Direct Measurement of Nitric Oxide and Oxygen Partitioning into Liposomes and Low Density Lipoprotein

Abstract

Nitric oxide ( ⅐ 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 ⅐ NO reactivity in the lipid milieu are its diffusion and solubility, but there is no data concerning the actual diffusion (D) and partition coefficients (K P ) of ⅐ NO in biologically relevant hydrophobic phases. Herein, a "equilibrium-shift" method was designed to directly determine the ⅐ NO and O 2 partition coefficients in liposomes and low density lipoprotein (LDL) relative to water. It was found that ⅐ NO partitions 4.4-and 3.4-fold in liposomes and LDL, respectively, whereas O 2 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 ⅐ NO diffuses ϳ2 times slower ). The influence of ⅐ NO and O 2 partitioning and diffusion in membranes and lipoproteins on ⅐ NO reaction with lipid radicals and auto-oxidation is discussed. Particularly, the 3-4-fold increase in O 2 and ⅐ NO concentration within biological hydrophobic phases provides quantitative support for the idea of an accelerated auto-oxidation of ⅐ NO in lipid-containing structures, turning them into sites of enhanced local production of oxidant and nitrosating species.Nitric oxide produced by the oxidation of L-arginine to Lcitrulline by ⅐ NO 1 synthases accomplishes important physiological regulatory functions related to vasodilation, neurotransmission, immune response, and regulation of cell respiration (1, 2). Despite being a free radical, ⅐ 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 ⅐ 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 (O 2 . ) 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 O 2 yields nitrogen dioxide ( ⅐ NO 2 ) and dinitrogen trioxide (N 2 O 3 ), strong oxidant and nitrosating species (8, 9). Although the reaction with O 2 is complex and not completely understood (8, 9), the rate law is second-order in ⅐ NO and first-order in O 2 , with a third-order rate constant k ϭ 0.8 -1.2 ϫ 10 7 M Ϫ2 s Ϫ1 (8, 9). On the other hand, ⅐ 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 ϭ 1-3 ϫ 10 9 M Ϫ1 s Ϫ1 , 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...

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