.9 μm/s, respectively) under anaerobic conditions in 50 mm phosphate buffer, pH 7.4. Fluorescence intensity (λex = 337 nm, λem = 396 nm) was recorded over time and expressed as a percentage of control fluorescence.View Large Image Figure ViewerDownload (PPT)Considering LDL a more viscous environment than that of aqueous solutions, one would expect a slower diffusion of ⋅NO through the particle; however, the apparent quenching rate constants (kNO) obtained in LDL are large (1.2–2.5 × 1010m−1 s−1), with values approximating those for quenching in solution. However, an important consideration is that the x axis values in Fig. 3refer to the ⋅NO concentration in the aqueous phase and it should be noted that ⋅NO will partition into the lipid phase (37Liu X. Miller M.J. Joshi M.S. Thomas D.D. Lancaster Jr., J.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2175-2179Google Scholar), resulting in an increased concentration of quencher in the region surrounding the fluorophore. While the partition coefficient of⋅NO in LDL is not known, a good estimate can be adapted from knowledge of ⋅NO partitioning into non-polar solvents, being 6–8 in n-octanol:water (38Malinski T. Radomski M.W. Taha Z. Moncada S. Biochem. Biophys. Res. Commun. 1993; 194: 960-965Google Scholar). Importantly, even when considering a ⋅NO concentration in LDL six times greater than in the aqueous phase, corrected kNO values would be as high as 0.2–0.4 × 1010m−1 s−1, indicating that LDL offers no significant barrier to the diffusion of ⋅NO. In addition, similar kNO values for PMTMA and PMChO were obtained for LDL, confirming that ⋅NO can easily diffuse to the hydrophobic core of LDL as well.The determination of the exact diffusion coefficients of ⋅NO in the surface and core of LDL is precluded by the lack of knowledge of the actual concentration of ⋅NO in the lipophilic environments where the bimolecular quenching process occurs. However, apparent diffusion coefficients (D′NO) can be estimated using the Einstein-Smoluchowsky equation according to previously (23Denicola A. Souza J.M. Radi R. Lissi E. Arch. Biochem. Biophys. 1996; 328: 208-212Google Scholar),DNO′=kNO×1034πRNEquation 4 where R is the sum of the molecular radii of the probe plus ⋅NO (i.e. 6.9 × 10−8 cm and 9.1 × 10−8 cm for PMTMA and PMChO plus ⋅NO, respectively) and N Avogadro's number. The apparent diffusion coefficients of ⋅NO in native LDL from this approach were 2.3 × 10−5 cm2 s−1 in the surface and 1.7 × 10−5 cm2s−1 in the hydrophobic core of LDL, i.e.2.0 × 10−5 cm2 s−1 on average, half of the value obtained for ⋅NO in aqueous buffers (23Denicola A. Souza J.M. Radi R. Lissi E. Arch. Biochem. Biophys. 1996; 328: 208-212Google Scholar), whereas the D′NO in erythrocyte plasma membrane is six times less (Table II). This result indicates that diffusibility of ⋅NO in LDL exceeds that of biomembranes.Nitric oxide induced ∼2-fold greater pyrene derivative quenching for oxidized LDL, compared with native LDL (Fig. 3 and Table II), possibly due to oxidative modification of the lipoprotein rendering a more open structure that exposes the fluorescent probe to the solvent. This hypothesis is supported by the much larger quenching of pyrene fluorescence in oxidized LDL by the water-soluble quenchers iodide and tryptophan in oxidized LDL (Table I).The concentrations of ⋅NO in the subendothelium of small arterioles have been estimated in the range of 250–500 nm(reviewed in Ref. 39Buerk D.G. Annu. Rev. Biomed. Eng. 2001; 3: 109-143Google Scholar), and these concentrations seem to be more than sufficient to exert antioxidant actions in LDL (17Rubbo H. Parthasarathy S. Barnes S. Kirk M. Kalyanaraman B. Freeman B.A. Arch. Biochem. Biophys. 1995; 324: 15-25Google Scholar, 40Trostchansky A. Batthyány C. Botti H. Radi R. Denicola A. Rubbo H. Arch. Biochem. Biophys. 2001; 395: 225-232Google Scholar). The fact that⋅NO can readily diffuse to native, as well as oxidized LDL (Fig.3) makes, at a first glance, less likely the possibility that LDL oxidation may be promoted by a poor diffusion of ⋅NO toward sites of ongoing lipid oxidation. Indeed, an impairment of⋅NO-mediated antioxidant actions within LDL may be primarily related to a deficit of ⋅NO bioavailability, which may in turn be due to decreased production or alternative reactions with radical species (e.g. superoxide (O⨪2), lipid, and protein radicals) (17Rubbo H. Parthasarathy S. Barnes S. Kirk M. Kalyanaraman B. Freeman B.A. Arch. Biochem. Biophys. 1995; 324: 15-25Google Scholar, 24Rubbo H. Radi R. Trujillo M. Telleri R. Kalyanaraman B. Barnes S. Kirk M. Freeman B.A. J. Biol. Chem. 1994; 269: 26066-26075Google Scholar, 41Kissner R. Nauser T. Bugnon P. Lye P.G. Koppenol W.H. Chem. Res. Toxicol. 1997; 10: 1285-1292Google Scholar, 42Eiserich J.P. Butler J. van der Vliet A. Cross C.E. Halliwell B. Biochem. J. 1995; 310: 745-749Google Scholar) or iron-containing enzymes such as lipooxygenase (43Coffey M.J. Natarajan R. Chumley P.H. Coles B. Thimmalapura P.-R. Nowell M. Kuhn H. Lewis M.J. Freeman B.A. O'Donnell V.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8006-8011Google Scholar). Enhanced O⨪2 production by vascular cells has been observed under pathophysiologically-relevant stimuli (e.g. angiotensin II, altered hemodynamic forces) (44Wattanapitayakul S.K. Weinstein D.M. Holycross B.J. Bauer J.A. FASEB J. 2000; 14: 271-278Google Scholar, 45Warnholtz A. Nickenig G. Schulz E. Macharzina R. Brasen J.H. Skatchkov M. Heitzer T. Stasch J.P. Griendling K.K. Harrison D.G. Bohm M. Meinertz T. Munzel T. Circulation. 1999; 99: 2027-2033Google Scholar, 46Cominacini L. Pasini A.F. Garbin U. Davoli A. Tosetti M.L. Campagnola M. Rigoni A. Pastorino A.M. Lo Cascio V. Sawamura T. J. Biol. Chem. 2000; 275: 12633-12638Google Scholar, 47Griendling K.K. Sorescu D. Ushio-Fukai M. Circ. Res. 2000; 86: 494-501Google Scholar). In the latter case, not only ⋅NO would be less capable of diffusing into LDL by virtue of its accelerated consumption, but it may be transformed into peroxynitrite (ONOO−), an oxidant capable of initiating LDL oxidation (8Guy R.A. Maguire G.F. Crandall I. Connelly P.W. Kain K.C. Atherosclerosis. 2001; 155: 19-28Google Scholar, 44Wattanapitayakul S.K. Weinstein D.M. Holycross B.J. Bauer J.A. FASEB J. 2000; 14: 271-278Google Scholar, 48Graham A. Hogg N. Kalyanaraman B. O'Leary V. Darley-Usmar V. Moncada S. FEBS Lett. 1993; 330: 181-185Google Scholar, 49White C.R. Brock T.A. Chang L.Y. Crapo J. Briscoe P. Ku D. Bradley W.A. Gianturco S.H. Gore J. Freeman B.A. Tarpey M.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1044-1048Google Scholar). However, it is important to recognize that there is heterogeneity in the size and fatty acid composition of LDL among individuals and there may be scenarios under which small decreases in ⋅NO diffusion maintained over long periods of time might facilitate the initiation of oxidation processes. In this context, the diffusion of ⋅NO to the small (<25 nm diameter) and dense LDL particles versusthat of the more common, large and buoyant ones that may have different proatherogenic potential (50Mykkanen L. Kuusisto J. Haffner S.M. Laakso M. Austin M.A. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2742-2748Google Scholar, 51Campos H. Moye L.A. Glasser S.P. Stampfer M.J. Sacks F.M. J. Am. Med. Assoc. 2001; 286: 1468-1474Google Scholar) should be studied. Additionally, the influence of fatty acids that diminish atherogenic risk (i.e. fatty acids contained in oil fish) (52Lancet. 1999; 354: 447-455Google Scholar, 53Lopez D. Caballero C. Sanchez J. Puig-Parellada P. Mitjavila M.T. Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H2929-2935Google Scholar) on⋅NO diffusion in LDL needs to be addressed. These considerations may help to better define the role of LDL structure and composition and its interactions with ⋅NO in the inhibition/promotion of atherogenesis.In summary, our work reveals that ⋅NO readily diffuses to the hydrophilic surface and hydrophobic core of LDL, supporting the concept that ⋅NO can serve as an antioxidant in the vascular compartment, thus protecting LDL from oxidation and accounting for one component of the anti-atherogenic actions of ⋅NO. Lipid accumulation in the vascular wall is a characteristic feature of the early pathogenesis of atherosclerosis, with associated lipoprotein oxidation representing a critical component of endothelial dysfunction and foam cell formation (1Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. N. Engl. J. Med. 1989; 320: 915-924Google Scholar, 2Chisolm G.M. Steinberg D. Free Radic. Biol. Med. 2000; 28: 1815-1826Google Scholar). Low density lipoprotein (LDL)1 is the major vehicle for cholesterol transport in human plasma and, in a modified-oxidized form, serves as the major source of cholesteryl ester deposited in atheroma (3Goldstein J.L. Hazzard W.R. Schrott H.G. Bierman E.L. Motulsky A.G. J. Clin. Invest. 1973; 52: 1533-1543Google Scholar, 4Steinberg D. J. Biol. Chem. 1997; 272: 20963-20966Google Scholar). Indeed, oxidized LDL becomes suitable for uptake by macrophages through the scavenger-receptor pathway, promoting the formation of cholesteryl ester-containing foam cells, one of the initial events in atheroma formation (5Goldstein J.L. Ho Y.K. Brown M.S. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1980; 255: 1839-1848Google Scholar, 6Brown M.S. Ho Y.K. Goldstein J.L. J. Biol. Chem. 1980; 255: 9344-9352Google Scholar, 7Steinbrecher U.P. Lougheed M. Kwan W.C. Dirks M. J. Biol. Chem. 1989; 264: 15216-15223Google Scholar, 8Guy R.A. Maguire G.F. Crandall I. Connelly P.W. Kain K.C. Atherosclerosis. 2001; 155: 19-28Google Scholar, 9Boullier A. Gillotte K.L. Horkko S. Green S.R. Friedman P. Dennis E.A. Witztum J.L. Steinberg D. Quehenberger O. J. Biol. Chem. 2000; 275: 9163-9169Google Scholar). The LDL particle (∼2.5 MDa) consists of an apolar core of cholesteryl esters and triglycerides, surrounded by a monolayer of phospholipids, unesterified cholesterol, and one molecule of apolipoprotein B-100 (4,536 amino acids, 550 kDa) (10Schuster B. Prassl R. Nigon F. Chapman M.J. Laggner P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2509-2513Google Scholar, 11Orlova E.V. Sherman M.B. Chiu W. Mowri H. Smith L.C. Gotto Jr., A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8420-8425Google Scholar). LDL is ∼22 nm in diameter and contains 42% cholesteryl ester, 6% triglyceride, 8% cholesterol, 22% phospholipid, and 22% apoB-100 by weight. Thus, each LDL particle would contain about 1600 molecules of cholesteryl ester, 170 of triglyceride, 700 of phospholipid, and 600 molecules of free cholesterol (12Esterbauer H. Gebicki J. Puhl H. Jurgens G. Free Radic. Biol. Med. 1992; 13: 341-390Google Scholar). About 50% of esterified fatty acids in the different lipid classes of LDL are polyunsaturated (12Esterbauer H. Gebicki J. Puhl H. Jurgens G. Free Radic. Biol. Med. 1992; 13: 341-390Google Scholar), an important attribute considering the sensitivity of these species to oxidative reactions in the lipoprotein particle. Cholesteryl esters are the most abundant lipid class in LDL, with cholesteryl linoleate being the principal oxidizable lipid in the hydrophobic core of LDL (12Esterbauer H. Gebicki J. Puhl H. Jurgens G. Free Radic. Biol. Med. 1992; 13: 341-390Google Scholar). 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