Reaction between the anesthetic agent propofol and the free radical DPPH˙ in semiaqueous media: Kinetics and characterization of the products

Free Radical Biology & Medicine 45 (2008) 1011–1018

Contents lists available at ScienceDirect

Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Reaction between the anesthetic agent propofol and the free radical DPPH ˙ in semiaqueous media: Kinetics and characterization of the products Ouided Friaa a,b, Vincent Chaleix b,c,1, Marc Lecouvey b,c, Daniel Brault a,b,⁎ a b c

Université Pierre et Marie Curie–Paris 6, UMR 7033, BIOMOCETI, F-75005 Paris, France CNRS, UMR 7033, BIOMOCETI, F-75005 Paris, France Université Paris 13, BIOMOCETI, F-93017 Bobigny, France

a r t i c l e

i n f o

Article history: Received 2 April 2008 Revised form 1 July 2008 Accepted 1 July 2008 Available online 8 July 2008 Keywords: Propofol Anesthetic Diphenylpicrylhydrazyl radical DPPH Kinetics pH Hydroalcoholic medium Mechanism Free radicals

a b s t r a c t

˙

The reaction of the free radical diphenylpicrylhydrazyl (DPPH ) with the anesthetic agent 2,6diisopropylphenol (propofol, PPF) was investigated in buffered hydroalcoholic media. The kinetics was followed using a stopped-flow system. DPPH was reduced to the hydrazine analogue DPPH–H with a measured stoichiometry (DPPH /PPF) of 2. The main product of the reaction, 3,5,3′,5′-tetraisopropyl-(4,4′)diphenoquinone (PPFDQ) was isolated by chromatography and its structure was fully characterized. The reaction mechanism was inferred from the stoichiometry, kinetics, and product identification. The first step, which primarily determines the kinetics, is the reaction of DPPH with PPF to produce DPPH–H and the PPF radical. The rate constant was found to be 31.8, 207, and 908 M− 1 s− 1 at pH 6.4, 7.4, and 8.4, respectively. The pH dependence is indicative of a higher reactivity of the phenolate form of PPF. Then, PPF radicals combine to form dipropofol, which is quickly oxidized to PPFDQ by the remaining DPPH . This reaction scheme is corroborated by numerical simulations of the kinetics. In the course of this study we also disclosed an unexpected effect, the photochemical degradation of PPFDQ. The need to compare antioxidants on a kinetics basis is again emphasized. In our hands, PPF presents a significantly weaker reactivity than Trolox. © 2008 Elsevier Inc. All rights reserved.

The anesthetic agent propofol (2,6-diisopropylphenol, PPF), the structure of which is shown in Fig. 1, was introduced with the brand name Diprivan 20 years ago. It is approved for the induction and maintenance of anesthesia in more than 50 countries. Its anesthetic properties as well as its pharmacokinetics have been detailed in various reviews during the past 2 decades [1–4]. Meanwhile, PPF has been found to reduce the myocardial damage caused by ischemia and reperfusion [5]. This beneficial effect might be due partly to the antioxidant properties of this molecule, which shares some common structural elements with vitamin E (α-tocopherol). As shown by ESR spectroscopy, PPF acts as an antioxidant by reacting with free radicals to form a phenoxyl type radical [6,7]. PPF also reacts with other oxidizing species such as singlet oxygen [8,9]. The antioxidant properties of PPF were demonstrated in various

˙

Abbreviations: DPPH , 2,2-diphenyl-1-picrylhydrazyl free radical; DPPH–H, 1,1-diphenyl-2-picrylhydrazine; PPF, diisopropylphenol; Tris, tris(hydroxymethyl) aminomethane; Bis–Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl) methane; PPFDQ, 3,5,3′,5′-tetraisopropyl-(4,4′)-diphenoquinone. ⁎ Corresponding author. Fax: +33 1 69 87 43 60. E-mail address: [email protected] (D. Brault). 1 Present address: Laboratoire de Chimie des Substances Naturelles EA 1069, Faculté des Sciences et Techniques, Université de Limoges, F-87060 Limoges, France. 0891-5849/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2008.07.001

˙

˙

˙

˙

biological model systems. PPF can replace α-tocopherol in a model involving lipid peroxidation of liver microsomes [10]. It was also found to inhibit membrane peroxidation in rat liver mitochondria [11]. However, the importance of the antioxidant effect of PPF at concentrations relevant to a clinical situation has been questioned on the basis of some conflicting results [12]. The discrepancies may arise from the fact that different systems were used to test the antioxidant capacity and, more precisely, differences in the reaction media. Indeed, PPF is a lipid-soluble compound and partitioning between various compartments in biological systems must be considered [13]. Data on the inhibition of lipid peroxidation in pure aqueous solutions [12] do not permit one to address the radical scavenging activity of PPF in real biological structures. Experiments carried out on suspensions of liver microsomes or other cellular organoids might represent a better approach [10,11,13–15]. The overall evaluation of the antioxidant capacity of PPF should also take into account the possible contributions of metabolites or reaction products [16,17]. The colored free radical diphenylpicrylhydrazyl (DPPH ; see structure in Fig. 1) has been extensively used to characterize phenolic antioxidants in solution [18–21]. However, the influence of the medium and possible role of the deprotonation of the phenolic group have been outlined [20,21]. In an earlier study on the

˙

1012

O. Friaa et al. / Free Radical Biology & Medicine 45 (2008) 1011–1018

Fig. 1. Chemical structures and absorption spectra of compounds relevant to this work. DPPH (——), DPPH–H (- - -), and propofol (— —).

˙

˙

mechanism of the reaction of DPPH with Trolox, a water-soluble αtocopherol analogue, we used mixtures of ethanol with various proportions of water buffered at different pH values to have control over both the polarity and the acidity of the medium [22]. This solvent system is used here for PPF with a focus on kinetics and product analysis, two complementary methods that were used to elucidate the reaction mechanism. In the course of this study, we also showed the photosensitivity of a product of the reaction, a property that should be taken into account in the interpretation of data obtained with spectroscopic methods involving a high-intensity light source. Materials and methods Materials

˙

The stable free radical DPPH , 95% pure, and PPF, 97% pure, were purchased from Sigma–Aldrich (Saint-Quentin Fallavier, France). The reduced form of the radical, 1,1-diphenyl-2-picrylhydrazine (DPPH–H), 98% pure, was purchased from Fluka Chemika (Buchs, Switzerland). Stock solutions of these compounds were made in analytical grade ethanol (Merck, VWR International, Fontenay-sousBois, France). Hydroalcoholic solutions were prepared by mixing ethanol with the desired amount (vol/vol) of ultrapure water furnished by a DirectQ3 apparatus from Millipore (Molsheim, France). The water component was buffered by using 10 mM tris(hydroxymethyl)aminomethane (Tris) for pH 7.4 and 8.4 or 10 mM bis(2-hydroxyethyl) iminotris(hydroxymethyl) methane (Bis-Tris) for pH 6.4. Tris and BisTris were purchased from Merck and Sigma–Aldrich, respectively. Solutions were normally equilibrated with air.

proportion, were mixed. Absorption changes at a single wavelength were recorded using a conventional photomultiplier over total time ranging from 0.4 to 150 s. Kinetic traces were composed of 2000 points and at least four signals were averaged. Time-resolved absorption spectra were collected in the range 200–740 nm using a photodiode array with a resolution of 2.17 nm. At the maximum scan speed, spectra were recorded every 2.56 ms. Then, at the best, the first spectrum can be recorded 3.76 ms after mixing. In experiments with the diode array, the monochromator was set at zero-order position. All experiments were carried out at 20°C. Kinetics was fitted by using the software furnished by either Applied Photophysics or Kaleidagraph from Synergy Software (Reading, PA, USA). Both use the Levenberg– Marquard algorithm for nonlinear curve fitting. Bimolecular rate constants were determined as the slope of the linear fit of the observed rate constants versus the reagent in excess (propofol). The molar extinction coefficients were determined from Beer’s law, which was obeyed for all the reagents in this study. The Kaleidagraph software was used for these linear plots. It returns the standard error value on the slope, which is indicated in the results. The Mathcad software (Mathsoft, Cambridge, MA, USA) was used for numerical simulations. NMR spectra were recorded on a Varian Gemini 200 apparatus with a frequency of 200 MHz for proton and 50.3 MHz for carbon 13. The chemical shifts (δ), expressed versus tetramethylsilane are given in parts per million. Melting points were measured in a capillary tube with a Stuart apparatus SMP3. Infrared spectra were recorded on KBr pellets with a Nicolet FT-IR Model 380 spectrometer. Preparation of 3,5,3′,5′-tetraisopropyl-(4,4′)-diphenoquinone (PPFDQ) Two liters of DPPH (1.55 × 10− 4 M) in a 1/1 mixture of ethanol and water buffered to pH 7.4 with Tris were mixed with 2 liters of PPF (1.55 × 10− 3 M) in the same medium and allowed to react under stirring in the dark for 1 h. The reaction was followed by thin-layer chromatography (TLC) on 0.2-mm-thick silica gel 60F254 foil (Merck) with a mixture of hexane and ethyl acetate (80/20, v/v) as eluant. Spot contours were delimited from their color or under UV light. After ethanol evaporation and successive extractions with chloroform the organic products were collected and separated by chromatography on a column (25 × 250 mm) of silica (silica 60 ACC 20–40 μm). Two passages were necessary to purify the PPFDQ. For the first column, the eluant was a mixture of hexane and ethyl acetate with ratios varying from 100/0 to 70/30. For the second column the acidity of the silica was neutralized by triethylamin and the eluant was hexane. After solvent elimination and drying, 5 mg of PPFDQ was obtained as a yellow product (yield 18.3%), Rf = 0.6 (eluant hexane/ethyl acetate, 80/20, v/v). Spectral data were consistent with those reported in the literature [8]: melting point = 202–203°C; IR (KBr pellet, cm− 1): 2961, 2869, 1589; UV–visible; λmax = 424 nm, ɛ423 = 68,000 M− 1 cm− 1 in methanol; 1H NMR (CDCl3): 1.22 ppm (doublet, J = 7.09 Hz, 24H, –CH3), 3.24 ppm (septet, J = 7.03 Hz, 4H, –CHisopropyl), 7.65 (singlet, 4H, _ CH–); 13C NMR (CDCl3): 185.5 (C1), 148.07 (C2), 135.93 (C4), 125.56 (C3), 27.19 (C5), 21.24 (C6).

˙

Results Instruments Physicochemical properties of propofol The pH of the aqueous solutions used to prepare the hydroalcoholic mixtures was measured with a Radiometer PHM240 apparatus (Radiometer Analytical, Villeurbanne, France). UV–visible absorption spectra were recorded using a UVIKON 923 spectrophotometer (BioTek, Kontron Instrument, Milano, Italy). Kinetics and time-resolved spectra were measured with an Applied Photophysics (Leatherhead, UK) stopped-flow apparatus (Model SX 18MV) equipped with a 150W xenon arc lamp. The mixing time was 1.2 ms. Equal volumes of DPPH and PPF solutions, made with the same ethanol/water

˙

Solutions of PPF in 1/1 ethanol/buffer mixtures were found to obey Beer’s law for concentrations at least up to 1.5 × 10− 4 M for all the pH values (6.4, 7.4, and 8.4) of the buffer component. The absorption maximum of PPF was found at 272 nm (Fig. 1) with a molar extinction coefficient of 1900 ± 50 M− 1 cm− 1 in 1/1 ethanol/buffer mixtures for all the pH values. These values are similar to those found in methanol [23]. The pKa of the OH/O− couple in PPF has been published as 11.0 [24], about 1 unit lower than the pKa (11.9) of Trolox [25].

O. Friaa et al. / Free Radical Biology & Medicine 45 (2008) 1011–1018

1013

˙ Upon reaction with PPF, DPPH is reduced to the substituted ˙ hydrazine form, DPPH–H. As shown in Fig. 2, the course of the reaction can be followed by the decrease in the absorption of DPPH at 524 nm. ˙ The stoichiometry of the reaction, defined as the number of DPPH ˙ molecules consumed per PPF molecule, was determined as described in our previous paper by using DPPH in excess [22]. Mixing of ˙ solutions of DPPH (7.5 × 10 M after mixing) and PPF (7.8 × 10 M ˙ after mixing) in 1/1 ethanol/buffer mixtures was carried out with the Stoichiometry of the reaction of DPPH with PPF

−5

−6

stopped flow. Spectral changes were monitored with the diode array detector. The stoichiometry of the reaction, n, was calculated by using the relation n = Δ(Abs)/(Δɛ × l × [PPF]), where Δ(Abs) is the absorbance change at 524 nm (DPPH maximum) recorded at the end of the reaction, Δɛ is the difference between the molar extinction coefficients of DPPH and DPPH–H, l is the optical length of the optical cell, and [PPF] is the propofol concentration. The stoichiometry was 2.0 ± 0.3 in 1/1 ethanol/pH 7.4 buffer mixtures. This value corresponds to the mean (± standard deviation) of four measurements on solutions containing DPPH and PPF in ratios between 10 and 20. The values measured for the other pH, 2.1 ± 0.4 and 2.2 ± 0.4 at pH 8.4 and 6.4, respectively, were similar within experimental uncertainty.

˙

˙

˙

˙

Kinetics of the reaction of DPPH with PPF in excess

˙

˙

The reaction between DPPH and PPF in excess (PPF/DPPH ≥10) was studied in 1/1 ethanol/buffer mixtures at three pH values. Experiments carried out in a monochromatic mode, i.e., those involving the monochromator set at a given wavelength and detection with the photomultiplier (see Materials and methods), are considered first. The slits of the monochromator were reduced to 0.1 mm, allowing a minimal light irradiation of the sample during the recording of kinetics. Typical decays of the absorption at 524 nm, which indicates consumption of DPPH , are given in Fig. 3a. These decays were tentatively fitted by monoexponentials leading to observed rate constants kobs. The decays were significantly accelerated when the PPF concentration was increased while keeping constant the initial DPPH concentration. This behavior is reminiscent of pseudofirst-order conditions [26] although the reaction mechanism is quite

˙

˙

˙

Fig. 3. Kinetics of the reaction between DPPH and PPF. (a) Absorbance change at 524 nm after mixing of DPPH (5.0 × 10− 5 M after mixing) with PPF in 1/1 ethanol/buffer mixtures at pH 7.4. The experiments were performed by using the stopped-flow system with the photomultiplier for signal detection. PPF concentrations after mixing were 6.25 × 10− 4 M (line 1) or 1.00 × 10− 3 M (line 2). The decays were reasonably fitted by monoexponentials leading to pseudo-first-order observed rate constants, kobs. (b) Linear correlation between the observed rate constant, kobs, and the PPF concentration for experiments carried out in 1/1 mixtures of ethanol with buffer at pH 6.4 (●), 7.4 (▴), and 8.4 (■). Rate constants derived from simulations of kinetics for pH 7.4 are indicated by open symbols. The bimolecular rate constants used in the simulation were kaf = 1.5 × 102 M− 1 s− 1, k1c = 7 × 103 M− 1 s− 1 (□) or kaf = 1.8 × 102 M− 1 s− 1, kar = 6 × 105 M− 1 s− 1, and k1c = 6 × 103 M− 1 s− 1 (○).

˙

complex (see below). As a matter of fact, the plot of the observed rate constants as a function of the PPF concentration (see Fig. 3b) is linear. Then, a bimolecular rate constant for the reaction between PPF and DPPH was tentatively derived from the slope. It was found to significantly depend on pH, with values of 31.8 ± 2.2, 207 ± 6, and 908 ± 30 M− 1 s− 1 at pH 6.4, 7.4, and 8.4, respectively. As shown in Fig. 2, a new compound clearly distinguishable from DPPH–H is formed from the reaction of DPPH with PPF. It is characterized by a marked absorption around 424 nm. The kinetics was analyzed in monochromatic mode at this wavelength on two time scales. As shown in Figs. 4a and 4b, the change in the absorption at 424 nm presents a sigmoid shape, suggesting a multistep formation of the product. A plateau is attained after about 50 s, a time that corresponds also to the disappearance of DPPH . No further change is observed at longer times as depicted in Fig. 4c. It is worth noting that the final absorption at 424 nm is significantly decreased when the PPF concentration is increased. These observations will be interpreted latter.

˙

˙

˙

˙

Reaction of DPPH with PPF in excess: time-resolved absorption spectra Spectra were recorded by using the photodiode array of the stoppedflow apparatus. Typical sets of spectra recorded after mixing DPPH and PPF solutions (7.7 × 10− 5 and 7.75 × 10− 4 M after mixing, respectively) in 1/1 ethanol/buffer mixtures at pH 7.4 are displayed in Fig. 5a. In this experiment, the slits of the monochromator were adjusted to 0.8 mm, a value providing a good signal-over-noise ratio. It should be kept in mind that this mode requires one to set the monochromator grating to zeroorder position to furnish a white light beam. Compared to the monochromatic analysis reported above with 0.1-mm slits, the light intensity received by the sample is several orders of magnitude higher. In keeping with the spectra shown in Fig. 2, the DPPH band at 524 nm decreases and that at 424 nm, corresponding to the product formed, increases. Quite well-defined isosbestic points were first observed at 343 and 457 nm. However, these isosbestic points were not maintained.

˙

˙

Fig. 2. UV–visible characterization of the reaction between DPPH and PPF. Absorption spectrum recorded about 3 min after mixing DPPH (7.7 × 10− 5 M after mixing) and PPF (7.7 × 10− 4 M after mixing) in 1/1 ethanol/pH 7.4 buffer mixtures (——). No further change is recorded over 1 h. Absorption spectra of DPPH 7.7 × 10− 5 M (- - -), DPPH–H 7.7 × 10− 5 M (— —), and DPPH–H 7.7 × 10− 5 M mixed with PPF, 7.31 × 10− 4 M (⋯) are shown for reference.

˙

˙

˙

1014

O. Friaa et al. / Free Radical Biology & Medicine 45 (2008) 1011–1018

This product was characterized by 1H and 13C NMR in CDCl3, UV– visible, and IR spectra and its melting point (see Materials and methods). The 1H NMR is shown in Fig. 6. In agreement with the literature [8,27,28], all the data allowed us to assign the product to 3,5,3′,5′-tetraisopropyl-(4,4′)-diphenoquinone, abbreviated PPFDQ to reference the starting material. The relatively low yield of PPFDQ (18.3%) could be explained by partial product degradation during the preparation due to the ambient light and to the acidity of the silica used for chromatography. Photochemical behavior of PPFDQ The possibility of photobleaching of PPFDQ under the conditions used for stopped-flow experiments with diode array detection was investigated. For that purpose, the optical fiber bundle used to convey the light from the xenon lamp to the optical cuvette of the stopped flow was disconnected and the light coming from the optical fibers used as a source. The front face of a standard 10-mm optical cuvette containing 2 ml of a solution of PPFDQ in 1/1 ethanol/buffer, pH 7.4, mixture was illuminated by the open-ended fiber bundle. The solution was gently mixed with a magnetic stirrer during the irradiation. Absorption spectra were recorded after various irradiation times. The results shown in Fig. 7 clearly indicate a photodegradation of PPFDQ within a time range consistent with the stopped-flow experiments. Discussion

˙

Fig. 4. Kinetics of the formation of PPFDQ, the main product of the reaction between DPPH and PPF followed by absorbance change at 424 nm: experimental and simulation results. The solvent system was the usual 1/1 ethanol/buffer, pH 7.4, mixture and the DPPH concentration 5.0 × 10− 5 M after mixing. (a) PPF concentration after mixing is 6.25 × 10− 4 M. The simulation with the set of rate constants kaf = 1.8 × 102 M− 1 s− 1, kar = 6 × 105 M− 1 s− 1, and k1c = 6 × 103 M− 1 s− 1 and known molar extinction coefficients is shown as a dashed line. (b) PPF concentration after mixing is 1 × 10− 3 M. The simulation with the same set of parameter as in (a) is shown as a dashed line. (c) Observation at a longer time scale. The PPF concentrations after mixing are 5.0 × 10− 4, 6.2 × 10− 4, 7.5 × 10− 4, 8.8 × 10− 4, and 1 × 10− 3 M.

˙ ˙

The mechanism of the reaction between DPPH and PPF can be established from its stoichiometry, its kinetics, and the characterization of its products. The stoichiometry, defined as the number of DPPH molecules consumed per PPF molecule, was 2.0 ± 0.3. In this

˙

After about 15 s, the band at 424 nm decreases and is no longer apparent after 150 s. The time profiles of these changes are shown in Fig. 5c. When the slits of the monochromator were adjusted to 0.4 mm, the isosbestic points were maintained for a longer time. The decrease of the 524 nm band was not modified, but the shape of the time profile for the 424 nm band was significantly changed. The maximum amplitude was higher and the rate of the subsequent decay lower than in the previous case. These results strongly suggest that the decay of the product characterized by the 424 nm band is due to a photochemical degradation when the diode array mode is used. This point will be confirmed below. The results obtained with 1/1 ethanol/buffer mixtures for the pH values 6.4 and 8.4 were similar.

˙

Characterization of the products of the reaction between DPPH and PPF in semiaqueous media To avoid any bias, the experiments aimed to characterize the products were performed with the concentrations also used for the kinetics studies with PPF in excess. Full experimental details are given under Materials and methods. In agreement with the kinetics reported above, as soon as the reactants were mixed, the coloration of the mixture changed. TLC analysis of the crude mixture showed the disappearance of DPPH after 5 min and the formation of two major products. One product was identified as DPPH–H from the comparison with an authentic commercial sample. After elimination of ethanol, extraction with chloroform, and a two-step column chromatography procedure, the second major product was isolated as a yellow powder.

˙

˙

Fig. 5. Stopped-flow study of the reaction between DPPH and PPF in 1/1 ethanol/buffer, pH 7.4, mixtures using the diode array module. (a, b) Time-resolved absorption spectra. The concentrations of DPPH and PPF were 7 × 10− 5 and 7.75 × 10− 4 M after mixing, respectively. Selected spectra are shown at 0.39, 1.17, 1.95, 2.72, 3.50, 5.06, 6.61, 8.95, 10.5, 15.2, 20.6, 30.7, 40.1, 50.2, 60.3, 80.5, 100.8, and 150.6 s after mixing. The arrows indicate the direction of the spectral changes. (c, d) Time dependence of the absorbance changes at 424 (——) and 524 nm (- - -). Each trace is derived from 200 points. The slits of the monochromator in zero-order position were adjusted to 0.8 (a, c) or 0.4 mm (b, d).

˙

O. Friaa et al. / Free Radical Biology & Medicine 45 (2008) 1011–1018

1015

˙

Fig. 6. Characterization of the major product of the reaction between PPF and DPPH by 1H NMR. The assigned structure, 3,5,3′,5′-tetraisopropyl-(4,4′)-diphenoquinone (PPFDQ) is shown. The carbon labels are those used for 13C NMR resonance attribution.

˙

reaction, DPPH is subjected to a one-electron reduction to the hydrazine form, DPPH–H. Propofol is oxidized to the diquinone PPFDQ, which was fully characterized as reported above. Then, the overall reaction can be written as: 4 DPPH] þ 2 PPFY4 DPPH−H þ PPFDQ: A detailed reaction mechanism is proposed in Fig. 8. In keeping with previous data on phenolic compounds, the first step (Fig. 8, line a) is likely to be the one-electron oxidation of PPF to the phenoxyl radical with concomitant reduction of DPPH to DPPH–H, which is signed by the decrease in the 524 nm DPPH absorption band. This step is written as a reversible process as suggested by recent studies [29]. The rate constants for the forward and reverse reactions are denoted kaf and kar, respectively.

˙ ˙

The second step (Fig. 8, line b) is shown as the dimerization of the radical formed to yield dipropofol, PPFD. In phenoxyl radicals, the free electron has a significant probability of being delocalized on the para and ortho carbons. Because of resonance stabilization, the lifetime of the radical is long enough to favor bimolecular dimerization, which is readily achieved at the unsubstituted para position in the case of propofol. A dimerization rate constant (2 ×kb) of about 3 × 109 M− 1 s− 1, near the diffusion limit, has been recently derived from laser flash photolysis experiments [23]. This mechanism has been found quite general for phenolic compounds. An alternative mechanism involving coupling of the radical with an extra phenol (or phenolate) molecule followed by further oxidation received little support [35]. As the reaction of DPPH with PPF is relatively fast, the steady-state concentration of radicals is expected to be sufficient to favor their self-reaction. The last reaction (Fig. 8, line c) is the oxidation of dipropofol to the diquinone, PPFDQ, by DPPH , accounting for the observed stoichiometry. It is assumed that this reaction corresponds to a sequence of two steps with the transitory formation of monooxidized dipropofol rather than a trimolecular process. The overall reaction scheme is supported by the experimental results obtained in the presence of PPF in excess and by simulations of kinetics as fully reported in the supplementary material. As mentioned above, the overall decay of DPPH can be approximated by a monoexponential. Although the fit was not perfect, the residuals, i.e., the difference between fitted and experimental values, did not exceed 2% of the overall amplitude of the signal. Moreover, the apparent rate constant thus derived (kobs) was found to linearly depend on the concentration of PPF that was in large excess (see Fig. 3b). This indicates that the overall observed kinetics is mainly governed by the first step. The sigmoid shape of the buildup of the band at 424 nm, which signals the formation of PPFDQ, is most interesting (Figs. 4a and 4b). Indeed, dipropofol with no electron delocalization on the two cycles does not absorb in this region [23]. The kinetics at 424 nm shows that PPFDQ is formed after a delay, in agreement with the sequence of the reactions shown in lines b and c. Noticeably, the overall amplitude of the band at 424 nm decreases with increasing PPF concentrations (Fig. 4b). At the highest PPF concentrations, DPPH is more rapidly consumed through the reaction shown in line a. The PPF radical can combine to form dipropofol. However, the quantity of DPPH required to oxidize dipropofol to PPFDQ becomes a limiting factor, which explains the decrease in the signal amplitude.

˙

˙

˙

Fig. 7. Photodegradation of PPFDQ in 1/1 ethanol/buffer mixtures at pH 7.4. The PPFDQ solution in a standard 1-cm optical cuvette was illuminated as described under Results to simulate conditions prevailing in the stopped-flow experiments. (——) Nonirradiated PPFDQ sample. Irradiation times were 60, 120, 180, 240, and 300 s, in the order shown by the arrows.

˙

˙

1016

O. Friaa et al. / Free Radical Biology & Medicine 45 (2008) 1011–1018

˙

Fig. 8. Postulated scheme of the reaction between DPPH and PPF.

˙

Nevertheless, the oxidation of dipropofol by DPPH is very fast as indicated by the DPPH decay that essentially displays a monophasic character. As a matter of fact, dipropofol has been found to be an effective antioxidant in various in vitro assays, including the DPPH test [36], and to inhibit lipid peroxidation about 100 times more efficiently than propofol [17]. Due to its complexity, it was not possible to obtain an analytical mathematical solution from the set of differential equations describing the time dependence of the concentration of the species involved. Instead, a program based on these differential equations and on rate constant estimates was written for the Mathcad software. For initial concentrations of DPPH and PPF, and guessed values for the reaction rate constants, the program simulates the concentrations of the various species as a function of time. The program and typical simulations are given in the supplementary material. In these simulations, the first step was considered either nonreversible (kar = 0) or reversible (kar N 0). The dimerization constant kb was taken as 1.5 × 109 M− 1 s− 1, in agreement with literature data [23]. The oxidation of dipropofol was assumed to proceed in two steps. The first consumes one molecule of DPPH with a rate constant k1c leading to a transitory monooxidized dipropofol form that is quickly oxidized by a second DPPH molecule to PPFDQ with a reaction rate constant k2c. The latter step was assumed to be diffusion controlled with a rate constant k2c of 2 × 109 M− 1 s− 1 (provided this constant is high enough, it has no influence on the simulation). The curves simulating the DPPH concentration were fitted to monoexponentials. Fluttering of the residuals was similar to those obtained for experimental curves. The exponential factor obtained from the simulation was compared to the experimental rate constant kobs and the set of the reaction rate constants kaf, kar, and k1c adjusted to obtain the best agreement over the whole range of the PPF concentrations. If step a is considered not reversible, the best adjustment (see Fig. 3b) was found with kaf = 1.5 × 102 M− 1 s− 1 and k1c = 7 × 103 M− 1 s− 1 for experiments carried out at pH 7.4. In addition, the program yielded the time dependence of the PPFDQ concentration. Knowing the molecular extinction coefficients of DPPH , DPPH–H (Fig. 1), and PPFDQ at 424 nm, it was

˙

˙

˙

˙

˙

˙

˙

possible to simulate absorption changes at this wavelength and to compare them to experimental data. The sigmoid character of the curves shown in Figs. 4a and 4b was reproduced. The best fit of experimental data was found, however, if it is assumed that step a is reversible. The set of values leading to the best simulation of the shape of the changes of absorbance at 424 nm and to a linear dependence of the rate constants versus the PPF concentrations were kaf = 1.8× 102 M− 1 s− 1, kar = 6 × 105 M− 1 s− 1, and k1c = 6 × 103 M− 1 s− 1. It can be noted that k1c is about 30 times larger than kaf, in agreement with the higher reactivity of dipropofol reported elsewhere [17]. The equilibrium constant for the first step, i.e., the ratio kaf/kar, is predicted to be 3 × 10− 4, a value comparable to those reported for other substituted phenols [29]. As the dimerization process drives the reaction to the right, consumption of DPPH goes to completion whatever the concentration of PPF, provided it remains in excess. It can be noted that the kaf value, 180 M− 1 s− 1, is not too far from the value of the slope (210 M− 1 s− 1) of the kobs versus PPF curve, which agrees with the fact that the first step largely governs the overall kinetics. An excellent agreement between experimental and simulated curves is observed in Figs. 3b, 4a, and 4b. The simulation also predicts the decrease in the final absorbance at 424 nm as a function of the PPF concentration depicted in Fig. 4c. By using the above-mentioned rate constant set, the ratio of absorbance values for PPF concentrations of 5 × 10− 4 and 1 × 10− 3 M is predicted to be 1.178, whereas the experimental value is 1.162. Other examples of oxidation of propofol via a radical mechanism have been reported. The PPF radical has been detected by EPR in the course of the reaction of propofol with peroxynitrite [7]. This reaction was found to yield oxidation products such as monomeric and dimeric diquinone (PPFDQ) [37]. Oxidation of PPF by singlet oxygen formed upon light excitation of photosensitizers was also found to yield similar oxidation products [8]. As depicted in Fig. 3b, the rate of the reaction of DPPH with PPF significantly increases with pH, the values of the rate constant being 30, 210, and 910 M− 1 s− 1 at pH 6.4, 7.4, and 8.4, respectively. Such pH dependence was previously observed for other phenols. It was

˙

˙

O. Friaa et al. / Free Radical Biology & Medicine 45 (2008) 1011–1018

attributed to the ionization of the OH group even if the amount of the phenolate form is low. In fact, the ionized form presents a much higher reactivity than the neutral form. Hence, besides solvent control by the formation of hydrogen bonds [30], a sequential proton loss electron transfer mechanism was recently proposed by Litwinienko and Foti [20,31–33]. Alternatively, electron and proton transfer could be concerted in the transition state [34]. In a study on the reaction of Trolox, a water-soluble α-tocopherol analogue also bearing a phenoltype OH group, with DPPH in the same solvent system [22], we found a 1.8 increase in the rate constants between pH 6.4 and 8.4. An increase of 30 times is found for PPF. As a matter of fact, the pKa of the phenol group for PPF (11.0) is almost 1 unit lower than that for Trolox (11.9). Although in both cases the amount of the ionized phenolate form is low in the pH range investigated, the proportion is relatively more important in the case of PPF, accounting for the observed results. In the course of our experiments with the stopped flow in the diode array mode, we disclosed an unexpected effect, the photochemical degradation of PPFDQ, the main product formed upon reaction of PPF with DPPH . The spectra displayed in Fig. 7 do show that the light intensity provided by the analyzing light beam of the stopped flow in this mode is sufficient to induce degradation. A misinterpretation of the data could have occurred in the absence of control of the effect of light intensity by changing the monochromator slit width. The antioxidant properties of propofol have been evaluated by various methods, but few of them allow real insight into the chemical mechanism and comparison with other antioxidants. As the protection against oxidative stress relies on a dynamic competition between scavenging of radical species and their harmful reaction, kinetics data are most valuable to understanding the action of antioxidants [38]. They are essential to compare antioxidants on a reactivity basis. This approach has been seldom followed in the case of propofol except by Rigobello et al., who measured a rate of reaction of DPPH with PPF in buffer/ethanol mixtures similar to our values [39]. These authors did not go into mechanistic details, however. Gulcin et al. [40] previously reported on the reaction of DPPH with PPF but these authors measured only the consumption of DPPH at a unique time largely exceeding the time scale investigated here. In this assay they found PPF to be somewhat more efficient than αtocopherol. However, their test provides information more on stoichiometry than on reactivity. The reactivity, estimated from the bimolecular rate constants, of propofol at pH 7.4 is found here to be about 90 times less than that of Trolox. But the reactivity of PPF was only a little lower (ratio of initial rate constants of about 0.7) than that of guaiacol, another known antioxidant (Friaa and Brault, unpublished results). The free para position in PPF makes dimerization much more favorable than in the case of the fully substituted butylated hydroxytoluene [41], for instance. As shown in the literature [16,17] and by our data, dipropofol possesses a high antioxidant activity. The formation of this metabolite requires that the concentration of PPF radicals is high enough to favor their bimolecular combination. Then, provided that the total PPF amount is sufficient, we could anticipate a superior antioxidant response to a high radical production resulting from an important oxidative stress. A last problem is to estimate the importance of the antioxidant effect of PPF at concentrations relevant to the clinical situation. Obviously, the plasma PPF concentration required to maintain anesthesia is of little use. Indeed, with an octanol/water partition coefficient of 6760 (Diprivan data sheet) and an affinity for liposomes of 7.2 × 103 M− 1 [9], the concentration of PPF in membranes is much higher. In fact, PPFDQ formation was also observed for PPF incorporated into liposomes when the system was subjected to an oxidative stress induced by singlet oxygen [9]. In conclusion, the present data on the reaction of an oxidizing radical with propofol furnish a set of kinetics and mechanistic information, which will be most certainly useful in the understanding of the antioxidant properties of this drug, which is now the most used anesthetic.

˙

˙

˙

˙ ˙

1017

Acknowledgments This work was supported by the charity organizations Fondation Marcel Bleustein-Blanchet pour la Vocation and Association pour la Recherche contre le Cancer (ARC), through Ph.D. grants to O.F. The stopped-flow apparatus was acquired thanks to a subsidy from ARC (Grant 7209). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.freeradbiomed.2008.07.001. References [1] Langley, M. S.; Heel, R. C. Propofol: a review of its pharmacodynamic and pharmacokinetic properties and use as an intravenous anaesthetic. Drugs 35:334–372; 1988. [2] Dundee, J. W.; Clarke, R. S. Propofol. Eur. J. Anaesthesiol. 6:5–22; 1989. [3] Bryson, H. M.; Fulton, B. R.; Faulds, D. Propofol: an update of its use in anaesthesia and conscious sedation. Drugs 50:513–559; 1995. [4] Trapani, G.; Altomare, C.; Liso, G.; Sanna, E.; Biggio, G. Propofol in anesthesia: mechanism of action, structure–activity relationships, and drug delivery. Curr. Med. Chem. 7:249–271; 2000. [5] Kato, R.; Foex, P. Myocardial protection by anesthetic agents against ischemia– reperfusion injury: an update for anesthesiologists. Can. J. Anaesth. 49:777–791; 2002. [6] Murphy, P. G.; Myers, D. S.; Davies, M. J.; Webster, N. R.; Jones, J. G. The antioxidant potential of propofol (2,6-diisopropylphenol). Br. J. Anaesth. 68:613–618; 1992. [7] Mouithys-Mickalad, A.; Hans, P.; Deby-Dupont, G.; Hoebeke, M.; Deby, C.; Lamy, M. Propofol reacts with peroxynitrite to form a phenoxyl radical: demonstration by electron spin resonance. Biochem. Biophys. Res. Commun. 249:833–837; 1998. [8] Heyne, B.; Kohnen, S.; Brault, D.; Mouithys-Mickalad, A.; Tfibel, F.; Hans, P.; Fontaine-Aupart, M. P.; Hoebeke, M. Investigation of singlet oxygen reactivity towards propofol. Photochem. Photobiol. Sci. 2:939–945; 2003. [9] Heyne, B.; Brault, D.; Fontaine-Aupart, M. P.; Kohnen, S.; Tfibel, F.; MouithysMickalad, A.; Deby-Dupont, G.; Hans, P.; Hoebeke, M. Reactivity towards singlet oxygen of propofol inside liposomes and neuronal cells. Biochim. Biophys. Acta 1724:100–107; 2005. [10] Aarts, L.; van der Hee, R.; Dekker, I.; de Jong, J.; Langemeijer, H.; Bast, A. The widely used anesthetic agent propofol can replace alpha-tocopherol as an antioxidant. FEBS Lett. 357:83–85; 1995. [11] Eriksson, O.; Pollesello, P.; Saris, N. E. Inhibition of lipid peroxidation in isolated rat liver mitochondria by the general anaesthetic propofol. Biochem. Pharmacol. 44:391–393; 1992. [12] Green, T. R.; Bennett, S. R.; Nelson, V. M. Specificity and properties of propofol as an antioxidant free radical scavenger. Toxicol. Appl. Pharmacol. 129:163–169; 1994. [13] Bao, Y. P.; Williamson, G.; Tew, D.; Plumb, G. W.; Lambert, N.; Jones, J. G.; Menon, D. K. Antioxidant effects of propofol in human hepatic microsomes: concentration effects and clinical relevance. Br. J. Anaesth. 81:584–589; 1998. [14] Musacchio, E.; Rizzoli, V.; Bianchi, M.; Bindoli, A.; Galzigna, L. Antioxidant action of propofol on liver microsomes, mitochondria and brain synaptosomes in the rat. Pharmacol. Toxicol. 69:75–77; 1991. [15] Murphy, P. G.; Bennett, J. R.; Myers, D. S.; Davies, M. J.; Jones, J. G. The effect of propofol anaesthesia on free radical-induced lipid peroxidation in rat liver microsomes. Eur. J. Anaesthesiol. 10:261–266; 1993. [16] Boisset, S.; Steghens, J. P.; Favetta, P.; Terreux, R.; Guitton, J. Relative antioxidant capacities of propofol and its main metabolites. Arch. Toxicol. 78:635–642; 2004. [17] Ogata, M.; Shin-Ya, K.; Urano, S.; Endo, T. Antioxidant activity of propofol and related monomeric and dimeric compounds. Chem. Pharm. Bull. (Tokyo) 53:344–346; 2005. [18] Blois, M. S. Antioxidant determination by the use of a stable free radical. Nature 181:1199–1200; 1958. [19] Brand-Williams, W.; Cuvelier, M. E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. Lebensm.-Wiss. u.-Technol. 28:25–30; 1995. [20] Foti, M. C.; Daquino, C.; Geraci, C. Electron-transfer reaction of cinnamic acids and their methyl esters with the DPPH(⁎) radical in alcoholic solutions. J. Org. Chem. 69:2309–2314; 2004. [21] Litwinienko, G.; Ingold, K. U. Abnormal solvent effects on hydrogen atom abstractions. 1. The reactions of phenols with 2,2-diphenyl-1-picrylhydrazyl (dpph⁎) in alcohols. J. Org. Chem. 68:3433–3438; 2003. [22] Friaa, O.; Brault, D. Kinetics of the reaction between the antioxidant Trolox and the free radical DPPH in semi-aqueous solution. Org. Biomol. Chem. 4:2417–2423; 2006. [23] Heyne, B.; Tfibel, F.; Hoebeke, M.; Hans, P.; Maurel, V.; Fontaine-Aupart, M. P. Photochemistry of 2,6-diisopropylphenol (propofol). Photochem. Photobiol. Sci. 5:1059–1067; 2006. [24] Demerseman, P.; Lechartier, J. -P.; Reynaud, R.; Cheutin, A.; Royer, R.; Rumpf, P. Recherche de relations entre les structures et les propriétés physicochimiques des alcoylphenols. Bull. Soc. Chim. Fr. 2559–2563; 1963.

1018

O. Friaa et al. / Free Radical Biology & Medicine 45 (2008) 1011–1018

[25] Steenken, S.; Neta, P. One-electron redox potentials of phenols: hydroxy- and aminophenols and related compounds of biological interest. J. Phys. Chem. 86:3661–3667; 1982. [26] Capellos, C.; Bielski, B. H. J. Kinetic Systems. Wiley–Interscience, New York; 1972. [27] Cardillo, G.; Cricchio, R.; Merlini, L. Reaction of ortho alkenyl- and alkylphenols with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ). Tetrahedron 27:1875–1883; 1971. [28] Menger, F. M.; Carnahan, D. W. Comparison of phenolic couplings on potassium permanganate and potassium manganate surfaces. J. Org. Chem. 50:3927–3928; 1985. [29] Foti, M. C.; Daquino, C. Kinetic and thermodynamic parameters for the equilibrium reactions of phenols with the dpph radical. Chem. Commun. (Cambridge) 3252–3254; 2006. [30] Valgimigli, L.; Banks, J. T.; Ingold, K. U.; Lusztyk, J. Kinetic solvent effects on hydroxylic hydrogen atom abstractions are independent of the nature of the abstracting radical: two extreme tests using vitamin E and phenol. J. Am. Chem. Soc. 117:9966–9971; 1995. [31] Litwinienko, G.; Ingold, K. U. Abnormal solvent effects on hydrogen atom abstraction. 3. Novel kinetics in sequential proton loss electron transfer chemistry. J. Org. Chem. 70:8982–8990; 2005. [32] Litwinienko, G.; Ingold, K. U. Abnormal solvent effects on hydrogen atom abstraction. 2. Resolution of the curcumin antioxidant controversy: the role of sequential proton loss electron transfer. J. Org. Chem. 69:5888–5896; 2004.

˙

˙

[33] Musialik, M.; Litwinienko, G. Scavenging of DPPH radicals by vitamin E is accelerated by its partial ionization: the role of sequential proton loss electron transfer. Org. Lett. 7:4951–4954; 2005. [34] Neta, P.; Huie, R. E.; Maruthamuthu, P.; Steenken, S. Solvent effects in the reactions of peroxyl radicals with organic reductants: evidence for proton-transfermediated electron transfer. J. Phys. Chem. 93:7654–7659; 1989. [35] Musso, H. Phenol oxidation reactions. Angew. Chem. Int. Ed. Engl. 2:723–735; 1963. [36] Gulcin, I.; Dastan, A. Synthesis of dimeric phenol derivatives and determination of in vitro antioxidant and radical scavenging activities. J. Enz. Inhib. Med. Chem. 22:685–695; 2007. [37] Cudic, M.; Ducrocq, C. Transformations of 2,6-diisopropylphenol by NO-derived nitrogen oxides, particularly peroxynitrite. Nitric Oxide 4:147–156; 2000. [38] Foti, M. C. Antioxidant properties of phenols. J. Pharm. Pharmacol. 59:1673–1685; 2007. [39] Rigobello, M. P.; Stevanato, R.; Momo, F.; Fabris, S.; Scutari, G.; Boscolo, R.; Folda, A.; Bindoli, A. Evaluation of the antioxidant properties of propofol and its nitrosoderivative. comparison with homologue substituted phenols. Free Radic. Res. 38:315–321; 2004. [40] Gulcin, I.; Alici, H. A.; Cesur, M. Determination of in vitro antioxidant and radical scavenging activities of propofol. Chem. Pharm. Bull. (Tokyo) 53:281–285; 2005. [41] Lambert, C. R.; Black, H. S.; Truscott, T. G. Reactivity of butylated hydroxytoluene. Free Radic. Biol. Med. 21:395–400; 1996.