Propofol Is a Peroxynitrite Scavenger

Peroxynitrite is a potent oxidant formed by the rapid reaction between nitric oxide and superoxide radical [1]. The peroxynitrite anion is relatively stable, but it can rapidly protonate to peroxynitrous acid, an unstable species, which decomposes with a half-life of about one second at pH 7.4 [1,2]. Peroxynitrite can serve as a precursor for other potent reactive species, including nitrogen dioxide, nitronium ion, and an intermediate with hydroxyl radical-like reactivity toward a variety of biological molecules [2]. Propofol (2, 6-diisopropylphenol) has been used extensively as a rapidly acting intravenous anesthetic with a high therapeutic index for both inducing and maintaining general anesthesia [3]. Recent in vitro studies have demonstrated that propofol has potent antioxidant properties very similar to those of alpha-tocopherol [4-6]. Propofol inhibits lipid peroxidation induced by oxidative stress in the rat liver microsomes [5,6], mitochondria, and brain synaptosomes [6]. Although propofol reacts primarily with organic or organoperoxyl radical species [7], its effect on peroxynitrite is not known. Chemiluminescence has been widely used for measuring the production of free radicals and reactive oxygen metabolites from enzyme, cell, or organ systems [8,9]. In the present study, we used the chemiluminescence method for the assessment of the scavenging activity of propofol against peroxynitrite. Methods Peroxynitrite Synthesis Peroxynitrite was prepared using a quenched flow reaction as described by Beckman et al. [10]. Briefly, an aqueous solution of 0.6 M sodium nitrite was rapidly mixed with an equal volume of 0.6 M H2 O2 containing 0.7 M HCl and immediately quenched with the same volume of 1.2 M NaOH. All reactions were performed on ice. Excess H2 O2 was removed by addition of manganese dioxide (MnO (2)) powder to the peroxynitrite solution. The mixture was shaken for 5 min, and then MnO2 was removed by passage over a cellulose acetate disposable filter. The solution was used fresh or frozen at -20 degrees C for as long as a week. The final concentration of peroxynitrite was determined spectrally in 1.2 M NaOH (epsilon (302) = 1670 M/cm). Dilutions of this peroxynitrite stock solution were made in distilled water before use. Peroxynitrite-Induced Chemiluminescence Luminol- and lucigenin-enhanced chemiluminescence were measured at 37 degrees C by using a chemiluminometer (BioOrbit, Turku, Finland). Phosphate-buffered saline (PBS, 10 mM KH2 PO4 and 150 mM NaCl, pH 7.4) was mixed with luminol (250 micro M prepared daily in 2 M NaOH and diluted with PBS) or lucigenin (250 micro M prepared daily in PBS) in a cuvette. After adding catalase (500 U/mL) to the cuvette to remove H2 O2 left after MnO2 treatment, peroxynitrite at 2 micro M or 0.1 mM was injected for luminol and lucigenin chemiluminescence, respectively. The chemiluminescence produced was measured continuously for 5 min and recorded in a computer by using the Luminometer 1250 program (version 1.12, BioOrbit). Duplicate assays were performed in all experiments. The effects of various concentrations of propofol (Diprivan [R]) and intralipid (Lipovenos [R] 10%, the solvent of propofol) were examined by addition immediately before the peroxynitrite. All compounds were obtained from Sigma Chemical Company (St. Louis, MO) except for H2 O2, which was purchased from Merck (Darmstadt, Germany). All the measurements correspond to the peak value of light emission, and the effects of propofol and intralipid were expressed as a percent change from the response to the peroxynitrite alone. Results are given as mean +/- SEM. Differences between the control and treated groups were analyzed by using an unpaired Student's t-test. A P < 0.05 was considered statistically significant. Results Peroxynitrite at a 2-micro M concentration produced 7909 +/- 163 mV (n = 21) in luminol chemiluminescence. However, lucigenin chemiluminescence was less sensitive, and only 973 +/- 170 mV (n = 9) was measured at a 0.1-mM concentration of peroxynitrite. Propofol showed a marked inhibition in both luminol-and lucigenin-enhanced peroxynitrite chemiluminescence. Propofol started to inhibit peroxynitrite chemiluminescence at 1 micro M and caused almost complete inhibition at 1 mM, with 50% inhibition concentration values of 14 and 24 micro M for lucigenin and luminol chemiluminescence, respectively (Figure 1 and Figure 2), which shows that there was a significant scavenging activity against peroxynitrite. Intralipid was tested at 1 mM, the highest propofol concentration used in this study, and caused a slight reduction (12% +/- 4%, n=14, P < 0.05 compared with 1 mM propofol) in luminol chemiluminescence. However, a small increase (30% +/- 10%, n = 8, P < 0.05 compared with 1 mM propofol) in lucigenin chemiluminescence was recorded in the presence of 1 mM intralipid.Figure 1: A, Typical recorder tracings representing the effects of propofol (1 mM) and intralipid (1 mM) on luminol-induced peroxynitrite (2 micro M) chemiluminescence. B, Effects of the same concentrations of propofol and intralipid on lucigenin-induced peroxynitrite (0.1 mM) chemiluminescence.Figure 2: Concentration-dependent effects of propofol on luminol-(solid bars) and lucigenin-(open bars) enhanced peroxynitrite chemiluminescence (CL). Numbers in parentheses indicate the number of experiments.Discussion In this study, we have shown that peroxynitrite reacts with luminol and lucigenin to yield chemiluminescence. These results agree with previous reports that show that luminol or lucigenin chemiluminescence can detect peroxynitrite generated by either chemical synthesis [11,12] or cultured endothelial cells [12]. Propofol, but not intralipid, produced concentration-dependent inhibition of peroxynitrite-induced chemiluminescence. We have shown for the first time in this study that propofol is a peroxynitrite scavenger. Peak plasma concentrations of propofol of 40-60 micro M occur with the induction of anesthesia, and during the maintenance of anesthesia, the anesthetic concentrations are 10-25 micro M [13]. The 50% inhibition concentration value of 14 micro M for propofol suggests that this peroxynitrite scavenging property can be of clinical importance. Intralipid did not reduce peroxynitrite chemiluminescence, which implies that the antioxidant effects of propofol in vivo are unlikely to be mediated by its solvent. Peroxynitrite is one of the potent reactive metabolites for the initiation of lipid peroxidation [14]. Therefore, propofol appears to inhibit lipid peroxidation in two ways: it reacts with lipid peroxyl radicals to form the relatively stable propofol phenoxyl radical [5,7], and it scavenges peroxynitrite, as shown in this study. Propofol also suppresses phagocytosis and the killing of bacteria by polymorphonuclear leukocytes [15]. Since peroxynitrite is a potent bactericidal agent [16], this suppression is likely to be mediated by the peroxynitrite-scavenging activity of propofol. The concentration of propofol that caused an observable inhibitory effect on peroxynitrite chemiluminescence is in the same range as the anesthetic concentrations, and it is therefore possible that propofol may act as an antioxidant during anesthesia. Controlled clinical studies might reveal whether this in vitro observation may be beneficial in disease states such as acute lung injury [17], in which peroxynitrite formation is thought to play an important role.