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J. Biol. Chem., Vol. 277, Issue 2, 932-936, January 11, 2002

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Diffusion of Nitric Oxide into Low Density Lipoprotein^*

Ana Denicola, Carlos Batthyány^§¶, Eduardo Lissi, Bruce A. Freeman^**, Homero Rubbo^§, and Rafael Radi^§

From the  Department of Physical Biochemistry, Facultad de Ciencias, Universidad de la República, 11400 Montevideo, Uruguay, the ^§ Department of Biochemistry, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay, the  Department of Chemistry, Universidad de Santiago de Chile, Santiago 2, Chile, and the ^** Department of Anesthesiology, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35233

Received for publication, July 13, 2001, and in revised form, October 15, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

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 (_o) of the excited probes. The apparent second order rate quenching constants of ^·NO (k_NO) for both probes were 2.6-3.8 × 10¹⁰ M¹ s¹ and 1.2 × 10¹⁰ M¹ s¹ 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 k_NO value of ~0.2 × 10¹⁰ M¹ s¹ 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 × 10⁵ cm² s¹, 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

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 (1, 2). 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 (3, 4). 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 (5-9).

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) (10, 11). 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 (12). About 50% of esterified fatty acids in the different lipid classes of LDL are polyunsaturated (12), 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 (12).

Human LDL contains a number of antioxidants that inhibit lipid oxidation, with -tocopherol the most abundant (~6 -tocopherol molecules per LDL particle), and other antioxidants (e.g. carotenoids, ubiquinol-10) present in much lower abundance (12). -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 (13-16).

Low density lipoprotein oxidation is inhibited by both chemically and cell-derived nitric oxide (^·NO) (17-21). Nitric oxide has multiple physicochemical qualities that make it a potentially more effective lipid antioxidant than -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) (22, 23) and (b) reacts to terminate propagation reactions catalyzed by lipid alkoxyl and peroxyl radical species (24-26). Thus, by virtue of its high reactivity with lipid radical species, ^·NO can spare lipophilic antioxidants (e.g. -tocopherol) from oxidation (27).

Nitric oxide production by vascular endothelium (28) 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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Chemicals-- Nitric oxide and argon were purchased from AGA SA (Montevideo, Uruguay). Pyrene derivatives 1-(pyrenyl)methyltrimethylammonium (PMTMA) and 1-(pyrenyl)methyl-3-(9-octadecenoyloxy)-22,23-bisnor-5-cholenate (PMChO) were from Molecular Probes (Eugene, OR). NOC-7 (3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine) was from Dojindo (Kumamoto, Japan). All other chemicals were of reagent grade and purchased from Sigma. Since PMChO is insoluble in water, ethanol was used as solvent when studies in solution were required.

LDL Preparation-- LDL from normolipidemic donors was obtained as previously (29). Briefly, it was first isolated from fresh plasma by ultracentrifugation (368,000 × g, 2.5 h) through a potassium bromide gradient (~5 mg/ml in protein) and further purified by size exclusion-high performance liquid chromatography on a column of Superose 6 HR 10/30 with UV detection at 280 nm (29). The LDL preparation obtained (native LDL, n-LDL) was ~1.5 mg/ml in protein and was stored at 4 °C under argon in the dark until used (within 2 days).

Oxidation of LDL-- High performance liquid chromatography-purified LDL was autoxidized as previously (30). LDL (0.5 mg/ml in 100 mM sodium phosphate buffer, pH 7.4) was transferred (8 ml) to a dialysis bag (Spectrapor tubing, number 4, MWCO: 12-14,000) and the bag was immersed in 250 ml of buffer. Oxygen was bubbled into the mixture through a plastic tube located outside of the dialysis bag, for up to 48 h at 37 °C. Oxidation was confirmed by measuring the formation of thiobarbituric acid-reactive substances, conjugated dienes, and the relative electrophoretic mobility of LDL in agarose gels (31).

Fluorescent Probe Incorporation-- The fluorescent probes (PMTMA and PMChO) were incorporated into LDL (0.5 mg/ml protein) by adding aliquots of an ethanolic stock solution probe (final ethanol concentration <0.1% and probe concentration 10⁶ M to avoid excimer formation) and incubating overnight at 4 °C. The excess fluorescent probe was removed by elution on a PD-10 column (Sephadex G-25, Amersham Bioscience, Inc.) equilibrated with 50 mM potassium phosphate buffer, pH 7.4. The absence of free fluorescent probe in LDL was confirmed by the lack of fluorescence quenching by 15 mM acrylamide. Indeed, acrylamide is extremely effective for the quenching of pyrene derivative fluorescence in aqueous environments, but it is totally ineffective when the probe is incorporated into lipid domains (32). The absence of excimers was confirmed by the lack of fluorescence emission at 470 nm (32). Once incorporated, LDL-PMTMA was used within a short period of time (<5 h) since leakage of the probe at longer times was observed. LDL-associated PMChO did not leak from LDL throughout a 5-h observation period.

Lifetime of Excited Probes (_o)-- Lifetimes of excited probes in solution and incorporated into native or oxidized LDL were measured by kinetics of fluorescence decay after excitation with a nitronite nitrogen laser in argon. Fluorescence decay was recorded in a Tektronik 7633 oscilloscope and fitted to Equation 1,

(Eq. 1)

where I represents fluorescence intensity, t is the time after excitation with the laser, and _o the lifetime of the excited fluorescent probe.

Preparation of ^·NO Solutions-- Stock ^·NO solutions were prepared by dissolving pure ^·NO gas anaerobically in aqueous solution as previously (23). Briefly, the gas sampling tube was filled with 50 mM phosphate buffer, pH 7.4, and deoxygenated by flushing with argon ~30 min. Then, ^·NO gas that had been previously washed with 5 M NaOH to eliminate contaminating nitrous oxide and nitrogen dioxide was bubbled for ~15 min, to achieve close to saturation levels, i.e. 1.7 mM at 37 °C. The final ^·NO concentration was measured before experiments with an electrochemical ^·NO sensor (WPI Model ISO-NO) and by the oxyhemoglobin reaction (33, 34). Importantly, since nitrite moderately quenches pyrene fluorescence (K_SV for PMTMA in erythrocyte membranes = 0.05 mM¹), alkaline washing of ^·NO gas was essential for accurate and reproducible results. A 100 mM stock NOC-7 solution was prepared in alkaline pH (2 mM potassium phosphate buffer, pH 11.0) to avoid its decomposition before addition to the reaction mixture containing LDL.

Fluorescence Quenching Analyses-- These experiments were performed by measuring steady-state fluorescence intensities of free and LDL-incorporated pyrene derivatives (_exc = 337 nm, _em = 396 nm) at different ^·NO concentrations in a Aminco-Bowman spectrofluorometer (Aminco SLM 2). Briefly, 2.5 ml of 50 mM potassium phosphate buffer, pH 7.4, were deoxygenated by bubbling argon for 15 min in an anaerobic fluorometer cuvette. Then 0.05-0.25 ml of LDL preparation (native or oxidized) was added through a rubber septum to obtain a final protein concentration of 0.03 mg/ml. The mixture was very gently bubbled with argon since extensive bubbling disrupted the LDL particle and tended to cause aggregates. The fluorescence intensity was recorded as I_o and then aliquots from a freshly prepared stock ^·NO solution were added with a gas-tight syringe through the rubber septum, gently mixed, and the decrease in fluorescence intensity registered as a function of ^·NO concentration. Extensive deoxygenation before ^·NO addition was critical since oxygen also quenches pyrene fluorescence (23). For quenching experiments in solution, the concentration of the pyrene derivative used was 4 µM in buffer or ethanol for PMTMA and in ethanol for PMChO. Additionally, a limited number of experiments were performed exposing PMChO-containing LDL to the ^·NO donor NOC-7 under anaerobic conditions. In this case, the fluorescence quenching by NOC-7-derived fluxes of ^·NO was followed continuously over a 5-min period. All experiments were conducted at 22 °C.

As previously (23), the apparent second-order quenching constants (k_NO) between the excited state probe and ^·NO were calculated from the slope of Stern-Volmer plots, K_SV (Equations 2 and 3),

(Eq. 2)

(Eq. 3)

where _o is the lifetime of the excited probe in the absence of quencher. In addition, pyrene fluorescence quenching experiments were performed using the water-soluble quenchers iodide (0 to 4 mM) and tryptophan (0 to 2 mM).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

The 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 (32). 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 (35) and susceptibility to quenching by highly hydrophilic quenchers (36). 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 the K_SV values of Table I are converted to k_NO (Equation 3) using _o 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.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Chemical structures of the pyrene derivatives. A, 1-(pyrenyl)methyltrimethylammonium, PMTMA, a positively charged pyrene derivative that locates on the surface of the LDL particle. B, PMChO, a cholesteryl ester derivative of pyrene that penetrates into the core of the LDL particle.

View larger version (7K): [in this window] [in a new window]   Fig. 2.   Emission spectra of PMTMA. A, emission spectrum of PMTMA (4 µM) in 50 mM phosphate 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 this table: [in this window] [in a new window]   Table II Lifetimes (O), Stern-Volmer slopes (KSV) and quenching rate constants by ·NO (kNO)

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 ₀ 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 (23). 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 (K_SV) the k_NO values 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 (23).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Stern-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 as KSV.

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 (t_1/2 ~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.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Time 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 (ex = 337 nm, em = 396 nm) was recorded over time and expressed as a percentage of control fluorescence.

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 (k_NO) obtained in LDL are large (1.2-2.5 × 10¹⁰ M¹ s¹), with values approximating those for quenching in solution. However, an important consideration is that the x axis values in Fig. 3 refer to the ^·NO concentration in the aqueous phase and it should be noted that ^·NO will partition into the lipid phase (37), 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 (38). Importantly, even when considering a ^·NO concentration in LDL six times greater than in the aqueous phase, corrected k_NO values would be as high as 0.2-0.4 × 10¹⁰ M¹ s¹, indicating that LDL offers no significant barrier to the diffusion of ^·NO. In addition, similar k_NO 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 (23),

(Eq. 4)

where R is the sum of the molecular radii of the probe plus ^·NO (i.e. 6.9 × 10⁸ cm and 9.1 × 10⁸ 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 × 10⁵ cm² s¹ in the surface and 1.7 × 10⁵ cm² s¹ in the hydrophobic core of LDL, i.e. 2.0 × 10⁵ cm² s¹ on average, half of the value obtained for ^·NO in aqueous buffers (23), 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. 39), and these concentrations seem to be more than sufficient to exert antioxidant actions in LDL (17, 40). 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 (O₂), lipid, and protein radicals) (17, 24, 41, 42) or iron-containing enzymes such as lipooxygenase (43). Enhanced O₂ production by vascular cells has been observed under pathophysiologically-relevant stimuli (e.g. angiotensin II, altered hemodynamic forces) (44-47). 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 (8, 44, 48, 49). 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 versus that of the more common, large and buoyant ones that may have different proatherogenic potential (50, 51) should be studied. Additionally, the influence of fatty acids that diminish atherogenic risk (i.e. fatty acids contained in oil fish) (52, 53) 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.

	ACKNOWLEDGEMENT

We thank Dr. Jack R. Lancaster, Jr. for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants R03 TW00999 and TW001493 (to B. A. F., A. D., H. R., and R. R.), Swedish Agency for Research Cooperation (Sweden), International Centre for Genetic Engineering and Biotechnology (Italy), and the Howard Hughes Medical Institute (to R. R.), Third World Academy of Sciences (Italy), and Comisión Sectorial de Investigación Cientifica, Universidad de la República (Uruguay) (to A. D.), and Pfizer-Fundación Manuel Pérez (to H. R. and C. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Partially supported by a fellowship from PEDECIBA, Uruguay.

International Research Scholar of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Avda. Gral. Flores 2125, 11800 Montevideo, Uruguay. E-mail: rradi@fmed.edu.uy.

Published, JBC Papers in Press, October 31, 2001, DOI 10.1074/jbc.M106589200

	ABBREVIATIONS

The abbreviations used are: LDL, low density lipoprotein; PMTMA, 1-(pyrenyl)methyltrimethylammonium; PMChO, 1-(pyrenyl)-methyl-3-(9-octadecenoyloxy)-22,23-bisnor-5-cholenate; NOC-7, 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine; n-LDL, native low density lipoprotein; ox-LDL, oxidized low density lipoprotein.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

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