In Vitro  Kinetic Evaluation of the Free Radical Scavenging Ability of Propofol

• Propofol has antioxidant and free radical scavenging activity

• The free radical 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^·+) is used to study the kinetics of free radical scavenging

• Propofol covalently reduced ABTS^·+to ABTS and several propofol-ABTS compounds in vitro 

• The free radical scavenging activity of propofol was a fast and stable dynamic process

PROPOFOL (2,6-diisopropylphenol) is a widely used intravenous anesthetic (fig. 1) that has been approved for use in more than 50 countries.1Propofol, also marketed as Diprivan™ by AstraZeneca Pharmaceuticals LP (Wilmington, DE), has been extensively used during the induction and maintenance of general anesthesia,2although the anesthetic properties of propofol have not been explored in detail over the past 20 yr.3,4Propofol has also been documented to inhibit hydroxyl radical generation,5have antioxidant6,7and free radical scavenging activities,8,–,11and enhance ischemic tolerance of middle-aged hearts by inhibiting lipid peroxidation.12The antioxidant capacity of blood has been observed to increase in patients with the administration of propofol.13,–,17In addition, propofol can attenuate peroxynitrite-mediated DNA damage and apoptosis in cultured astrocytes.18Bayona et al.  reported that propofol has a neuroprotective effect on cerebral ischemia.19Propofol prevents cardiac fibroblast proliferation by interfering with the generation of reactive oxygen species and involves the activation of the protein kinase B-endothelial nitric oxide synthase-nitric oxide pathway.20Furthermore, propofol has been reported to reduce oxidative stress by inhibiting a nicotinamide adenine dinucleotide phosphate oxidase subunit.21 

The redox regulation in vitro  and in vivo  mainly includes the transfer of electrons and hydrogen atoms.22The compounds, 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, see structure and absorption spectra in fig. 1) and 2,2-diphenyl-1-picrylhydrazyl (DPPH), are used most often to probe the kinetics of radical scavenging activities.1,23,–,26The reaction between antioxidants and the free radical DPPH^·, which is widely used for assessing the ability of an antioxidant to transfer labile hydrogen atoms, has kinetic characteristics.1The colored free radical 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS^·+), which is also widely used to assess the ability of an antioxidant to transfer electron, has been extensively used to characterize antioxidants in solution.26,27Although the ABTS^·+has been previously used to assess propofol's antioxidant capacity,28a kinetic evaluation of ABTS^·+scavenging was not adequately described.

To investigate the reaction between propofol and free radicals, we studied the interaction kinetics of propofol with the synthetic stable radical ABTS^·+, which has been used extensively to investigate the kinetics of free radical scavenging by transferring electrons.24,25,29Kinetic analyses enabled us to demonstrate significant differences in reactivity between the subunits, such as propofol and ABTS^·+.

Propofol of 97% purity and ABTS were purchased from Sigma–Aldrich (St. Louis, MO). Propofol injection (Limengxin™) was purchased from Xi'an Libang Pharmaceutical Co., Ltd. (Xi'an, Shaanxi Province, China). Propofol injection (Diprivan™) was purchased from AstraZeneca S.p.A. (Basiglio, MI, Italy). Propofol with 1% Fresenius (Jing'an™) was purchased from Fresenius Kabi (Bad Homburg v.d.H., Germany, packing in Beijing Fresenius). Acetonitrile and methanol (for high performance liquid chromatography, 99.8% purity) were purchased from Fisher Scientific (Pittsburgh, PA). Other chemicals and reagents were products by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

A stock solution of ABTS^·+was prepared following the procedure of Re et al.  29with minor modifications. Specifically, the ABTS^·+solution was prepared by incubating 2.8 mM potassium persulfate with 7 mM ABTS in deionized water for at least 5 h. The solution was kept in the dark before use, and used within 24 h. The ABTS^·+stock solution was diluted 125-fold with 5 mM phosphate-buffered saline (PBS, pH 7.2) to yield a standard working solution containing approximately 36 μM ABTS^·+and 18 μM ABTS. Propofol was dissolved in methanol or dimethyl sulfoxide (DMSO) to prepare a solution with a concentration of 1 mM propofol. The reaction between ABTS^·+and propofol with a ratio of ABTS working solution:propofol of 249:1 (v/v) was followed by monitoring specific absorbance changes of ABTS and its radical form (ABTS^·+), blanking with PBS. After measuring the absorbance at time zero, propofol was added to the reaction solution. The absorbance was measured using a Cary 50 Ultraviolet-Visible Spectrophotometer (Varian Corporation, Palo Alto, CA) and analyzed by the Cary WinUV software (Varian Corporation).

The reaction between ABTS^·+and propofol was monitored by determining the reaction mixture using a Cary 50 Ultraviolet-Visible Spectrophotometer, blanking with PBS. The concentrations of ABTS and ABTS^·+were determined by absorbance scanning using the extinction coefficients of ε³⁴⁰= 4.8 × 10⁴M⁻¹cm⁻¹and ε⁴¹⁵= 3.6 × 10⁴M⁻¹cm⁻¹for ABTS and ABTS^·+, respectively.27The reaction rate of propofol and ABTS^·+during the first 10 min was obtained by the Cary WinUV software to analyze the absorbance values.

In kinetic experiments, PBS was added to a working solution of ABTS^·+based on the various ABTS^·+concentrations needed to produce different ABTS^·+/ABTS ratios and ABTS^·+/propofol ratios. The initial rate of ABTS^·+scavenging by propofol during the first 10 s of the reaction was estimated through a linear regression analysis of the ABTS concentration obtained by absorbance measurements using the Cary WinUV software. The reaction between ABTS^·+and propofol was prepared using a ratio of ABTS working solution:propofol of 249:1 (v/v). The total consumption of ABTS^·+and formation of ABTS products during the initial, rapid reaction phase were calculated by estimating the absorbance through linear regression analysis. The number of propofol of moles turned over for scavenging ABTS^·+were determined by a nonlinear regression analysis of initial reaction rate using different ABTS^·+to propofol ratios.

To determine the ABTS^·+scavenging activity of different propofol sources for clinical applications, we measured the free radical scavenging activity of the following three different propofol sources: Limengxin™ (propofol injection), Diprivan™ (propofol injection), and Jing'an™ (propofol 1% Fresenius). At the same time, the propofol (97% purity) was dissolved in Intralipid (Sino-Swed Pharmaceutical Corp. Ltd., Wuxi, Jiangsu, China) to obtain a 10 mg/ml propofol solution, as Diprivan™. The three clinically used propofol products and previously mentioned propofol solution were dissolved in methanol or DMSO to prepare a 0.1 mg/ml propofol solution. The reaction between ABTS^·+and propofol used a ratio of ABTS working solution:propofol of 99:1 (v/v). The reaction was monitored by measuring changes in the absorbance of ABTS and ABTS^·+, which was obtained by Cary 50 Ultraviolet-Visible Spectrophotometer using the software Cary WinUV.

After at least 5 h, analytical high-performance lipid chromatography (HPLC) measurements were performed on the ABTS^·+solution, containing 7 mM ABTS with 2.8 mM potassium persulfate in deionized water, following the procedure of Osman et al.  30,31with minor modifications. The ABTS^·+solution (500 μl) was incubated either with 60 μl propofol (100 mM, dissolved in methanol) for 30 min, after which the reaction was stopped by the addition of sodium azide (0.2 mM, final concentration). The reaction was then incubated at room temperature for 2 h. After a 10-fold dilution with deionized water, 20 μl of the reaction was loaded onto an analytical reversed-phase Econosphere C¹⁸(250 × 4.6 mm, 5 μm) column (Alltech Associates, Inc., Bannockburn, IL) using an analytical HPLC pump (Waters Corporation, Milford, MA). Eluent A was deionized water and eluent B was acetonitrile. The following gradient was used with a flow rate of 0.8 ml/min: 100% A for 4 min, 5% of B for 1 min, followed by a linear gradient 15–20% B in A (5–10 min), a linear gradient 20–30% B in A (10–15 min), a linear gradient 30–32% B in A (15–17 min), a linear gradient 32–35% B in A (17–20 min), a linear gradient 35–45% B in A (20–25 min), a linear gradient 45–55% B in A (25–27 min), a linear gradient 55–60% B in A (27–30 min), an isocratic 60% B in A (30–35 min), a linear gradient 60–45% B in A (35–37 min), a linear gradient 45–35% B in A (37–40 min), a linear gradient 35–15% B in A (40–45 min), a linear gradient 15–5% B in A (45–48 min), and 100% A (48–50 min).

An UltiMate3000 HPLC (Dionex Corporation, Sunnyvale, CA) and 3200 Q TRAP LC/MS/MS System (AB SCIEX, Foster City, CA) were used for liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) analysis following the procedure of Osman et al.  30,31with minor modifications. The reaction solution as described previously was diluted 200-fold with deionized water. The solution was injected directly into the liquid chromatography-mass spectrometry (LC-MS) spectrometer operating in positive mode with a flow rate of 0.8 ml/min, and the following gradient operations were the same as previously described. When the positive ion mode was applied, the following electrospray ionization source parameters were used: source voltage + 5.5 kV, capillary temperature 480°C, and sheath gas (nitrogen) flow of 55 (arbitrary units). For full-scan mass spectrometry (MS) analyses, spectra were acquired in the range of m /z  50–1500.

The kinetic data were plotted and fitted to the nonlinear Hill regression equation with rectangular hyperbolae using Origin 8.0 (OriginLab Ltd., Northampton, MA). All results represent the mean ± SD for six or seven independent experiments. The independent Student t  test was used to compare the means of two samples such as the reaction rate of propofol on ABTS^·+scavenging ability at each time point. To correct for multiple comparisons, an adjusted value of P < 0.01 was considered as statistically significant and indicated with asterisks as described in the figure legends.

The antioxidant capacity is important for the free radical scavenging activity of propofol and can be evaluated by using ABTS^·+.24The final concentrations of propofol and ABTS^·+used in this study were 4 μM and 36 μM, respectively. Full-band scanning (from 260 nm to 900 nm) of the solutions at different time points showed a decrease in the ABTS^·+-specific 415 nm and 735 nm absorption peaks and a corresponding increase of the ABTS-specific absorption peak at 340 nm (fig. 2, and see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 1, which illustrates wavelength scanning of DMSO and propofol (dissolved in DMSO) during ABTS^·+scavenging). Our data demonstrated that DMSO alone showed slow scavenging activity with detection by ABTS^·+, whereas methanol has no scavenging activity. However, propofol significantly boosted this scavenging process, indicating that propofol has fast and stable dynamic ABTS^·+scavenging activity.

To further confirm the dynamic scavenging activity of propofol on ABTS^·+, we measured the ABTS^·+scavenging process at 340 nm, and 415 nm, respectively. As shown in figure 3A and Supplemental Digital Content 1, http://links.lww.com/ALN/A846, figure 2A (which illustrates the kinetics of the scavenging activities of 4 μM propofol on ABTS^·+), the sample showed distinctive dynamic ABTS^·+scavenging activity. The ABTS^·+scavenging reaction of propofol could be divided into the following two steps: a fast reaction phase (approximately 5 s) and a slow reaction phase (after 5 s). The second phase was regarded as specific and needed to be further explored. After 5 s, ABTS^·+molecules had been consumed continuously, indicating that propofol may play a continuous role in the human body. Figure 3B and Supplemental Digital Content 1, http://links.lww.com/ALN/A846, figure 2B (which illustrates the kinetics of the scavenging activities of 4 μM propofol on ABTS^·+) illustrated how the rate of ABTS^·+scavenging and the total formation of ABTS were calculated. The scavenging rate by propofol on ABTS^·+was determined at different initial concentrations of ABTS^·+by calculating the changes in absorbance using ε⁴¹⁵. The rate of ABTS formation was independent of the concentrations of initial ABTS and reacted ABTS. However, the scavenging activities of propofol dissolved in methanol or DMSO were not equal. The scavenging activity of propofol dissolved in methanol seemed to be higher than in DMSO, indicating that DMSO might attenuate the free radical scavenging activity of propofol.

To determine the process of ABTS formation and ABTS^·+loss, the amount of total ABTS^·+and ABTS was calculated. Propofol reacted with ABTS^·+and converted the ABTS^·+(dark green in color) into colorless compounds, such as ABTS. The amount of the converted ABTS or reduced ABTS^·+was quantified by measuring the increase in absorbance at 340 nm or a decrease in absorbance at 415 nm. Our data showed that the rate of ABTS formation and ABTS^·+consumption rate of propofol are clearly different during the first 120 s of the reaction (fig. 4, and see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 3, which illustrates the rate of propofol on ABTS^·+scavenging ability). The ABTS^·+consumption rate of propofol dissolved in methanol was estimated to be 1.8742 ± 0.0869 μM/s in the first 5 s. When dissolved in DMSO, the ABTS^·+consumption rate of propofol was 1.7417 ± 0.3623 μM/s in the first 5 s. Therefore, our data indicated that propofol has a rapid rate of ABTS^·+consumption. The rate of ABTS^·+consumption by propofol was higher than ABTS formation during the reaction, suggesting that ABTS^·+scavenging activity of propofol is gradually increased.

To further probe the ABTS^·+scavenging kinetics of propofol, the reaction rate of the total consumption of ABTS^·+and ABTS formation was examined during the first 10 s of the reaction (fig. 5A, and see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 4A, which illustrates reaction rate and turnover number of ABTS^·+/ABTS at various ABTS^·+concentrations). Both the scavenging rate of ABTS^·+and the ABTS formation rate increased to a maximum concentration with increasing initial concentrations of ABTS^·+. The line in figure 5indicated that the scavenging rate of ABTS^·+exceeded the rate of ABTS formation. The Vmax  and Km  values for the decrease in the concentration of ABTS^·+when propofol was dissolved in methanol were calculated by the nonlinear Hill regression equation to be 1.4396 μM/s and 8.0509 μM, respectively. The Vmax  and Km  values in DMSO were 1.2935 μM/s and 8.0712 μM, respectively. The total ABTS formation and ABTS^·+consumption were then determined as functions of ABTS^·+scavenging ability of propofol through the initial ABTS^·+concentration within 10 s (fig. 5B, and see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 4B, which illustrates reaction rate and turnover number of ABTS^·+/ABTS at various ABTS^·+concentrations). From the data, we were able to estimate that approximately 36–37 ABTS^·+molecules could be scavenged by 10 propofol molecules when dissolved in methanol. In addition, 23–24 ABTS^·+molecules were converted by 10 propofol molecules during the first 10 s of the reaction when dissolved in methanol. Approximately 32–33 ABTS^·+molecules could be scavenged by 10 propofol molecules and 20–21 ABTS^·+molecules were converted by 10 propofol molecules during the first 10 s of the reaction when dissolved in DMSO. These data suggest that the ABTS^·+scavenging activity of propofol may be related to the solvent.

Using the ABTS^·+scavenging approach, we further examined the free radical scavenging activity of the following three propofol products widely used in clinical applications: Limengxin™, Diprivan™, and Jing'an™. Our data demonstrated that ABTS^·+could be significantly reduced by all three clinically used propofol products (see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 5, which illustrates wavelength scanning of three different propofol products widely used in clinical applications during ABTS^·+scavenging process). DMSO was observed to have a slow scavenging activity of ABTS^·+, but all three propofols significantly boosted this scavenging process, whereas methanol has no scavenging activity. These results indicate that all three clinical propofols have dynamic ABTS^·+scavenging activities.

In addition, all of the three above-mentioned propofol samples showed strong ABTS^·+scavenging abilities. There is no significant difference among these three propofols, dissolved in the same solvent, on the rate of ABTS^·+quenching (see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 6, which illustrates the dynamic scavenging activities of three different propofol products widely used in clinical applications on ABTS^·+). These data further indicate that the antioxidant activity of three different propofols widely used in clinical applications remained unchanged.

The HPLC chromatogram shows peaks corresponding to the products of the reaction between propofol and ABTS^·+(see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 7, which illustrates reversed-phase HPLC analysis of proposed products at 254 nm). Four peaks with retention times of 9.84, 16.18, 16.94 and 36.56 min could be detected. There was no product formation observed in the absence of the ABTS^·+radicals, indicating that propofol is stable under our experimental conditions (data not shown). Moreover, under the experimental conditions, the formation of degradation products was negligible in the absence of propofol (data not shown).

The masses of reaction products were readily determined in the positive ion mode. The base peaks of reaction product with retention times of 16.57, 18.51, 36.58 and 46.06 min in total ion chromatography (see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 8, which illustrates the full-scan electrospray ionization MS analysis of the products of the reaction between propofol and ABTS^·+) exhibited m /z  values of 453, 690 (see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 8B), 677 (see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 8C), 436, 453 (see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 8D), and 258 (see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 8E), respectively. These base peaks were selected and subjected to fragmentation to produce the MS/MS spectra. Fragmentation of the base peak of reaction products with retention times of 16.57 min yielded positive ions with m /z  of 453 and 690, respectively. At the same time, corresponding to successive increase of the MS fragments 22 (NaN³was added to stop the reaction, so there were Na⁺ions in the solution), LC-MS/MS fragmentation analysis of the major positive ion (m /z  453 and 690) yielded the other two positive ions (m /z  475 and 712), respectively (see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 8B). Fragmentation of the base peak of reaction products with retention times of 18.51 min yielded positive ions with m /z  of 677. Similar to the retention times of 16.57 min, corresponding to successive increase of the MS fragments 22, LC-MS/MS fragmentation analysis of the major positive ion (m /z  677) yielded the other ions (m /z  699) (see Supplemental Digital Content, http://links.lww.com/ALN/A846, fig. 8C). Although the two positive ions (m/a 339 and 350) were half of m /z  677 and 699, this means that the m /z  677 and 699 may carry two electric charges. Fragmentation of the base peak of reaction products with retention times of 36.58 min yielded positive ions with m /z  of 436. However, corresponding to successive increase of MS fragments 16 (the ABTS was diammonium salt used in this work, so there was NH³in the solution), LC-MS/MS fragmentation analysis of the major positive ion (m /z  436) yielded the other ions (m /z  453) (see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 8D). In addition, the fragmentation of retention times of 46.06 min yielded positive ions with m /z  of 258 (see Supplemental Digital Content 1, http://links.lww.com/ALN/A846, fig. 8E).

The products of propofol with ABTS were analyzed following previously described methods24,30,–,32with some minor modifications. The reaction product may be similar with phloroglucinol because of the structure of hydroxybenzene between propofol and phloroglucinol. These results, together with the HPLC analysis and LC-MS spectra of the reaction products, are consistent with the structures proposed in figure 6. In short, there are five major degradation products of the reactions between propofol and ABTS^·+, including ABTS-propofol, a part of the ABTS molecule, and combination of propofol with a part of the ABTS molecule (fig. 6).

The structure of propofol, a hindered phenolic, is similar to those of tocopherols, butylated hydroxytoluene, butylated hydroxyanisole, and α-tocopherol.33The antioxidant activities of butylated hydroxytoluene, butylated hydroxyanisole, and α-tocopherol have been reported.34,35The potential protective effect of propofol may be mediated by its antioxidant properties, which have been shown to play a protective role in apoptosis and ischemia–reperfusion injury.36However, the kinetics of free radicals scavenging ability of propofol has not been analyzed in previous studies.

In this paper, we have studied the reaction of propofol with ABTS^·+in vitro . Propofol is known to rapidly (in approximately 5 s) reduce ABTS^·+and produce ABTS. The structure of propofol contains a phenolic hydroxyl group, which is important for free radical scavenging activities. Our data strongly demonstrate that propofol has a fast radical scavenging rate. In addition, we show that there are five major degradation products of reactions between propofol and ABTS^·+, including ABTS-propofol, a part of the ABTS molecule, and combination of propofol with a part of the ABTS molecule. Thus, our data provided evidence that enables a better understanding of the beneficial effect of propofol in the body. In this study, the concentration of the propofol is 4 μM (0.7132 mg/l), whereas the dosage of propofol is 2 mg/kg at least in clinical practice. The concentration of the propofol being studied is less than the clinical relevance. Because propofol has clear antioxidant and free radical scavenging properties, the drug concentration in the body will have this activity.

From our data, we can see that the three clinically used propofol products still have the scavenging free radical abilities. In addition, all of the three above-mentioned propofols have strong ABTS^·+scavenging abilities. However, we cannot draw the conclusion that the Diprivan™ formulation of propofol has an enhanced scavenging ability toward ABTS^·+compared with the Limengxin™ and Jing'an™ formulations. Different formulations may contain additional components such as lecithin, which is reported to have its own antioxidant activity.37,38 

In summary, our studies demonstrated that propofol rapidly reacts with ABTS^·+. Furthermore, we propose a reaction experiment for radical scavenging by propofol based on the aforementioned dynamic experiments. Our current results suggest the following: (1) the rapid and stable radical scavenging capacity is an important characteristic of propofol; (2) the scavenging activity of propofol has the kinetics characteristic; and (3) the ABTS^·+scavenging ability of propofol is gradually increased during the time course of the reaction. Furthermore, the rate of ABTS^·+scavenging activity by propofol displayed significant kinetics. The kinetic feature of propofol for scavenging ABTS^·+in vitro  indicated that propofol might also play a role in the beneficial effect by scavenging free radicals resulting from surgical injury to the body.