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Originally published In Press as doi:10.1074/jbc.M413699200 on January 4, 2005

J. Biol. Chem., Vol. 280, Issue 10, 8850-8854, March 11, 2005
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Direct Measurement of Nitric Oxide and Oxygen Partitioning into Liposomes and Low Density Lipoprotein*

Matías Möller{ddagger}§, Horacio Botti§||, Carlos Batthyany§||, Homero Rubbo§||**, Rafael Radi§||{ddagger}{ddagger}, and Ana Denicola{ddagger}§§§

From the {ddagger}Laboratorio de Fisicoquímica Biológica, Facultad de Ciencias, the ||Departamento de Bioquímica, Facultad de Medicina, and the §Center for Free Radical and Biomedical Research, Universidad de la República, 11400 Montevideo, Uruguay

Received for publication, December 6, 2004 , and in revised form, December 29, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (·NO) has been proposed to play a relevant role in modulating oxidative reactions in lipophilic media like biomembranes and lipoproteins. Two factors that will regulate ·NO reactivity in the lipid milieu are its diffusion and solubility, but there is no data concerning the actual diffusion (D) and partition coefficients (KP) of ·NO in biologically relevant hydrophobic phases. Herein, a "equilibrium-shift" method was designed to directly determine the ·NO and O2 partition coefficients in liposomes and low density lipoprotein (LDL) relative to water. It was found that ·NO partitions 4.4- and 3.4-fold in liposomes and LDL, respectively, whereas O2 behaves similarly with values of 3.9 and 2.9, respectively. In addition, actual diffusion coefficients in these hydrophobic phases were determined using fluorescence quenching and found that ·NO diffuses ~2 times slower than O2 in the core of LDL and 12 times slower than in buffer . The influence of ·NO and O2 partitioning and diffusion in membranes and lipoproteins on ·NO reaction with lipid radicals and auto-oxidation is discussed. Particularly, the 3–4-fold increase in O2 and ·NO concentration within biological hydrophobic phases provides quantitative support for the idea of an accelerated auto-oxidation of ·NO in lipid-containing structures, turning them into sites of enhanced local production of oxidant and nitrosating species.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide produced by the oxidation of L-arginine to L-citrulline by ·NO1 synthases accomplishes important physiological regulatory functions related to vasodilation, neurotransmission, immune response, and regulation of cell respiration (1, 2). Despite being a free radical, ·NO is a weak redox intermediate, and its reactivity is restricted to paramagnetic species, including metals, metalloproteins (3, 4), and other radical molecules (5, 6).

The reactions of ·NO with other radicals can be classified as oxidant or antioxidant, depending on the reactivity of the products. Nitric oxide reacts with the superoxide anion radical () at diffusion-controlled rates to yield peroxynitrite, a strong oxidant that can induce lipid and protein oxidation and nitration (6, 7). Similarly, the reaction with O2 yields nitrogen dioxide (·NO2) and dinitrogen trioxide (N2O3), strong oxidant and nitrosating species (8, 9). Although the reaction with O2 is complex and not completely understood (8, 9), the rate law is second-order in ·NO and first-order in O2, with a third-order rate constant k = 0.8–1.2 x 107 M–2 s–1 (8, 9).

On the other hand, ·NO has been proposed as an important antioxidant in vivo (10–12), mainly because it reacts with organic peroxyl radicals at near diffusion-limited rates (k = 1–3 x 109 M–1 s–1, see Refs. 5 and 13) effectively inhibiting lipid peroxidation chain reactions (7, 10–12, 14–17). This antioxidant activity may be specially relevant in low density lipoprotein (LDL), because oxidatively modified lipoproteins can be recognized by scavenger receptors on macrophages and lead to macrophage lipid loading and eventually to atherosclerosis (18, 19).

There is an increasing interest in the reactions of ·NO and derived reactive species in hydrophobic milieu. In addition to inhibiting lipid oxidation, ·NO has been shown to inhibit intramembrane peptide nitration (20), to regulate mitochondrial respiration (2) and to be consumed more rapidly in the presence of membranes (21). Two key physicochemical parameters that can influence on ·NO reactivity in membranes are its diffusion, affecting the number of effective collisions in that medium, and its partition, that determines the concentration of ·NO achieved in that hydrophobic milieu. We have previously estimated the diffusion coefficients of ·NO in liposomes, erythrocyte plasma membranes and LDL (22, 23), although the values obtained were apparent because the solubility of ·NO in these hydrophobic environments was unknown. Nitric oxide is ~6–9 times more soluble in organic solvents than in water (24, 25), and it has been proposed that it is more soluble in membranes as well. The values reported in organic solvents were used to support the concept of increased ·NO antioxidant efficacy in membranes and lipoproteins (10–12) and used to explain the accelerated reaction with O2 in membranes (21) that would lead to locally enhanced nitrosative stress (21, 26). Therefore, the essential question to be answered concerning ·NO reactions in lipid milieu is how much ·NO actually partitions into biomembranes and lipoproteins? Membranes and lipoproteins pose constraints to solute dissolution compared with the organic solvents currently referenced in the literature (24, 25) because of its organized structure and lateral packing (27), thus the direct determination of ·NO partition coefficients (KP) between biologically relevant lipid phases and water is mandatory.

We developed a new method for measuring KP (the ratio between solute concentrations in hydrophobic and aqueous phases at equilibrium), which we have called "equilibrium-shift," that allowed us to determine KP of ·NO as well as O2 in two biologically relevant lipid systems, LDL and liposomes. In addition, these results were used to calculate actual diffusion coefficients for ·NO and O2 in these hydrophobic environments using fluorescence quenching (22, 23).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—·NO, N2, and argon were purchased from AGA SA (Montevideo, Uruguay). Egg yolk phosphatidylcholine (EYPC) was from Avanti%20Polar%20Lipids">Avanti Polar Lipids (Pelham, AL). The pyrene probe 1-(pyrenyl)methyl-3-(9-octadecenoyloxy)-22,23-bisnor-5-cholenate (PMChO) was from Molecular Probes (Eugene, OR). All other reagents were of analytical grade and purchased from Sigma.

LDL Purification and Liposome Preparation—LDL was isolated from normolipidemic donors by double ultracentrifugation using a KBr gradient (28) and then desalted using a PD-10 column (Amersham, Biosciences) previously equilibrated with 0.10 M sodium phosphate, 0.10 mM DTPA, pH 7.4. This buffer composition was used throughout all this work. The LDL was stored in the dark at 4 °C under argon and used within 48 h. The protein concentration was determined at 280 nm ({epsilon}280 = 1.05 mg–1 ml cm–1, Ref. 29). Multilamellar liposomes were prepared from 2 ml of 20 mg/ml chloroformic EYPC solution as previously (7), in buffer.

Oxygen Partition Coefficient Determination—The samples were used at high concentrations, up to 40 mg/ml EYPC and 9 mg/ml protein LDL in buffer. The samples (300 µl) were placed in 1.8-ml septum-sealed vials and flushed with water-humidified O2 for 3 h in a temperaturecontrolled water bath. Control (distilled water) and blank samples (buffer) were included in every assay. The O2 concentration of the sample ([O2]S) was determined using a thermostatized Clark-type electrode (YSI, model 5300), injecting a sample aliquot to 1.6 ml of deoxygenated distilled water (15 min N2 bubbling). The electrode was calibrated using air-saturated water ([O2]25w = 246 µM; [O2]37w = 199 µM, Ref. 30).

Nitric Oxide Partition Coefficient Determination—The samples (100 µl in 1.8-ml septum-sealed vials) were deoxygenated by flushing water-humidified N2 for 3 h, then flushed with deoxygenated 5 M NaOH-washed ·NO for 10 min and left for 4–5 h under very gentle agitation to equilibrate. An aliquot was withdrawn from the gas phase to confirm equilibration (see below), and another aliquot was withdrawn from the buffer or lipid suspension to determine the lipid/water ·NO partitioning. The latter was measured by injecting 15 µl of the sample into 3.00 ml of deoxygenated water (15 min N2 bubbling) in a 3.5-ml thermostatized chamber, and the ·NO release was registered using a ·NO selective electrode (ISO-NO, WPI Inc. Sarasota, FL). Calibration of the electrode was done using nitrite in iodide acidic solution as indicated by the manufacturer. It is worth mentioning that the greatest dilution used (1:200) draws most of the lipid-dissolved ·NO into the aqueous phase. To confirm that ·NO in the samples had equilibrated between gas and suspensions, the gas phase [·NO] in the sample vials was measured, knowing that the ratio of [·NO] between gas and liquid water at 25 °C is 20.9 at equilibrium (25). Briefly, 30 µl were withdrawn from the gas phase of the sample vials using a gas-tight syringe and injected into a septum-sealed vial with aerated 500 µl of 10 mM NaOH. Nitrite, the ·NO main oxidation product, was analyzed by the method of Griess (31).

Rationale for Partition Coefficient Determination—The partition coefficient for O2 and ·NO between the hydrophobic and the aqueous phase was determined measuring the increase in apparent solubility in lipid phase-containing suspensions relative to buffer alone. The total O2 and ·NO concentration increase in solutions with lipid phases can be described by the following equation, derived from the mass conservation relationship (exemplified for O2, Ref. 32),

(Eq. 1)
where [O2]T and [O2]aq stand for total and aqueous O2 concentrations, KP is the O2 partition coefficient between the hydrophobic and the aqueous phase, i.e. KP = [O2]h/[O2]aq, and {alpha}h is the hydrophobic fractional volume, the ratio between hydrophobic and total volumes.

The O2 electrode only measures the [O2] at the aqueous phase, and hence [O2]T cannot be measured directly. To solve this problem, a dilution step into deoxygenated water is introduced where part of the O2 that is dissolved into the membrane or lipoprotein goes into aqueous solution, and the released O2 is determined electrochemically. The [O2] in both lipid and aqueous phases changes because of this dilution. It follows from Equation 1 that if the sample is diluted by a factor of "b," the measured [O2] would be,

(Eq. 2)
where the measured [O2]S is not just [O2]T/b, because part of the O2 is still retained within the lipid phase after dilution (provided KP > 1, and b is low). To calculate [O2]T we assume that at saturation the solubility of O2 in the aqueous phase will be the same for all samples, and it will be mainly dependent on the buffer salts concentration and little affected by the polar groups from the lipid phase. If [O2] in the aqueous phase is constant for any {alpha}h prior to dilution, it can be calculated from [O2] measurements in buffer samples ([O2]B). Equation 1 can thus be rearranged to yield,

(Eq. 3)
and then we can use this relation to replace [O2]T in Equation 2 and rearrange it to get a function that relates KP to {alpha}h and to the measured [O2],

(Eq. 4)
where [O2]S is the measured [O2] in the different samples, [O2]B is the measured [O2] in buffer, and b is the dilution factor, which was 6.9 for most assays. The left term in Equation 4 can be regarded as the corrected relative increase in O2 solubility, and a plot of the measured corrected relative increase in [O2] against {alpha}h can yield KP from the slope.

The greater sensitivity of the ·NO electrode allowed the use of smaller amounts of sample, i.e. a greater dilution into the electrode chamber (b = 200). Considering [·NO]B x b » [·NO]S, Equation 4 simplifies greatly and factoring KP out,

(Eq. 5)
as this method was derived for measuring O2 and ·NO concentrations from re-equilibration after dilution, it was called equilibrium-shift method.

The partition coefficients were determined in 0.1 M phosphate and 0.1 mM DTPA buffer, because LDL aggregates in pure distilled water and to avoid salting out effects by the polar groups of the lipid particles. The corrected partition coefficients between liposomes or LDL and water were then calculated using the relative solubilities of each gas in buffer and water, KP(h/w) = KP(h/w) x [·NO]B/[·NO]w, where [·NO]B/[·NO]w = 0.90 and [O2]B/[O2]w = 0.92.

Hydrophobic Fractional Volume ({alpha}h) Calculation—The {alpha}h was calculated for liposomes taking into account the EYPC concentration (in g/ml), the density (d) and the hydrophobic fraction (Fh) of the bilayer, {alpha}h = [EYPC] x Fh/d, where d = 1.00 g/ml, and Fh = 0.75 excluding the polar phosphocholine headgroups, assuming that O2 and ·NO dissolve mainly in the acyl chain region (33–35). For LDL, a molar lipid volume of 2.12 x 103 liter/mol was calculated considering the molar volume of the LDL (2.5 x 103 liter/mol), the mass of the apoB-100 (5.13 x 105 g/mol) and its specific volume (0.74 ml/g, Ref. 36). This volume was then multiplied by the molar concentration of LDL to derive {alpha}h, {alpha}h = [apoB-100] (mol/liter) x 2.12 x 103 (liter/mol).

O2 Diffusion in LDL Studied by Pyrene Probe Fluorescence Quenching—Oxygen-quenching experiments were performed as described before for ·NO (22), using PMChO-incorporated LDL as the fluorescent probe and O2-saturated water.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
O2 Partition between Lipid and Aqueous Phases—The partition coefficient of O2 between LDL and buffer was determined by the equilibrium-shift method described above, using up to 9 mg/ml (in protein) LDL. Saturation of the sample was achieved within 1.5 h at 25 °C without agitation (data not shown), but 3 h was used to assure that the lipid sample was equilibrated with O2. The O2 load was dependent on LDL concentration, and the KP between LDL and buffer was determined from the slope of the equilibrium-shift plot (Equation 4, Fig. 1A), KP = 3.2 ± 0.4 and the corrected value for water 2.9 ± 0.3. It was observed that O2 partition between dimyristoyl phosphatidylcholine liposomes and water increases dramatically from the gel to the liquid crystalline state (37). However, the core lipids of LDL exhibit a broad phase transition temperature (20–40 °C, Ref. 38), and no difference in O2 partition was observed for LDL at 37 °C (data not shown).



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  		FIG. 1.
O2 partition between LDL or liposomes and buffer. LDL (A) and EYPC liposomes (B) in 0.1 M sodium phosphate, 0.1 mM DTPA, pH 7.4, were saturated with O2, and then the [O2] was determined electrochemically after diluting the sample in deoxygenated water. The ordinates, CRI [O2], stand for corrected relative increase in [O2] (see "Equation 4"). The x axis {alpha}h is the hydrophobic fractional volume. The slope of this plot is KP – 1 (Equation 4), thus KP = 3.2 ± 0.4 (r = 0.849; n = 13; p < 0.001) for LDL (A); KP = 4.2 ± 0.4 (r = 0.940; n = 10; p < 0.0001) for EYPC liposomes (B).

 
The same assays were conducted with EYPC liposomes, using up to 40 mg/ml EYPC. The measured O2 concentration increased with higher liposome content, ~0.33 µM O2 ml/mg egg-PC. The KP for O2 between liposomes and buffer was 4.2 ± 0.4 (Fig. 1B, Table I), and the corrected value for liposomes/water was 3.9 ± 0.4. Furthermore, the linearity observed for the equilibrium-shift plots (Fig. 1) indicates that the polar groups in the lipid particles do not yield a significant salting out of the O2, otherwise, a hyperbolic shape should have been observed. Therefore, the assumption made for proposing the equilibrium-shift method was accomplished under these experimental conditions.


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  		TABLE I
Partition (KP) and diffusion (D) coefficients for O2 and ·NO in lipid and aqueous systems at 25 °C

 
·NO Partition between Lipid and Aqueous Phases—Fig. 2 shows the ·NO-electrode records for water, buffer, and LDL samples flushed with ·NO. Water samples (Fig. 2, A) show the greatest [·NO], 6.9 ± 0.3 µM (n = 9), which after correcting for the dilution factor (b = 200) yields the water [·NO] = 1.39 mM. Buffer salts (Fig. 2, B) lead to the salting out of ·NO, thus a lower [·NO] was measured, 6.2 ± 0.2 µM (n = 8). On the other hand, LDL in buffer (Fig. 2, C) showed a greater [·NO] than buffer alone, 6.7 ± 0.1 µM for {alpha}h = 32 ± 1 x 10–3 (n = 5), and 6.5 ± 0.1 µM for {alpha}h = 16 ± 1 x 10–3 (n = 4), indicating that the ·NO partitioning between LDL and buffer was greater than one. The differences in ·NO concentration between buffer and LDL samples were significant for both LDL concentrations and buffer (p < 0.05). The KP for ·NO between LDL and buffer was calculated using Equation 5, 3.8 ± 0.8, and the corrected value for LDL/water was 3.4 ± 0.7 (Table I). EYPC liposomes at 40 mg/ml showed an 11% increase in [·NO] relative to buffer (n = 5), to yield a KP = 4.8 ± 0.5 and a corrected value for water of 4.4 ± 0.5.



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  		FIG. 2.
·NO partition in LDL. The [·NO] in ·NO-flushed water (A), buffer (B), and LDL samples (C) was determined electrochemically, injecting a 15-µl sample into 3000 µl of deoxygenated distilled water. The determined [·NO]s were 6.9 ± 0.3 (n = 9), 6.2 ± 0.2 (n = 8), and 6.7 ± 0.1 µM (n = 5) for water, buffer, and 15.2 µM LDL, respectively. The ·NO partition coefficient between LDL and buffer, according to Equation 5, is 3.8 ± 0.8, and the corrected value for water is 3.4 ± 0.7.

 
To confirm that equilibration was achieved, the [·NO] at the gas phase was measured. It was highly reproducible between assays, yielding 28 ± 2 mM ·NO, and the ratio [·NO]gas/[·NO]w was 20 ± 2, in agreement with the expected result of 20.9 (25).

·NO and O2 Diffusion in LDL—The diffusion of ·NO in LDL has been previously studied using fluorescence quenching of pyrene derivatives incorporated at the surface and into the core of LDL (22). Herein, the effect of O2 in the LDL core was tested and quantified in the same way by use of the highly hydrophobic PMChO (22). The bimolecular quenching rate constants (kQ) were determined from the Stern-Volmer relationship,

(Eq. 6)
where I0 and I are the fluorescence intensities measured in the absence and presence of quencher Q (·NO or O2), and {tau}0 is the lifetime of the fluorescent pyrene. This measured rate constant is termed apparent (kappQ), because the total quencher concentration ([Q]T) is used in the Stern-Volmer relationship, whereas the probe is located in the core of the particle and sensing [Q] at this hydrophobic phase where quenching reactions are taking place. The higher solubility of ·NO and O2 in LDL increases the frequency of collisions with the fluorescent probe in the lipid phase; thus, kappQ overestimates kQ. Using the KP values obtained herein for ·NO and O2 in LDL (Table I), the real kNO and kO2 can be calculated kQ = kappQ/KP (provided that the LDL concentration is low, as used in this work). This relation can then be used to calculate diffusion coefficients (DQ) by means of the Einstein-Smoluchowski's equation (22),

(Eq. 7)
where R is the sum of the molecular radii of the quencher and the fluorescent probe (9.1 x 10–8 cm, Ref. 22) and N is the Avogadro's number (neglecting the diffusion of the probe, Ref. 22). This relation was used to determine the real diffusion coefficients of O2 and ·NO in the core of LDL and the interior of a lipid bilayer, using our recent (22) and previous (23) data. The results are summarized in Table I. The diffusion of O2 in LDL resulted to be slightly higher than that of ·NO, in agreement with previous experiments using EYPC liposomes (23). It can also be appreciated that the diffusion of these molecules in a lipid milieu is slightly hindered, yielding on average, a 10 times lower diffusion than in water.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This work contributes to the understanding of ·NO reactions in lipid milieu, by filling an important blank in the physical properties of ·NO in biological systems, namely the actual partition and diffusion coefficients in lipoproteins and model membranes. A new method was developed and successfully used for measuring the KP of O2 and ·NO in LDL and EYPC liposomes. The KP of ·NO between lipid and aqueous phases was determined for the first time using this equilibrium-shift method, demonstrating that ·NO concentrates 4.4–3.4-fold in membranes and LDL, respectively, and similar results were obtained with O2 (Table I). Evidencing the difficulties in measuring KP using amphiphilic lipid phases, the KP of O2 between membranes and water has only been measured in dimyristoyl phosphatidylcholine liposomes and erythrocyte plasma membranes (37, 39). Smotkin et al. (37), found KP = 3.7 ± 0.6 for O2 between dimyristoyl phosphatidylcholine liposomes in the liquid crystalline state and water, measuring total O2 concentration by a modified Winkler technique. This value accounts for the entire bilayer, including polar phosphocholine headgroups. However, there is evidence indicating that O2, ·NO, and other apolar molecules, are less favorably dissolved in this region of the membrane (33–35, 40) and should therefore be excluded from the hydrophobic volume. We assumed a 0.75 bilayer hydrophobic fraction, thus the corrected KP from Smotkin et al. (37) would be 4.9 ± 0.8, which is in agreement within experimental errors with that found by ourselves (Table I). More complex membranes, such as erythrocyte plasma membranes, dissolve O2 in a similar fashion, with KP = 5 ± 4 (39).

LDL was expected to dissolve greater amounts of O2 into its very hydrophobic triglyceride and cholesterol ester-rich core than membranes. The lower KP value found for O2 in LDL compared with liposomes, 3.9 versus 2.9, could be due to a more ordered distribution and a higher packing of the cholesterol esters and triglycerides in the core of LDL compared with liposomes bilayers. This ordered core structure has been suggested by cryoelectron microscopy and other structural studies (36, 41).

Nitric oxide was found to partition into LDL in a similar fashion to O2, KP = 3.4 ± 0.7, whereas the partition in liposomes was slightly higher, 4.4 ± 0.4 (Table I). These values are lower than those earlier estimated, i.e. 6.5 in 1-octanol/water (24) or 9, based on the ·NO solubility in different organic solvents (21, 25). Compared with organic solvents, lipid membranes and lipoproteins show additional constraints to O2 or ·NO dissolution related to the acyl-chain ordering that suggest a lower value for KP than in an equivalent isotropic phase (27, 42, 43). For instance, a more negative entropy was reported for the transference of hexane or noble gases to membranes than to amorphous hydrocarbons (27, 43), indicating a resistance to dissolution. The exclusion effect can be seen for O2 in dimyristoyl phosphatidylcholine liposomes, where its KP decreases 3–4 times below the transition temperature (37). In the liquid crystalline state of membranes this effect will be less severe but still greater than in hydrocarbons.

Besides the partitioning effect, another factor that can influence the reaction rates in the lipid milieu are the diffusional properties of the reactants. It can be observed that O2 diffusion is nearly twice that of ·NO (Table I) and that both molecules diffuse in the core of LDL ~10 times slower than in water. The behavior of ·NO and O2 in the bilayers of EYPC liposomes proved to be very similar to that in LDL (Table I). These small molecules do not follow Stokes-Einstein law, meaning that their diffusion is not inversely related to the viscosity of the solvent (44). This property was previously evidenced for O2 diffusion in water and n-alkanes (45, 46) as well as for small non-electrolyte diffusion through lipid membranes (47). For instance, the viscosity at the core of LDL is expected to be higher than at the interior of membranes, and hence more than 100-times greater than water (0.89 versus 60–100 mPas for water (30) and membranes (48–50), respectively). However, ·NO and O2 diffusion in LDL is only 10-(and not 100-) times slower than in water. A consequence of this is that the diffusion of ·NO and O2 in lipoproteins and membranes will be less sensitive to changes in viscosity because of varying cholesterol content or fatty acid unsaturation degree, whereas on the other hand, the increase in lipid packing induced by cholesterol can lead to solute exclusion and a lower ·NO or O2 solubility in a lipid milieu.

The product of partition and diffusion coefficients KP x D is a good indicator of the rate at which diffusion-controlled reactions will occur in a lipid milieu (51), because it accounts for both the diffusion and the concentration in the lipid phase that will increase the frequency of collisions within that compartment. In a membrane or lipoprotein undergoing lipid oxidation, it is expected that O2 will react faster than ·NO with lipid derived alkyl radicals because of its higher KP x D (see Table I) and usually higher physiological concentration, yielding lipid peroxyl radicals. These peroxyl radicals will then react with ·NO in nearly diffusion-controlled reaction with k = 1–3 x 109 M–1 s–1 (5, 13). Although this reaction is expected to occur at slower rates in lipid phases than in solution (lower KP x D), it should still represent an important antioxidant pathway (10, 12, 52) leading to the formation of nitroso- and nitrolipids (7, 16, 53).

On the other hand, the 3–4-fold increase in O2 and ·NO concentration within biological lipid phases could lead to an accelerated auto-oxidation of ·NO, turning lipid phases into sites of enhanced local production of the oxidant and nitrosating species ·NO2 and N2O3 (21, 26). The oxidation, nitration and nitrosation of membrane proteins, as well as of polyunsaturated fatty acids by ·NO-derived species, is expected to be enhanced by this partitioning effect. It is worth mentioning that some of these modified lipids have recently been shown to display novel bioactivities, including vasorelaxing and anti-inflammatory activities (54, 55). Future studies will be aimed to define the extent and relevance of these ·NO/O2 reactions in biologically relevant hydrophobic phases.


   FOOTNOTES
 
* This work was supported in part by Grant R03 TW001493 from the National Institutes of Health (to H. R. and A. D.) and grants from the Wellcome Trust and PDT (Uruguay) (to H. R.) and the Howard Hughes Medical Institute (to R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Partially supported by fellowships from Programa de Desarrollo de Ciencias Basicas and Comisión Sectional de Investigacion Cientifica (Uruguay). Back

** A fellow of the John Simon Guggenheim Memorial Foundation. Back

{ddagger}{ddagger} An International Research Scholar of the Howard Hughes Medical Institute. Back

§§ To whom correspondence should be addressed: Laboratorio de Fisicoquímica Biológica, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay. E-mail: denicola{at}fcien.edu.uy.

1 The abbreviations used are: ·NO, nitric oxide; LDL, low density lipoprotein; EYPC, egg yolk phosphatidylcholine; PMChO, 1-(pyrenyl)-methyl-3-(9-octadecenoyloxy)-22,23-bisnor-5-cholenate; DTPA, diethylenetriaminepentaacetic acid. Back


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



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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