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ABSTRACT: Unlike the nanoparticles with surface plasmon resonance, the optical response of polystyrene microparticles (PSMPs) is insensitive to the chemical components of the surrounding medium under the wavelength-dependent differential interference contrast (DIC) microscopy. This fact is exploited for the measurement of the refractive index of cytoplasm in this study. PSMPs of 400 nm in diameter were loaded into the cell to contact cytoplasm seamlessly, and the refractive index information of cytoplasm could be extracted by DIC microscopy operated at 420 nm illumination wavelength through the contrast analysis of PSMPs images.
Electrophoresis 03/2013; · 3.30 Impact Factor
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ABSTRACT: CE features superior separation efficiency, small solvent consumption, as well as the ability to analyze most biomolecules with an open tube fused-silica column. When coupled with MS, the separation power of CE is enhanced by adding another separation dimension based on mass-to-charge ratios. CE-MS reduces the dependence on CE separation so that faster analysis can be achieved. It also yields higher sensitivity as well as the capability for analyte identification and structural elucidation. The use of CE-MS for biomolecule analysis has increased significantly in the last 5 years. New methods are being developed for large molecules, while analyses of smaller molecules are moving toward the study in more complex tissues and other matrices. In this article, the applications of CE-ESI-MS for complex samples in 2007-2011 are reviewed. The applications are categorized according to the types of analytes studied, including the analysis for proteins and peptides, carbohydrates, and small biomolecules. Sample preparation methods, coatings for capillary inner wall, online processing strategies, and other aspects are also reviewed in each category.
Proteomics 08/2012; 12(19-20):2991-3012. · 4.43 Impact Factor
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ABSTRACT: Current prostate cancer (PCa) diagnosis based on prostate-specific antigen (PSA) has been gradually losing its credibility over the last decade due to contradictory results in published literature and clinical practice. Recently, a group of potential PCa biomarkers in urine, particularly sarcosine, was found to increase significantly as the cancer progressed to metastasis. We report a simple, robust, and reproducible CE-ESI-MS/MS method for the determination of sarcosine and other representative potential biomarkers in pooled urine. The pooled urine was obtained from 20 healthy adult volunteers between the ages of 23-30 years old. A solid phase extraction (SPE) technique was optimized for maximum recovery of sarcosine. With no derivatization step, excellent resolution between sarcosine and its isomers (α-alanine and β-alanine) was achieved. A separate non-SPE method was also developed for quantitative determination of highly concentrated urinary metabolites. CE separation was performed on a positively-charged, polyethyleneimine (PEI)-coated capillary using 0.4-2% formic acid in 50% methanol. Precision for intra- and inter-day standard addition calibration of sarcosine were found to be within 15%, whereas intra-day precisions for the rest of the metabolites varied from 0.03 to 13.4%. Acceptable intra-day and inter-day accuracies, ranging from 80 to 124%, were obtained for sarcosine and the other metabolites.
Journal of chromatography. A 07/2012; · 4.19 Impact Factor
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ABSTRACT: Asymmetric dimethylarginine (ADMA) is an inhibitor of nitric oxide synthase and a risk factor for cardiovascular events. We have developed a new derivatization method to enable baseline separation of the regio-isomers, ADMA, and symmetric dimethylarginine (SDMA), within 15 min on a C18 reverse phase column. Reacting naphthalene-2,3-dicarboxaldehyde with ADMA and SDMA in the presence of 2-mercaptoethanol produces corresponding 2,3-dihydro-benzo[f]isoindol-1-ones that are more stable than previously reported ortho-phthaldialdehyde and 2-mercaptoethanol derivatives. LC-MS/MS quantitation of these derivatives can be used to determine ADMA and SDMA concentrations in the plasma of patients to receive on-pump coronary artery bypass grafting (CABG) surgery. The LOD, LOQ and lower LOQ (LLOQ) of this method were determined to be 2.6, 8.7, and 25 nM for ADMA, and 2.5, 8.3, and 25 nM for SDMA, respectively, with consumption of only 50 μL of plasma. The relative standard deviations and relative errors of the intraday and interday determinations, as measurements of reproducibility and accuracy, are all within 15%. The ADMA and SDMA concentrations in patient plasma are 298.1 ± 11.2 nM (mean ± S.E.M., n = 123) and 457.7 ± 19.8 nM (mean ± S.E.M., n = 123), respectively. Upon unblinding of our clinical trial, these predetermined values might explain patient clinical outcomes associated with on-pump CABG surgery, as ADMA is known to inhibit nitric oxide production. Furthermore, this derivatization reaction in conjunction with LC-MS/MS analysis may open a venue to explore alternative chemical labeling modes for LC-MS/MS applications, such as analysis of other amino acids, metabolites, and peptides containing primary amine group(s).
Electrophoresis 07/2012; 33(12):1911-20. · 3.30 Impact Factor
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ABSTRACT: An atmospheric ion lens incorporated into an electrospray ion source for capillary electrophoresis-mass spectrometry (CE-MS) is found to extend the stable operational regions for both flow rates and electrospray ionization (ESI) voltages. The stable operating conditions for the ESI source with and without the ion lens were characterized. The results showed that the stable operation region was widest when the voltage difference between the sprayer and the ion lens ranges from 2.6 to 2.8 kV, and under these condition, the CE-MS interface can be adapted to a broader range of electroosmotic and modifier flow rates. Modeling of the electric field in the electrospray ion source with the ion lens suggests that the extension of the stable region is attributed to the flatter equipotential surfaces around the sprayer tip and higher electric field strengths in the rest of the interface region.
Electrophoresis 04/2012; 33(8):1322-30. · 3.30 Impact Factor
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ABSTRACT: A sensitive and robust LC-MS/MS method was developed to quantify basal adenosine concentrations in human plasma of patients undergoing on-pump coronary artery bypass grafting (CABG) surgery. A strong cation exchange (SCX) monolithic cartridge was used to enrich analyte, improve robustness, and reduce biological complexity. A simple modifier-free mobile phase was employed to improve sensitivity and reproducibility. This method exhibits consistent precision and accuracy, and the RSDs or REs of all the intraday and interday determinations were within 10%. The calibration curve was linear across the examined dynamic range from 1nM to 500nM (r(2)=0.996). LOD and LOQ were determined to be 0.257nM and 0.857nM respectively, while LLOQ was below 10nM. This method was used to monitor changes of adenosine levels in patient plasma drawn intraoperatively during on-pump CABG surgery. The analysis of 84 patients revealed that the mean concentration of adenosine in coronary sinus plasma after cardiopulmonary bypass (CPB) is higher than that in coronary sinus before CPB (p=0.0024; two-tailed t-test) and that in radial artery plasma after CPB (p=0.0409; two-tailed t-test). These findings suggest that the equilibrium between adenosine production and elimination has favored the elevation of adenosine basal level during on-pump CABG surgery and the change is specific to heart tissues. Evaluation of adenosine with a sensitive and robust analytical method has important implications on providing consistent results and meaningful insights into adenosine regulation, as well as its steady state and sustained action on the heart. Relating patient characteristics or clinical outcomes with basal adenosine concentration can be used to optimize the CABG-CPB maneuver by regulating adenosine level via pharmacological intervention, and differentiating adenosine's contribution to cardioprotection from other modulatory factors.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 02/2012; 885-886:30-6. · 2.78 Impact Factor
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ABSTRACT: We have developed a simple, sensitive, and robust liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method to determine free 3-nitrotyrosine concentrations in human plasma of patients receiving on-pump coronary artery bypass grafting surgery. A one-step solid-phase extraction protocol was optimized to enrich the analyte at low nanomolar concentrations. The processed samples were analyzed by LC-MS/MS with a 2.1 × 100 mm Kinetex PFP column and a triple quadrupole mass spectrometer. The method was validated for 3-nitrotyrosine concentrations close to real patient plasma levels. The relative standard deviations or relative errors of the intraday and interday determinations were all within 10%. Limit of detection and limit of quantitation were determined to be 0.034 nM and 0.112 nM, respectively, while lower limit of quantitation was below 0.625 nM. No deterioration of the column performance was noticed after running a large number of patient samples. The results showed that the 3-nitrotyrosine concentrations in coronary sinus plasma samples were elevated after cardiopulmonary bypass (CPB) procedure. The pre-CPB and post-CPB concentrations of 3-nitrotyrosine in patient plasmas were 1.494 ± 0.107 nM and 2.167 ± 0.177 nM (mean ± SEM), respectively. Application of this method to more patients in clinical studies may help validate 3-nitrotyrosine as a meaningful biomarker for nitrosative stress and link patient characteristics, clinical outcomes, and cardioprotective treatments to endogenous nitrosative stress levels.
Electrophoresis 02/2012; 33(4):697-704. · 3.30 Impact Factor
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ABSTRACT: Myocardial ischemia-reperfusion injury is an important cause of impaired heart function in the early postoperative period subsequent to cardiac surgery. Reactive oxygen species (ROS) generation increases during both ischemia and reperfusion and it plays a central role in the pathophysiology of intraoperative myocardial injury. Unfortunately, the cellular source of these ROS during ischemia and reperfusion is often poorly defined. Similarly, individual ROS members tend to be grouped together as free radicals with a uniform reactivity towards biomolecules and with deleterious effects collectively ascribed under the vague umbrella of oxidative stress. This review aims to clarify the identity, origin, and progression of ROS during myocardial ischemia and reperfusion. Additionally, this review aims to describe the biochemical reactions and cellular processes that are initiated by specific ROS that work in concert to ultimately yield the clinical manifestations of myocardial ischemia-reperfusion. Lastly, this review provides an overview of several key cardioprotective strategies that target myocardial ischemia-reperfusion injury from the perspective of ROS generation. This overview is illustrated with example clinical studies that have attempted to translate these strategies to reduce the severity of ischemia-reperfusion injury during coronary artery bypass grafting surgery.
Pharmacology [?] Therapeutics 11/2011; 133(2):230-55. · 8.56 Impact Factor
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ABSTRACT: A capillary electrophoresis mass spectrometry (CE-MS) interface utilizing a flow-through microvial is used to ensure the electric continuity and supply the catholyte and mobilizer solutions during the capillary isoelectric focusing (cIEF) and mobilization process. The flow-through microvial provides a stable chemical environment and helps to improve the ionization efficiency without significantly diluting the analyte. The CE-MS interface facilitates the transfer of the mobilized cIEF effluent to the site of electrospray ionization, and the gaseous ions can be detected directly by a mass spectrometer. It also allows for complete focusing and mobilization processes to be performed automatically in programmed sequences with commercial CE systems. Two different strategies, using either a part of the capillary or the flow-through microvial of the CE-MS interface as the catholyte reservoir for bare fused silica capillaries or neutral coated capillaries, respectively, were developed for automated cIEF-electrospray ionization (ESI)-MS. Reasonable separation efficiency was achieved using proper concentration of carrier ampholytes and suitable strategies of electroosmotic/electrophoretic mobilization.
Analytical Chemistry 11/2011; 83(22):8748-55. · 5.86 Impact Factor
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ABSTRACT: Peri-implant infections have been reported as one of the major complications that lead to the failure of orthopedic implants. An ideal solution to the peri-implant infection is to locally deliver antimicrobial agents through the implant surface. The rising problem of infections caused by multiple antibiotic-resistant bacteria makes traditional antibiotics less desirable for the prevention of peri-implant infections. One of the promising alternatives is the family of antimicrobial peptides (AMPs). In this study, we report the local delivery of AMPs through the nanotubular structure processed on titanium surface. Self-organized and vertically oriented TiO(2) nanotubes, about 80 nm in diameter and 7 μm thick, were prepared by the anodization technique. HHC-36 (KRWWKWWRR), one of the most potent broad-spectrum AMPs, was loaded onto the TiO(2) nanotubes via a simple vacuum-assisted physical adsorption method. Antimicrobial activity testing against Gram-positive bacterium, Staphylococcus aureus, demonstrated that this AMP-loaded nanotubular surface could effectively kill the bacteria (˜99.9% killing) and reduce the total bacterial number adhered to the surface after 4 h of culture. In vitro AMP elution from the nanotubes was investigated using liquid chromatography-mass spectrometry (LC-MS). The release profiles strongly depended on the crystallinity of the TiO(2) nanotubes. Anatase TiO(2) nanotubes released significantly higher amounts of AMP than amorphous nanotubes during the initial burst release stage. Both followed almost the same slow release profile from 4 h up to 7 days. Despite the differences in release kinetics, no significant difference was observed between these two groups in bactericidal efficiency. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2011.
Journal of Biomedical Materials Research Part A 11/2011; · 2.63 Impact Factor
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ABSTRACT: A method for adapting widely used CE conditions for the separation of fluorescently labeled carbohydrates to permit online ESI-MS detection is presented. Reverse polarity separations were performed in bare fused-silica capillaries with an acidic BGE. Under these conditions, negatively charged 8-aminopyrene 1,3,6-trisulfonate-labeled carbohydrates migrate forward against the EOF, which is towards the capillary inlet. Therefore, the CE-MS interface must simultaneously back-fill the capillary, in order to maintain the CE circuit, and provide a stable forward flow at the sprayer tip to support the electrospray process. This was achieved using a junction-at-the-tip interface, which provides a flow of solution to the junction formed by the capillary terminus and the inner wall of the emitter needle tip. Because the flow rate required for this arrangement is much less than in conventional sheath flow interfaces, dilution of the analytes is minimized. Optimized separation conditions permit baseline resolution of glucose oligomers containing up to 15 glucose units, while longer oligomers, up to 33 glucose units, were observed as resolved peaks in the negative ion mode mass spectrum.
Electrophoresis 07/2011; · 3.30 Impact Factor
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ABSTRACT: When coupling capillary electrophoresis with postcolumn detection methods, such as mass spectrometry, the presence of postcolumn band broadening must be considered. The band broadening effects introduced by junction-at-the-tip CE-MS interfaces using a postcolumn micro flow-through vial are investigated by studying the hydrodynamic flow patterns and mass transport process inside the micro vial at the end of the CE separation capillary. Simulation results obtained by solving the Navier-Stokes and mass balance equations provide insights into the velocity field and concentration distribution of the analytes in the micro vial and demonstrate that, with a low flow rate of chemical modifier solution, the laminar flow streams confine the analyte molecules to the central part of the micro vial and thus maintain major features of the peak shapes. Peaks detected by UV and MS under similar experimental conditions were compared to verify the numerical prediction that the main features of the UV peak can be retained in the MS peak. Experiments also show that band broadening can be minimized when an appropriate chemical modifier flow rate is selected.
Analytical Chemistry 06/2011; 83(12):4916-23. · 5.86 Impact Factor
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ABSTRACT: Mass conservation is the guiding principle for analyte distribution at channel intersections of microfluidic devices, where analyte migration is mainly driven by an applied electric field, and in electro-fluid-dynamic (EFD) devices, where multiple fields and pressures can be applied simultaneously on the same channel network. This paper introduces another type of conservation, the conservation of effective volumetric flow rate, at channel intersections when the conductivity of the solution in the intersecting channels is maintained constant. This conservation principle provides an additional criterion needed to describe analyte migration in channels connecting to a common intersection and to predict how analyte is distributed into individual channels in the channel network of EFD devices, when multiple voltages and pressures are applied. The theoretical bases of effective volumetric flow rate balance are discussed, and the potential use of this principle in conjunction with the principle of mass conservation to predict the migration behavior of analytes is demonstrated. Junctions of different geometry in EFD devices are used to demonstrate the validity of these equations, and the measured velocities and numbers of microbeads in each channel agree with the predicted values.
Analytical Chemistry 02/2011; 83(4):1189-92. · 5.86 Impact Factor
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ABSTRACT: Patients with diabetes experience increased cardiovascular complications after cardiac surgery. Hyperglycaemia predicts increased mortality after myocardial infarction and may influence cardiovascular risk in humans. Impaired prosurvival phosphatase and tensin homologue on chromosome 10 (PTEN)-Akt signaling could be an important feature of the diabetic heart rendering it resistant to preconditioning. This study was designed to evaluate for differences and relationships of myocardial PTEN-Akt-related signaling and baseline glycaemic control marker in type 2 diabetic and non-diabetic patients undergoing coronary artery bypass surgery.
Methods
Right atrial biopsies and coronary sinus blood were obtained from 18 type 2 diabetic and 18 non-diabetic patients intraoperatively. Expression and phosphorylation of Akt, eNOS, Bcl-2 and PTEN were evaluated by western blot. Plasma 15-F2t-isoprostane concentrations were evaluated by liquid chromatography-mass spectrometry.
Results
PTEN expression and 15-F2t-isoprostane concentrations were significantly higher in diabetic patients. Increased fasting blood glucose levels correlated with increased coronary sinus plasma 15-F2t-isoprostane concentrations. Increased cardiac 15-F2t-isoprostane generation was highly correlated with myocardial PTEN expression. Bcl-2 expression and eNOS phosphorylation were significantly lower in diabetic compared to non-diabetic patients. Akt phosphorylation tended to be lower in diabetic patients, however this tendency failed to reach statistical significance.
Conclusion
The current results suggest that prosurvival PTEN-Akt signaling is impaired in the diseased diabetic myocardium. Hyperglycaemia and increased oxidative stress may contribute to this phenomenon. These findings strengthen the understanding of the underlying biologic mechanisms of cardiac injury in diabetic patients, which could facilitate development of new treatments to prevent cardiovascular complications in this high-risk population.
Clinical Endocrinology 01/2011; 74(6):705-713. · 3.17 Impact Factor
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ABSTRACT: Because electric field distribution is determined by emitter size and shape, sprayer tip geometry determines the optimum liquid flow rate that can be processed by the electrospray ionization interface. Electric field is the highest at the sharpest edge of an electrode; therefore, for a beveled tip, the field is highest at the very tip, and for a conventional symmetrically tapered tip, the field is the highest around the rim of the electrode. Electrospray performance as a function of flow rate was investigated using both continuous infusion and peak-based analysis. The sharpest symmetrical emitter gave the most stable electrospray ionization (ESI) at flow rates ≤0.10 μL/min, while beveled emitters provided significantly better performance at expanded flow rates up to 1 μL/min. The use of beveled emitters offers the potential for increased versatility in electrospray ionization interfaces.
Analytical Chemistry 10/2010; 82(20):8377-81. · 5.86 Impact Factor
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ABSTRACT: Adipocere is a postmortem decomposition product consisting of mostly a mixture of free fatty acids (FFAs) that are formed because of the hydrolysis of triglycerides in adipose tissues. This article describes a simple and robust method for the extraction, identification, and quantification of FFA commonly found in adipocere using gas chromatography–mass spectrometry (GC/MS). This method was applied to analyze tissues from Kwäday Dän Ts’ìnchí, ancient remains discovered in a retreating glacier in the Tatshenshini-Alsek Park, British Columbia, Canada in August 1999. The lyophilized tissues were grinded and extracted with hexane. The trimethylsilyl fatty acid derivatives were analyzed by GC/MS, and the relative abundances of myristic acid, palmitic acid, oleic acid, and stearic acid were determined. Milligram per gram levels of saturated fatty acids were found in the tissues of the ancient remains, while the levels of unsaturated fatty acids, such as palmitoleic acid, were found to be negligible. The results provided further evidence of the existence of adipocere found during forensic examination of the Kwäday Dän Ts’ìnchí ancient remains.
Journal of Forensic Sciences 06/2010; 55(4):1039 - 1043. · 1.23 Impact Factor
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ABSTRACT: Adipocere is a postmortem decomposition product consisting of mostly a mixture of free fatty acids (FFAs) that are formed because of the hydrolysis of triglycerides in adipose tissues. This article describes a simple and robust method for the extraction, identification, and quantification of FFA commonly found in adipocere using gas chromatography-mass spectrometry (GC/MS). This method was applied to analyze tissues from Kwäday Dän Ts'ìnchí, ancient remains discovered in a retreating glacier in the Tatshenshini-Alsek Park, British Columbia, Canada in August 1999. The lyophilized tissues were grinded and extracted with hexane. The trimethylsilyl fatty acid derivatives were analyzed by GC/MS, and the relative abundances of myristic acid, palmitic acid, oleic acid, and stearic acid were determined. Milligram per gram levels of saturated fatty acids were found in the tissues of the ancient remains, while the levels of unsaturated fatty acids, such as palmitoleic acid, were found to be negligible. The results provided further evidence of the existence of adipocere found during forensic examination of the Kwäday Dän Ts'ìnchí ancient remains.
Journal of Forensic Sciences 04/2010; 55(4):1039-43. · 1.23 Impact Factor
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ABSTRACT: An interface for CE-ESI-MS that decouples both the electrical and the solution flow rate requirements of the separation and ionization processes is presented. The interface uses a tapered and beveled stainless steel hollow needle surrounding the separation capillary terminus so that the inside of the electrode acts as the CE outlet vial and the outside tip acts as the electrospray emitter. No capillary pre-treatment is required, enabling the use of capillaries with any type of surface modification. A chemical modifier solution is introduced through a second capillary connected to the needle via a tee junction and can be used to improve the compatibility of the CE BGE with electrospray. The flow rate of modifier solution can be as low as 0.1 microL/min, much less than that in a typical sheath-flow interface, thus minimizing dilution of the CE effluent in order to maximize sensitivity. The presence of the modifier solution also allows the use of neutral-coated capillaries for protein analysis by CE-MS without using an assisting pressure, despite the absence of EOF under these conditions. The interface is easily integrated into a commercial CE instrument, such that all operations can be carried out by the automated controls. Compared with a commercial sheath-flow CE-MS interface operating under optimized conditions, LODs for amino acids were, on average, improved fivefold.
Electrophoresis 03/2010; 31(7):1130-7. · 3.30 Impact Factor
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ABSTRACT: Mixing of two solutions into one is a spontaneous process with a net increase in entropy. However, the reverse of the mixing process is usually not possible unless certain conditions are met. A continuous solution stream containing a mixture of two compounds can be separated into two channels, each containing a pure compound, thus reversing the mixing process using a two-dimensional microfluidic electro-fluid-dynamic (EFD) device. When the electric field is strategically applied in the interconnecting channels of an EFD device, the pressure required to direct an analyte into a certain channel can be calculated by using the solutions of electric field and fluid dynamics in the mass balance equation. If the pressure and electric potential at various inlets and outlets satisfy these predetermined conditions, the reverse of a mixing process is observed. Conventional microfluidic devices have been used to introduce samples from interconnecting channels or efficiently mix different solutions into a single channel. The EFD devices expand the spatial separation of analytes from one dimension to two using both the differential migration behavior of analytes and the velocity field distribution in different channel geometries. The devices designed according to these basic physicochemical principles can be used for complete processing of minute samples and to obtain pure chemical species from complex mixtures.
Analytical Chemistry 02/2010; 82(6):2182-5. · 5.86 Impact Factor
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ABSTRACT: Propofol is a widely used intravenous anesthetic agent with antioxidant properties secondary to its phenol based chemical structure. Treatment with propofol has been found to attenuate oxidative stress and prevent ischemia/reperfusion injury in rat heart. Here, we report that propofol protects cardiac H9c2 cells from hydrogen peroxide (H(2)O(2))-induced injury by triggering the activation of Akt and a parallel up-regulation of Bcl-2. We show that pretreatment with propofol significantly protects against H(2)O(2)-induced injury. We further demonstrate that propofol activates the PI3K-Akt signaling pathway. The protective effect of propofol on H(2)O(2)-induced injury is reversed by PI3K inhibitor wortmannin, which effectively suppresses propofol-induced activation of Akt, up-regulation of Bcl-2, and protection from apoptosis. Collectively, our results reveal a new mechanism by which propofol inhibits H(2)O(2)-induced injury in cardiac H9c2 cells, supporting a potential application of propofol as a preemptive cardioprotectant in clinical settings such as coronary bypass surgery.
Biochemical and Biophysical Research Communications 09/2009; 389(1):105-11. · 2.48 Impact Factor
