Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2015 www.elsevier.com/locate/jbiosc
Imaging the oxidation effects of the Fenton reaction on phospholipids at the interface between aqueous phase and thermotropic liquid crystals Minmin Zhang and Chang-Hyun Jang* Department of Chemistry, Gachon University, Seongnam-Si, Gyeonggi-Do 461-701, South Korea Received 13 June 2014; accepted 18 December 2014 Available online xxx
The lipid peroxidation process has attracted much attention because of the growing evidence of its involvement in the pathogenesis of age-related diseases. Herein, we report a simple, label-free method to study the oxidation of phospholipids by the Fenton reaction at the interface between an aqueous phase and immiscible liquid crystals (LCs). The different images produced by the orientation of 4-cyano-40 -pentylbiphenyl (5CB) corresponded to the presence or absence of oxidized 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG). The oxidation effects of the Fenton reaction on DOPG were evaluated by monitoring the orientational response of liquid crystals upon contact with the oxidized DOPG solutions. DOPG was oxidized into chain-changed products containing hydroxy, carbonyl, or aldehyde groups, resulting in the rearrangement of the phospholipid layer. This induced the orientational transition of LCs from homeotropic to planar states; therefore, a dark to bright optical shift was observed. This shift was due to the Fenton reaction preventing DOPG to induce the orientational alignment of LCs at the aqueous/LC interface. We also used an ultraviolet spectrophotometer to confirm the effects of oxidation on phospholipids by the Fenton reaction. Using this simple method, a new approach for investigating phospholipid oxidation was established with high resolution and easy accessibility. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Oxidation; Phospholipids; Liquid crystals; Orientational transition; Fenton reaction]
Phospholipids are the most abundant lipids in cell membranes. Because of their amphiphilic nature, they form spherical lipid bilayers known as liposomes or vesicles (1,2). They are important in mammalian cell biology because they act as lipid mediators by providing a permeable barrier as well as substrates for synthesis (3,4). The role of lipid oxidation in disease is still a subject of considerable research, and within this area, there is growing interest in the roles of oxidized phospholipids, which have been detected in pathological conditions, often at raised levels compared to those from subjects with normal physiology (5). There is substantial evidence that the oxidized phospholipids of oxidized lowdensity lipoprotein accumulate in vivo and play an important role in cardiovascular disease (6e8). The Fenton reaction is defined as the oxidation of organic substrates by a mixture of hydrogen peroxide and ferrous iron (9). The oxidizing intermediates involved in Fenton reactions are thought to cause damage to phospholipids and play a significant role in the aging process and a variety of diseases, such as cancer (10). The Fenton reaction is a key reaction in the oxidation of phospholipids. Notably, this reaction is thought to occur in heart diseases, such as ischemia and reperfusion (11). Many researchers doubt that the significance and existence of the Fenton reaction in biological systems is due to the low concentrations of H2O2 and free iron. They
* Corresponding author. Tel.: þ82 31 750 8555; fax: þ82 31 750 8774. E-mail address: [email protected] (C.-H. Jang).
also suggest that the high and undiscriminating reactivity of hydroxyl radicals produce great limitations on diffusion and cause more wide-ranging damage to biomolecules (10). Assays for identifying and characterizing the oxidation of phospholipids have been the subject of many studies. Development of these assays include: (i) separation of phospholipid oxidation products by mass spectrometry (12), (ii) biological functions of oxidized phospholipids (13), (iii) identification of new families of bioactive phospholipids generated by immune cells (3), and (iv) measurement of the oxidation of phospholipids by physicochemical tests (14). A. Reis et al. (12) used mass spectrometry coupled with high performance liquid chromatography (HPLC) separation techniques (HPLC-MS) to study phosphatidylcholine peroxidation products. They developed a reverse-phase liquid chromatography method coupled to electrospray mass spectrometry for the separation of glycero-phosphatidylcholine peroxidation products formed by the Fenton reaction. In addition, Domingues et al. (15) used tandem mass spectrometry with a method of soft ionization (electrospray and matrix-assisted laser desorption ionization) to characterize changes in fatty acyl chain. Qian and Buettner (16) also used electron paramagnetic resonance spin trapping to examine iron-induced free radical oxidations. All of these large facility-based methods are time consuming and require sophisticated instrumentations. In the past decade, long-range orientational anchoring of liquid crystals has become one of the most considerable tools in the field of biological assays, particularly based on the effects of interfacial
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.12.016
Please cite this article in press as: Zhang, M., and Jang, C.-H., Imaging the oxidation effects of the Fenton reaction on phospholipids at the interface between aqueous phase and thermotropic liquid crystals, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2014.12.016
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interactions of biomolecules. The property of birefringence of LCs, due to their anisotropic nature, allows the orientational change of LCs to be observed under a polarized optical microscopy, which is visible with naked eye. We recently have shown that phospholipid monolayer spontaneously assembled at the aqueous/liquid crystal interface is correlated to the orientation of LCs (17e19). The biological enzyme activity involved in combining phospholipids is demonstrated to trigger orientational transition of LCs that can be readily visualized under crossed polarizers. This correlation provides principles to transduce and amplify biological enzymatic events that occur at these interfaces. In the present study, we developed an LC-based approach for the label-free characterization of oxidized phospholipids by the Fenton reaction occurring at the interface between aqueous phase and immiscible liquid crystals. We attempted to explore the activities of iron(II) and hydrogen peroxide towards 1,2-dioleoyl-snglycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG) membrane at the aqueous/LC interface and their correlation to the orientational transitions of LCs. The impacts of different catalysts and pH on the Fenton oxidation were also explored. To the best of our knowledge, an LC has never been utilized to report the effects of oxidation of phospholipids by the Fenton reaction before. This study suggests the applicability and superiority of a label-less and low-cost LC-based approach for identifying phospholipids oxidized by the Fenton reaction and for screening their potential uses. MATERIALS AND METHODS Materials Tris buffered saline (TBS; 0.05 M Tris, 0.138 M NaCl, and 0.0027 M KCl, pH 8.0), sulfuric acid, hydrogen peroxide (30% w/v), octyltrichlorosilane (OTS), DOPG, sodium chloride, hydrochloric acid, sodium hydroxide, chloroform, ethanol, methanol, methylene dichloride, and capillary tubes were purchased from SigmaeAldrich (USA). Liquid crystal, 40 -pentyl-4-cyanobiphenyl (5CB), was purchased from EM industries (Hawthorne, NY, USA). Copper specimen grids (50 meshes, 500 mm pitch, 420 mm hole, 80 mm bar, 25 mm 5 mm thickness) were obtained from Gilder Grids (Grantham, UK). Premium glass microscope slides were purchased from Fisher Scientific (Pittsburgh, PA, USA). Ultrapure water, with a resistivity of 18.2 MU cm, was obtained from a Milli-Q system (Millipore, Bedford, MA, USA). Treatment of glass microscope slides with OTS Glass microscope slides were cleaned in accordance with a previously described procedure (17). Briefly, the slides were immersed in the piranha solution [70% (v/v) sulfuric acid and 30% (v/v) hydrogen peroxide] for 30 min at w80 C (warning: piranha solution reacts strongly with organic compounds and should be handled with extreme caution; do not store the solution in closed containers). The slides were then rinsed with water, ethanol, and methanol and dried under a stream of gaseous nitrogen, after which they were heated at 120 C overnight prior to OTS deposition. A 0.5 mM OTS solution was prepared and the piranha-cleaned slides were immersed in 0.5 mM OTS in a heptane solution at room temperature for 30 min. The samples were then rinsed with methylene dichloride and dried under nitrogen. Next, films of nematic LC [4-cyano-40 -pentylbiphenyl (5CB)] were deposited into the pores of gold grids on octadecyltrichlorosilane (OTS)ecoated glass substrates, the OTS-coated glass caused the LC to assume a perpendicular (homeotropic) orientation at the glass surface. Any sample that did not exhibit homeotropic anchoring of 5CB was rejected. Preparation of a glass slide-supported LC optical cell A glass slidesupported LC optical cell was prepared as previously reported (19). Simply put, OTS-treated glass slides were fixed to the bottom of an eight-well chamber slide with silicone mounting medium. Subsequently, transmission electron microscopy grids (50 meshes and 25 mm 5 mm thickness) were placed onto the slide. The copper grid was then impregnated with 2 mL of 5CB using a Hamilton syringe. By heating to its isotropic phase (>35 C), excess 5CB was removed from the LCs with a 20-mL capillary tube. The grids containing LCs were then immersed into aqueous solutions of interest. Each assay was performed at least six times independently. Preparation of phospholipid vesicles LCs, decorated with phospholipid membrane, were prepared from DOPG phospholipid solution following previously reported procedures (20). Simply put, phospholipids dissolved in chloroform (50 mg/mL) were dried under a stream of nitrogen and desiccated under vacuum for at least 3 h. Dried phospholipids were resuspended with Tris-buffered saline (TBS, consisting of 0.05 M Tris and adjusted to a pH of 5.8 prior to use). The final concentration of DOPG phospholipid solution was 1 mM. The turbidity of the resulting solution represented the presence of large multilamellar vesicles. The phospholipid suspension was then sonicated three times each for 5 min to clarify
J. BIOSCI. BIOENG., using a probe sonicator. Then, the solution was filtered twice with a 0.22-mm filter and typically used within 2 d after the preparation. Prior to the formation of the DOPG membrane, 400 mL of DOPG solution was added to one of the wells supported on the glass slide. The DOPG membrane was then formed by touching a copper grid impregnated with 5CB to the DOPG solution in the well. Preparation of oxidized phospholipid vesicles The phospholipid vesicles prepared as described above were diluted to a final concentration of 0.5 mM and the mixture was vortexed. Oxidative treatments using Fe(Ⅱ) and H2O2 were performed as per the procedure detailed in a previous publication (21). 5 mL FeSO4 solution (50 mM) and 500 mL of hydrogen peroxide (H2O2) at a certain concentration are added to 500 mL of the phospholipid vesicles. This mixture was then left to react at room temperature for 2 h under continuous shaking. LC-based technique for testing the oxidation of phospholipid Prior to the identification of the oxidation effects of hydrogen peroxide on phospholipids, 400 mL of a solution containing oxidized phospholipids (in 0.05 M Tris, 0.138 M NaCl, and 0.0027 M KCl; the pH was adjusted prior to use) was added to individual wells. 5CB (confined in copper grids) was then immersed in these oxidized phospholipid solutions. Optical examination of LC texture The optical texture of the LCs was examined using a Nikon eclipse LV100 POL microscope equipped with crossed polarizers in transmission mode. The sample was placed on a rotating stage between the crossed polarizers. All optical images were captured using a digital camera (DS2Mv, Nikon, Tokyo, Japan) mounted on the microscope with a resolution of 1600 1200 pixels, a gain of 1.00, and a shutter speed of 1/10 s. Determination of phospholipid oxidation by UV/Vis spectroscopy The percent oxidation of the phospholipid was determined using Beer’s Law by calculating the peroxidized phospholipid concentration from the absorbance at 235 nm. An aliquot (50 mL) of the reaction mixture was taken out and diluted with ethanol to 500 mL. The absorbance at 235 nm was measured with a Varian 50 Bio UV/Vis spectrophotometer.
RESULTS AND DISCUSSION LC-based method for imaging oxidation effects of the Fenton reaction on bioactive phospholipids The oxidation of lipids has been a topic of interest in biochemical and food sciences for a long time. The basic principles of non-enzymatic free radical effects on phospholipids are well established, although questions about detailed mechanisms remain unanswered. In this LC-based technique, the phospholipid monolayer at the aqueous/LC interface was used as the oxidation substrate. Phospholipids, because of their amphiphilic nature, form spherical lipid bilayers known as liposomes and vesicles. When phospholipid vesicles are exposed to a hydrophobic surface, they fuse with the surface and form a planar membrane, leading to the formation of a lipid monolayer. Previous studies have demonstrated that the presence and organization of amphiphiles at the interface between liquid crystals (LCs) and an immiscible aqueous phase is coupled to the orientational ordering of the LCs. Brake et al. (30) reported that contact the interface of a thermotropic LC with an aqueous solution of phospholipid resulted in the spontaneous assembly of a monolayer of phospholipid at the aqueous/LC interface, corresponding to a dark image in the optical response. Fig. 1 shows the schematic illustration of the experimental system. A phospholipid monolayer was prepared by exposing the 5CB-filled copper grid to an aqueous solution containing 500 mM of DOPG. Following the exposure, the initially bright optical images of 5CB became uniformly dark (Fig. 1A), indicating the self-assembly of DOPG at the aqueous/LC interface. Films of 5CB laden with DOPG were then washed with TBS buffer five times and were stable for more than a week. However, when 5CB films were exposed to phospholipids oxidized by the Fenton reaction, the optical images of 5CB evolved from dark to bright colors (Fig. 1B). This observation is in accordance with previous studies demonstrating that the orientational changes of LCs correspond to the formation of the phospholipid monolayer at aqueous/LC interfaces (22). We also examined the optical response of LCs after introducing the Tris buffer solution (TBS) into the chamber containing LCs and observed a bright appearance (Fig. 2A), which was attributed to the planar orientation of LC molecules at the aqueous/LC interface.
Please cite this article in press as: Zhang, M., and Jang, C.-H., Imaging the oxidation effects of the Fenton reaction on phospholipids at the interface between aqueous phase and thermotropic liquid crystals, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2014.12.016
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FIG. 1. Schematic illustration of orientational transitions of 5CB at the aqueous/LC interface laden with DOPG (A) before and (B) after oxidation by the Fenton reaction.
Previous work has shown that oxidized phospholipids are generated when cellular phospholipids containing unsaturated fatty acids undergo oxidative attacks, resulting in the addition of an oxygen atom to the sn-2 fatty acid residue or fragmentation of the sn-2 fatty acid chain (23). Common products are phospholipids with esterified isoprostane-like structures and chainchanged products, which cannot facilitate the alignment of 5CB at the aqueous/LC interface (24). This oxidative attack can occur by the Fenton reaction, which mimics in vivo oxidative stress conditions. The Fenton reaction oxidizes organic substrates using iron(II) and hydrogen peroxide. This reaction was first described by H. J. H. Fenton, who observed the oxidation of tartaric acid by H2O2 in the presence of ferrous ions (9). The hydroxyl radicals generated from the Fenton reaction are much more reactive; they react rapidly with various biological molecules. As a result, hydroxyl radicals have a low degree of selectivity in reactions. In addition, in acidic hypoxic conditions, the increased solubility of ferrous ions may be responsible for the modification of phospholipids by the Fenton reaction (25,26). These findings indicated that finding a simple and fast way to investigate the oxidation effects of the Fenton reaction on phospholipids is important. We first exposed the 5CB films to a partially oxidized phospholipid solution, which was generated by incubating phospholipids with 500 mL of 100 mM H2O2 and 5 mL of 50 mM FeSO4 at room temperature for 2 h. The optical image of 5CB was bright but intermediate (Fig. 2B), indicating the interfacial partial oxidation of DOPG by the Fenton reagent. On the other hand, when we exposed 5CB films to a completely oxidized phospholipid solution generated
by incubating phospholipids with 500 mL of 200 mM H2O2 and 5 mL of 50 mM FeSO4 under the same conditions, the optical image of 5CB became completely bright (Fig. 2C), indicating a planar orientation of LCs at the aqueous/LC interface. These findings highlight the feasibility of using this LC technique to report the oxidation effects of the Fenton reaction on bioactive phospholipids. Reactivity of DOPG induced by ferrous ion- and ferric ioncatalyzed oxidation Ferrous ions have been speculated to play a vital role in the production of OH in the Fenton reaction. The presence of ferrous ions was responsible for the generation of OH. The degradation or oxidation of organic compounds took place sequentially in solution once the radicals diffused near the surface (27). Previous studies have demonstrated that oxidation of phospholipids via Fenton reaction is initiated by free radicals (5,24). Free radical-mediated chain reaction is initiated by the generation of carbon-centered radicals and hydroperoxides of phospholipids. Carbon-centered radicals rapidly react with molecular oxygen, producing peroxyl radicals. These Peroxyl radicals react with bisallylic methylene groups in other phospholipids molecules, leading to the transformation of peroxyl radicals to hydroperoxides and generation of new carbon centered radicals. Phospholipids hydroperoxides in turn produce reactive alkoxyl and hydroxyl radicals via ferrous catalyzed Fenton reactions, further propagating the chain reaction. The rate at which the OH was generated by ferrous ion-catalyzed oxidation was 3e4 orders of magnitude higher than that by ferric ion-catalyzed oxidation (28). To test the reactivity of DOPG by
FIG. 2. Cross-polarized optical images of 5CB at an aqueous/LC interface after being exposed to (A) Tris buffer solution, (B) partially oxidized DOPG solution, and (C) completely oxidized DOPG solution.
Please cite this article in press as: Zhang, M., and Jang, C.-H., Imaging the oxidation effects of the Fenton reaction on phospholipids at the interface between aqueous phase and thermotropic liquid crystals, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2014.12.016
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ferrous and ferric ion-catalyzed oxidation, 5CB films confined in a copper grid were immersed in an aqueous solution containing 400 mL of the oxidized DOPG. This DOPG solution was pre-treated with a ferrous ion-catalyzed reagent, which was composed of 500 mL of 200 mM H2O2 and 5 mL of 50 mM FeSO4. A bright appearance (Fig. 3A) in the optical response of 5CB was observed immediately, suggesting that LCs were in a planar state at the aqueous/LC interface. In contrast, when the oxidized DOPG solution pre-treated with a ferric ion-catalyzed reaction was transferred into the 5CB film chamber, the optical images of 5CB remained dark (Fig. 3B), indicating that LCs were in a homeotropic state at the aqueous/LC interface. These results indicated that ferrous ions are more effective than ferric ions. Ferrous ion-catalyzed Fenton reactions oxidized phospholipids into a few chain-changed products, which could not sustain the homeotropic alignment of 5CB at the aqueous/LC interface, resulting in a dark to bright appearance in the optical image. For ferric ion-catalyzed reactions, the reaction might be too weak to fully oxidize the phospholipids. There were still sufficient DOPG molecules in the aqueous solution to penetrate into the LCs and self-assemble at the aqueous/LC interface, resulting in a dark image coupled to LCs in a homeotropic state. These results are in good agreement with a previous study in which both ferrous and ferric ions catalyzed the decomposition of hydroperoxide producing free radicals, which accelerated the oxidation reaction (21). However, the ferrous ion-catalyzed reaction was much faster than the ferric ion-catalyzed reaction and the amount of radicals formed in the initial stage was determined from the concentration of ferrous ions. As a control experiment, 5CB films confined to a copper grid were exposed to an aqueous solution of 400 mL DOPG. This DOPG solution was pre-treated with 50 mM FeSO4 or 50 mM FeCl3 in the absence of H2O2. Both exhibited dark optical images in the optical response of 5CB (Fig. 3C and D), indicating that ferrous or ferric ions alone cannot cause phospholipid oxidation.
J. BIOSCI. BIOENG., We also used UV/Vis spectroscopy to confirm the oxidation of phospholipids (Fig. 3E). The absorption at 235 nm of a conjugated diene was used to determine the concentration of hydroperoxides. The oxidation in ferrous ion-catalyzed reactions proceeded noticeably faster than that in ferric ion-catalyzed reactions. The absorbance of both ferrous ion- and ferric ion-catalyzed oxidation show a trend of ascending followed by leveling off. Based on the above results, the ferrous ion-catalyzed Fenton reaction with a concentration of 50 mM was selected for further investigation. pH-dependence of the ferrous ion-catalyzed oxidation of phospholipids After we confirmed that the catalyzed Fenton reaction took place, we sought to find the optimum pH for phospholipid oxidation. A series of oxidized DOPG solutions formed by the Fenton reaction at different pH values were transferred into the LC film chamber. When 400 mL of 500 mM oxidized DOPG solution with a pH of 5.8 was introduced into the 5CB film chamber, the bright appearance (Fig. 4A) in the optical response was displayed, indicating a planar orientation of LCs at the aqueous/LC interface. This result was the same as the optical response of the LC described above (Fig. 3A). However, the LCs adopted a dark appearance (Fig. 4B and C) when they were introduced into oxidized DOPG solutions containing the Fenton reagent at pH of 7.0 and 8.0 in the 5CB film chamber, corresponding to a homeotropic alignment of LCs at the aqueous/LC interface. The same dark image was obtained (Fig. 4D) in the control experiment, in which 5CB films were immersed into a pure 500 mM DOPG aqueous solution at a pH of 5.8. The dark appearance of 5CB resulted from pure DOPG without any pretreatment with the Fenton reaction can remain stable for a least one week. Based on these findings, we confirmed that the pH of buffer could influence the oxidation effects of the Fenton reaction on DOPG molecules. High pH triggers iron to precipitate and H2O2 decomposition. Therefore, the oxidation effects on phospholipids were negligible, resulting in the optical response of 5CB at aqueous/LC interface to remain dark. A previous report
FIG. 3. Cross-polarized optical images of 5CB at an aqueous/LC interface after being exposed to (A) DOPG solution oxidized by ferrous-ion catalyzed oxidation, (B) DOPG solution oxidized by Ferric-ion catalyzed oxidation, (C) an aqueous mixture of DOPG only with FeSO4, (D) an aqueous mixture of DOPG only with FeCl3, and (E) observed UV/Vis absorbance of ferrous ion- and ferric ion-catalyzed oxidation reactions at 235 nm as a function of oxidation time. The solid lines serve to guide the eye.
Please cite this article in press as: Zhang, M., and Jang, C.-H., Imaging the oxidation effects of the Fenton reaction on phospholipids at the interface between aqueous phase and thermotropic liquid crystals, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2014.12.016
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FIG. 4. Cross-polarized optical images of 5CB at an aqueous/LC interface after being exposed to a series of DOPG solutions oxidized by the Fenton reaction at the following pH values: (A) pH 5.8, (B) pH 7.0, (C) pH 8.0, and (D) after being exposed to 0.5 mM DOPG solution at pH 5.8.
showed that maintaining phospholipid dispersion pH values around 6.5 is beneficial for physicochemical stability (29). Considering these two conditions, a pH of 5.8 was determined to be optimal for experimental use. Detection limit of H2O2 measured by LC-based method To determine the limit of detection of the LC-based technique, the Fenton reagent, 50 mM FeSO4 was mixed with H2O2 at different concentrations and pre-incubated with 500 mM DOPG before the aqueous mixtures were transferred into the 5CB film chamber. The incubation was performed at room temperature for 2 h under continuous shaking. H2O2 concentration at 100 mM or greater yielded a bright optical image of 5CB (Fig. 5AeC). When the concentration of H2O2 was 50 mM, a bright but intermediate optical appearance was observed (Fig. 5D). However, at an H2O2 concentration of 25 mM, only partially bright domains in the optical images of 5CB were observed (Fig. 5E). When the bulk concentration of H2O2 decreased to 2.5 mM or lower, no obvious
optical response of LCs was observed (Fig. 5F), indicating that the LCs adopted a homeotropic state at the aqueous/LC interface. To the best of our knowledge, there has been no previous report on using LC-based aqueous systems to detect oxidation effects of ferrous ion-catalyzed oxidation on phospholipids. LC-based detection systems can detect phospholipid oxidation rapidly using simple analytical devices. The detection limit of H2O2 in this LC-based detection system was w25 mM. In addition, different tilt angles and birefringence of 5CB were represented by the continuous color changes in the optical response. Based on a past study by Brake et al., the quantification of the color changes was a potential way to quantify the interfacial event accompanying LC orientational transitions, which, in our case, corresponds to the oxidation effects of the Fenton reaction on the phospholipid monolayer (30). We also used UV/Vis spectroscopy to confirm the oxidation level of phospholipids (Fig. 6). The accumulation of hydroperoxides in ferrous ion-catalyzed oxidation at pH 5.8 was consistent with the
FIG. 5. Cross-polarized optical images of 5CB at an aqueous/LC interface after being exposed to a series of DOPG solutions oxidized by the Fenton reaction with the following concentrations of H2O2: (A) 250 mM, (B) 200 mM, (C) 100 mM, (D) 50 mM, (E) 25 mM, and (F) 2.5 mM.
Please cite this article in press as: Zhang, M., and Jang, C.-H., Imaging the oxidation effects of the Fenton reaction on phospholipids at the interface between aqueous phase and thermotropic liquid crystals, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2014.12.016
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FIG. 6. Observed UV/Vis absorbance of Fenton reaction at 235 nm as a function of different concentrations of H2O2. The solid lines serve to guide the eye.
qualitative observation reported above. H2O2 concentrations of 100 mM or above exhibited strong absorption, whereas the concentration of H2O2 decreased to 50 mM or lower, such that the difference in the absorption between concentrations was trivial. This simple and quick method is comparable or even better to other previously reported methods used to investigate phospholipid oxidation. Compared to the existing assays, which require time-consuming and complicated operations of large-scale instrumentation, the LC-based method reported here is simple and can rapidly detect the presence of analytes. Conclusions We have demonstrated an LC-based approach for the real-time and label-free characterization of phospholipid oxidation by the Fenton reaction. Ferrous ion-catalyzed oxidation was used in this study as an effective catalyst. The optimum pH for phospholipid oxidation in this LC detection system was also investigated. The oxidation of the phospholipid monolayer, which can self-assemble at the aqueous/LC interface, induced orientational changes of LCs. Consequently, the optical signals that represented the oxidation level of phospholipids were observed in a real-time manner. The detection limit of H2O2, a vital component of the Fenton reagent, was also determined. Our work demonstrates the potential for the development of a simple and cost-effective LC-based device for the detection of phospholipid oxidation by the Fenton reaction. Monitoring lipid peroxidation products in phospholipids formed under oxidative stress conditions may provide a new strategy for studying oxidative stress signaling and disease states as well as new insight into the pathogenesis process. ACKNOWLEDGMENTS This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI13C0891) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF2013R1A1A1A05008333). References 1. Striebel, C., Brecht, A., and Gauglitz, G.: Characterization of biomembranes by spectral ellipsometry, surface plasmon resonance and interferometry with regard to biosensor application, Biosens. Bioelectron., 9, 139e146 (1994). 2. Heyse, S., Vogel, H., Sänger, M., and Sigrist, H.: Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes, Protein Sci., 4, 2532e2544 (1995).
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J., and Domingues, P.: Separation of peroxidation products of diacylphosphatidylcholines by reversed-phase liquid chromatography-mass spectrometry, Biomed. Chromatogr., 19, 129e137 (2005). 13. McIntyre, T. M.: Biologically active oxidized phospholipids, J. Biol. Chem., 274, 25189e25192 (1999). 14. Corliss, G. A. and Dugan, L. R., Jr.: Phospholipid oxidation in emulsions, Lipids, 5, 846e853 (1970). 15. Domingues, M. R. M., Reis, A., and Domingues, P.: Mass spectrometry analysis of oxidized phospholipids, Chem. Phys. Lipids, 156, 1e12 (2008). 16. Qian, S. Y. and Buettner, G. R.: Iron and dioxygen chemistry is an important route to initiation of biological free radical oxidations an electron paramagnetic resonance spin trapping study, Free Radic. Biol. Med., 26, 1447e1456 (1999). 17. Zhang, M. and Jang, C. H.: Liquid crystal based optical sensor for imaging trypsin activity at interfaces between aqueous phases and thermotropic liquid crystals, Bull. Korean Chem. Soc., 34, 2973e2977 (2013). 18. Hu, Q. and Jang, C. H.: Liquid crystal-based imaging of enzymatic reactions at aqueous-liquid crystal interfaces decorated with oligopeptide amphiphiles, Bull. Korean Chem. Soc., 31, 1262e1266 (2010). 19. Hu, Q. Z. and Jang, C. H.: Using liquid crystals to report molecular interactions between cationic antimicrobial peptides and lipid membranes, Analyst, 137, 567e570 (2012). 20. Brake, J. M. and Abbott, N. L.: Coupling of the orientations of thermotropic liquid crystals to protein binding events at lipid-decorated interfaces, Langmuir, 23, 8497e8507 (2007). 21. Kawakatsu, M., Terao, J., and Matsushita, S.: Phospholipid oxidation catalyzed by ferrous ion and ascorbic acid, Agric. Biol. Chem., 48, 1275e1279 (1984). 22. Brake, J. M., Daschner, M. K., and Abbott, N. L.: Formation and characterization of phospholipid monolayers spontaneously assembled at interfaces between aqueous phases and thermotropic liquid crystals, Langmuir, 21, 2218e2228 (2005). 23. Frank, S. and Kostner, G.: Lipoproteinserole in health and diseases, pp. 409e430. InTech, Croatia (2012). 24. Ashraf, M. Z., Kar, N. S., and Podrez, E. A.: Oxidized phospholipids: biomarker for cardiovascular diseases, Int. J. Biochem. Cell Biol., 41, 1241e1244 (2009). 25. Sergent, O., Morel, I., and Cillard, J.: Involvement of metal ions in lipid peroxidation: biological implications, pp. 251e287, in: Sigel, A. and Sigel, H. (Eds.), Metal ions in biological systems, vol. 36. Marcel Dekker, New York (1999). 26. Comporti, M., Signorini, C., Buonocore, G., and Ciccoli, L.: Iron release, oxidative stress and erythrocyte ageing, Free Radic. Biol. Med., 32, 568e576 (2002). 27. Yang, X. J., Xu, X. M., Xu, J., and Han, Y. F.: Iron oxychloride (FeOCl) an efficient fenton-like catalyst for producing hydroxyl radicals in degradation of organic contaminants, J. Am. Chem. Soc., 135, 16058e16061 (2013). 28. Pignatello, J. J., Oliveros, E., and MacKay, A.: Advanced oxidation processes for organic contaminant destruction based on the Fenton reaction and related chemistry, Crit. Rev. Environ. Sci. Technol., 36, 1e84 (2006). 29. Werling, J., Graham, S., Owen, H., Nair, L., Gonyon, T., and Carter, P. W.: Physicochemical stability of phospholipid-dispersed suspensions of crystalline itraconazole, Eur. J. Pharm. Biopharm., 69, 1104e1113 (2008). 30. Brake, J. M., Daschner, M. K., Luk, Y. Y., and Abbott, N. L.: Biomolecular interactions at phospholipid-decorated surfaces of liquid crystals, Science, 302, 2094e2097 (2003).
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Imaging the oxidation effects of the Fenton reaction on phospholipids at the interface between aqueous phase and thermotropic liquid crystals Minmin Zhang and Chang-Hyun Jang* Department of Chemistry, Gachon University, Seongnam-Si, Gyeonggi-Do 461-701, South Korea Received 13 June 2014; accepted 18 December 2014 Available online xxx
The lipid peroxidation process has attracted much attention because of the growing evidence of its involvement in the pathogenesis of age-related diseases. Herein, we report a simple, label-free method to study the oxidation of phospholipids by the Fenton reaction at the interface between an aqueous phase and immiscible liquid crystals (LCs). The different images produced by the orientation of 4-cyano-40 -pentylbiphenyl (5CB) corresponded to the presence or absence of oxidized 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG). The oxidation effects of the Fenton reaction on DOPG were evaluated by monitoring the orientational response of liquid crystals upon contact with the oxidized DOPG solutions. DOPG was oxidized into chain-changed products containing hydroxy, carbonyl, or aldehyde groups, resulting in the rearrangement of the phospholipid layer. This induced the orientational transition of LCs from homeotropic to planar states; therefore, a dark to bright optical shift was observed. This shift was due to the Fenton reaction preventing DOPG to induce the orientational alignment of LCs at the aqueous/LC interface. We also used an ultraviolet spectrophotometer to confirm the effects of oxidation on phospholipids by the Fenton reaction. Using this simple method, a new approach for investigating phospholipid oxidation was established with high resolution and easy accessibility. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Oxidation; Phospholipids; Liquid crystals; Orientational transition; Fenton reaction]
Phospholipids are the most abundant lipids in cell membranes. Because of their amphiphilic nature, they form spherical lipid bilayers known as liposomes or vesicles (1,2). They are important in mammalian cell biology because they act as lipid mediators by providing a permeable barrier as well as substrates for synthesis (3,4). The role of lipid oxidation in disease is still a subject of considerable research, and within this area, there is growing interest in the roles of oxidized phospholipids, which have been detected in pathological conditions, often at raised levels compared to those from subjects with normal physiology (5). There is substantial evidence that the oxidized phospholipids of oxidized lowdensity lipoprotein accumulate in vivo and play an important role in cardiovascular disease (6e8). The Fenton reaction is defined as the oxidation of organic substrates by a mixture of hydrogen peroxide and ferrous iron (9). The oxidizing intermediates involved in Fenton reactions are thought to cause damage to phospholipids and play a significant role in the aging process and a variety of diseases, such as cancer (10). The Fenton reaction is a key reaction in the oxidation of phospholipids. Notably, this reaction is thought to occur in heart diseases, such as ischemia and reperfusion (11). Many researchers doubt that the significance and existence of the Fenton reaction in biological systems is due to the low concentrations of H2O2 and free iron. They
* Corresponding author. Tel.: þ82 31 750 8555; fax: þ82 31 750 8774. E-mail address: [email protected] (C.-H. Jang).
also suggest that the high and undiscriminating reactivity of hydroxyl radicals produce great limitations on diffusion and cause more wide-ranging damage to biomolecules (10). Assays for identifying and characterizing the oxidation of phospholipids have been the subject of many studies. Development of these assays include: (i) separation of phospholipid oxidation products by mass spectrometry (12), (ii) biological functions of oxidized phospholipids (13), (iii) identification of new families of bioactive phospholipids generated by immune cells (3), and (iv) measurement of the oxidation of phospholipids by physicochemical tests (14). A. Reis et al. (12) used mass spectrometry coupled with high performance liquid chromatography (HPLC) separation techniques (HPLC-MS) to study phosphatidylcholine peroxidation products. They developed a reverse-phase liquid chromatography method coupled to electrospray mass spectrometry for the separation of glycero-phosphatidylcholine peroxidation products formed by the Fenton reaction. In addition, Domingues et al. (15) used tandem mass spectrometry with a method of soft ionization (electrospray and matrix-assisted laser desorption ionization) to characterize changes in fatty acyl chain. Qian and Buettner (16) also used electron paramagnetic resonance spin trapping to examine iron-induced free radical oxidations. All of these large facility-based methods are time consuming and require sophisticated instrumentations. In the past decade, long-range orientational anchoring of liquid crystals has become one of the most considerable tools in the field of biological assays, particularly based on the effects of interfacial
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.12.016
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interactions of biomolecules. The property of birefringence of LCs, due to their anisotropic nature, allows the orientational change of LCs to be observed under a polarized optical microscopy, which is visible with naked eye. We recently have shown that phospholipid monolayer spontaneously assembled at the aqueous/liquid crystal interface is correlated to the orientation of LCs (17e19). The biological enzyme activity involved in combining phospholipids is demonstrated to trigger orientational transition of LCs that can be readily visualized under crossed polarizers. This correlation provides principles to transduce and amplify biological enzymatic events that occur at these interfaces. In the present study, we developed an LC-based approach for the label-free characterization of oxidized phospholipids by the Fenton reaction occurring at the interface between aqueous phase and immiscible liquid crystals. We attempted to explore the activities of iron(II) and hydrogen peroxide towards 1,2-dioleoyl-snglycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG) membrane at the aqueous/LC interface and their correlation to the orientational transitions of LCs. The impacts of different catalysts and pH on the Fenton oxidation were also explored. To the best of our knowledge, an LC has never been utilized to report the effects of oxidation of phospholipids by the Fenton reaction before. This study suggests the applicability and superiority of a label-less and low-cost LC-based approach for identifying phospholipids oxidized by the Fenton reaction and for screening their potential uses. MATERIALS AND METHODS Materials Tris buffered saline (TBS; 0.05 M Tris, 0.138 M NaCl, and 0.0027 M KCl, pH 8.0), sulfuric acid, hydrogen peroxide (30% w/v), octyltrichlorosilane (OTS), DOPG, sodium chloride, hydrochloric acid, sodium hydroxide, chloroform, ethanol, methanol, methylene dichloride, and capillary tubes were purchased from SigmaeAldrich (USA). Liquid crystal, 40 -pentyl-4-cyanobiphenyl (5CB), was purchased from EM industries (Hawthorne, NY, USA). Copper specimen grids (50 meshes, 500 mm pitch, 420 mm hole, 80 mm bar, 25 mm 5 mm thickness) were obtained from Gilder Grids (Grantham, UK). Premium glass microscope slides were purchased from Fisher Scientific (Pittsburgh, PA, USA). Ultrapure water, with a resistivity of 18.2 MU cm, was obtained from a Milli-Q system (Millipore, Bedford, MA, USA). Treatment of glass microscope slides with OTS Glass microscope slides were cleaned in accordance with a previously described procedure (17). Briefly, the slides were immersed in the piranha solution [70% (v/v) sulfuric acid and 30% (v/v) hydrogen peroxide] for 30 min at w80 C (warning: piranha solution reacts strongly with organic compounds and should be handled with extreme caution; do not store the solution in closed containers). The slides were then rinsed with water, ethanol, and methanol and dried under a stream of gaseous nitrogen, after which they were heated at 120 C overnight prior to OTS deposition. A 0.5 mM OTS solution was prepared and the piranha-cleaned slides were immersed in 0.5 mM OTS in a heptane solution at room temperature for 30 min. The samples were then rinsed with methylene dichloride and dried under nitrogen. Next, films of nematic LC [4-cyano-40 -pentylbiphenyl (5CB)] were deposited into the pores of gold grids on octadecyltrichlorosilane (OTS)ecoated glass substrates, the OTS-coated glass caused the LC to assume a perpendicular (homeotropic) orientation at the glass surface. Any sample that did not exhibit homeotropic anchoring of 5CB was rejected. Preparation of a glass slide-supported LC optical cell A glass slidesupported LC optical cell was prepared as previously reported (19). Simply put, OTS-treated glass slides were fixed to the bottom of an eight-well chamber slide with silicone mounting medium. Subsequently, transmission electron microscopy grids (50 meshes and 25 mm 5 mm thickness) were placed onto the slide. The copper grid was then impregnated with 2 mL of 5CB using a Hamilton syringe. By heating to its isotropic phase (>35 C), excess 5CB was removed from the LCs with a 20-mL capillary tube. The grids containing LCs were then immersed into aqueous solutions of interest. Each assay was performed at least six times independently. Preparation of phospholipid vesicles LCs, decorated with phospholipid membrane, were prepared from DOPG phospholipid solution following previously reported procedures (20). Simply put, phospholipids dissolved in chloroform (50 mg/mL) were dried under a stream of nitrogen and desiccated under vacuum for at least 3 h. Dried phospholipids were resuspended with Tris-buffered saline (TBS, consisting of 0.05 M Tris and adjusted to a pH of 5.8 prior to use). The final concentration of DOPG phospholipid solution was 1 mM. The turbidity of the resulting solution represented the presence of large multilamellar vesicles. The phospholipid suspension was then sonicated three times each for 5 min to clarify
J. BIOSCI. BIOENG., using a probe sonicator. Then, the solution was filtered twice with a 0.22-mm filter and typically used within 2 d after the preparation. Prior to the formation of the DOPG membrane, 400 mL of DOPG solution was added to one of the wells supported on the glass slide. The DOPG membrane was then formed by touching a copper grid impregnated with 5CB to the DOPG solution in the well. Preparation of oxidized phospholipid vesicles The phospholipid vesicles prepared as described above were diluted to a final concentration of 0.5 mM and the mixture was vortexed. Oxidative treatments using Fe(Ⅱ) and H2O2 were performed as per the procedure detailed in a previous publication (21). 5 mL FeSO4 solution (50 mM) and 500 mL of hydrogen peroxide (H2O2) at a certain concentration are added to 500 mL of the phospholipid vesicles. This mixture was then left to react at room temperature for 2 h under continuous shaking. LC-based technique for testing the oxidation of phospholipid Prior to the identification of the oxidation effects of hydrogen peroxide on phospholipids, 400 mL of a solution containing oxidized phospholipids (in 0.05 M Tris, 0.138 M NaCl, and 0.0027 M KCl; the pH was adjusted prior to use) was added to individual wells. 5CB (confined in copper grids) was then immersed in these oxidized phospholipid solutions. Optical examination of LC texture The optical texture of the LCs was examined using a Nikon eclipse LV100 POL microscope equipped with crossed polarizers in transmission mode. The sample was placed on a rotating stage between the crossed polarizers. All optical images were captured using a digital camera (DS2Mv, Nikon, Tokyo, Japan) mounted on the microscope with a resolution of 1600 1200 pixels, a gain of 1.00, and a shutter speed of 1/10 s. Determination of phospholipid oxidation by UV/Vis spectroscopy The percent oxidation of the phospholipid was determined using Beer’s Law by calculating the peroxidized phospholipid concentration from the absorbance at 235 nm. An aliquot (50 mL) of the reaction mixture was taken out and diluted with ethanol to 500 mL. The absorbance at 235 nm was measured with a Varian 50 Bio UV/Vis spectrophotometer.
RESULTS AND DISCUSSION LC-based method for imaging oxidation effects of the Fenton reaction on bioactive phospholipids The oxidation of lipids has been a topic of interest in biochemical and food sciences for a long time. The basic principles of non-enzymatic free radical effects on phospholipids are well established, although questions about detailed mechanisms remain unanswered. In this LC-based technique, the phospholipid monolayer at the aqueous/LC interface was used as the oxidation substrate. Phospholipids, because of their amphiphilic nature, form spherical lipid bilayers known as liposomes and vesicles. When phospholipid vesicles are exposed to a hydrophobic surface, they fuse with the surface and form a planar membrane, leading to the formation of a lipid monolayer. Previous studies have demonstrated that the presence and organization of amphiphiles at the interface between liquid crystals (LCs) and an immiscible aqueous phase is coupled to the orientational ordering of the LCs. Brake et al. (30) reported that contact the interface of a thermotropic LC with an aqueous solution of phospholipid resulted in the spontaneous assembly of a monolayer of phospholipid at the aqueous/LC interface, corresponding to a dark image in the optical response. Fig. 1 shows the schematic illustration of the experimental system. A phospholipid monolayer was prepared by exposing the 5CB-filled copper grid to an aqueous solution containing 500 mM of DOPG. Following the exposure, the initially bright optical images of 5CB became uniformly dark (Fig. 1A), indicating the self-assembly of DOPG at the aqueous/LC interface. Films of 5CB laden with DOPG were then washed with TBS buffer five times and were stable for more than a week. However, when 5CB films were exposed to phospholipids oxidized by the Fenton reaction, the optical images of 5CB evolved from dark to bright colors (Fig. 1B). This observation is in accordance with previous studies demonstrating that the orientational changes of LCs correspond to the formation of the phospholipid monolayer at aqueous/LC interfaces (22). We also examined the optical response of LCs after introducing the Tris buffer solution (TBS) into the chamber containing LCs and observed a bright appearance (Fig. 2A), which was attributed to the planar orientation of LC molecules at the aqueous/LC interface.
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FIG. 1. Schematic illustration of orientational transitions of 5CB at the aqueous/LC interface laden with DOPG (A) before and (B) after oxidation by the Fenton reaction.
Previous work has shown that oxidized phospholipids are generated when cellular phospholipids containing unsaturated fatty acids undergo oxidative attacks, resulting in the addition of an oxygen atom to the sn-2 fatty acid residue or fragmentation of the sn-2 fatty acid chain (23). Common products are phospholipids with esterified isoprostane-like structures and chainchanged products, which cannot facilitate the alignment of 5CB at the aqueous/LC interface (24). This oxidative attack can occur by the Fenton reaction, which mimics in vivo oxidative stress conditions. The Fenton reaction oxidizes organic substrates using iron(II) and hydrogen peroxide. This reaction was first described by H. J. H. Fenton, who observed the oxidation of tartaric acid by H2O2 in the presence of ferrous ions (9). The hydroxyl radicals generated from the Fenton reaction are much more reactive; they react rapidly with various biological molecules. As a result, hydroxyl radicals have a low degree of selectivity in reactions. In addition, in acidic hypoxic conditions, the increased solubility of ferrous ions may be responsible for the modification of phospholipids by the Fenton reaction (25,26). These findings indicated that finding a simple and fast way to investigate the oxidation effects of the Fenton reaction on phospholipids is important. We first exposed the 5CB films to a partially oxidized phospholipid solution, which was generated by incubating phospholipids with 500 mL of 100 mM H2O2 and 5 mL of 50 mM FeSO4 at room temperature for 2 h. The optical image of 5CB was bright but intermediate (Fig. 2B), indicating the interfacial partial oxidation of DOPG by the Fenton reagent. On the other hand, when we exposed 5CB films to a completely oxidized phospholipid solution generated
by incubating phospholipids with 500 mL of 200 mM H2O2 and 5 mL of 50 mM FeSO4 under the same conditions, the optical image of 5CB became completely bright (Fig. 2C), indicating a planar orientation of LCs at the aqueous/LC interface. These findings highlight the feasibility of using this LC technique to report the oxidation effects of the Fenton reaction on bioactive phospholipids. Reactivity of DOPG induced by ferrous ion- and ferric ioncatalyzed oxidation Ferrous ions have been speculated to play a vital role in the production of OH in the Fenton reaction. The presence of ferrous ions was responsible for the generation of OH. The degradation or oxidation of organic compounds took place sequentially in solution once the radicals diffused near the surface (27). Previous studies have demonstrated that oxidation of phospholipids via Fenton reaction is initiated by free radicals (5,24). Free radical-mediated chain reaction is initiated by the generation of carbon-centered radicals and hydroperoxides of phospholipids. Carbon-centered radicals rapidly react with molecular oxygen, producing peroxyl radicals. These Peroxyl radicals react with bisallylic methylene groups in other phospholipids molecules, leading to the transformation of peroxyl radicals to hydroperoxides and generation of new carbon centered radicals. Phospholipids hydroperoxides in turn produce reactive alkoxyl and hydroxyl radicals via ferrous catalyzed Fenton reactions, further propagating the chain reaction. The rate at which the OH was generated by ferrous ion-catalyzed oxidation was 3e4 orders of magnitude higher than that by ferric ion-catalyzed oxidation (28). To test the reactivity of DOPG by
FIG. 2. Cross-polarized optical images of 5CB at an aqueous/LC interface after being exposed to (A) Tris buffer solution, (B) partially oxidized DOPG solution, and (C) completely oxidized DOPG solution.
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ferrous and ferric ion-catalyzed oxidation, 5CB films confined in a copper grid were immersed in an aqueous solution containing 400 mL of the oxidized DOPG. This DOPG solution was pre-treated with a ferrous ion-catalyzed reagent, which was composed of 500 mL of 200 mM H2O2 and 5 mL of 50 mM FeSO4. A bright appearance (Fig. 3A) in the optical response of 5CB was observed immediately, suggesting that LCs were in a planar state at the aqueous/LC interface. In contrast, when the oxidized DOPG solution pre-treated with a ferric ion-catalyzed reaction was transferred into the 5CB film chamber, the optical images of 5CB remained dark (Fig. 3B), indicating that LCs were in a homeotropic state at the aqueous/LC interface. These results indicated that ferrous ions are more effective than ferric ions. Ferrous ion-catalyzed Fenton reactions oxidized phospholipids into a few chain-changed products, which could not sustain the homeotropic alignment of 5CB at the aqueous/LC interface, resulting in a dark to bright appearance in the optical image. For ferric ion-catalyzed reactions, the reaction might be too weak to fully oxidize the phospholipids. There were still sufficient DOPG molecules in the aqueous solution to penetrate into the LCs and self-assemble at the aqueous/LC interface, resulting in a dark image coupled to LCs in a homeotropic state. These results are in good agreement with a previous study in which both ferrous and ferric ions catalyzed the decomposition of hydroperoxide producing free radicals, which accelerated the oxidation reaction (21). However, the ferrous ion-catalyzed reaction was much faster than the ferric ion-catalyzed reaction and the amount of radicals formed in the initial stage was determined from the concentration of ferrous ions. As a control experiment, 5CB films confined to a copper grid were exposed to an aqueous solution of 400 mL DOPG. This DOPG solution was pre-treated with 50 mM FeSO4 or 50 mM FeCl3 in the absence of H2O2. Both exhibited dark optical images in the optical response of 5CB (Fig. 3C and D), indicating that ferrous or ferric ions alone cannot cause phospholipid oxidation.
J. BIOSCI. BIOENG., We also used UV/Vis spectroscopy to confirm the oxidation of phospholipids (Fig. 3E). The absorption at 235 nm of a conjugated diene was used to determine the concentration of hydroperoxides. The oxidation in ferrous ion-catalyzed reactions proceeded noticeably faster than that in ferric ion-catalyzed reactions. The absorbance of both ferrous ion- and ferric ion-catalyzed oxidation show a trend of ascending followed by leveling off. Based on the above results, the ferrous ion-catalyzed Fenton reaction with a concentration of 50 mM was selected for further investigation. pH-dependence of the ferrous ion-catalyzed oxidation of phospholipids After we confirmed that the catalyzed Fenton reaction took place, we sought to find the optimum pH for phospholipid oxidation. A series of oxidized DOPG solutions formed by the Fenton reaction at different pH values were transferred into the LC film chamber. When 400 mL of 500 mM oxidized DOPG solution with a pH of 5.8 was introduced into the 5CB film chamber, the bright appearance (Fig. 4A) in the optical response was displayed, indicating a planar orientation of LCs at the aqueous/LC interface. This result was the same as the optical response of the LC described above (Fig. 3A). However, the LCs adopted a dark appearance (Fig. 4B and C) when they were introduced into oxidized DOPG solutions containing the Fenton reagent at pH of 7.0 and 8.0 in the 5CB film chamber, corresponding to a homeotropic alignment of LCs at the aqueous/LC interface. The same dark image was obtained (Fig. 4D) in the control experiment, in which 5CB films were immersed into a pure 500 mM DOPG aqueous solution at a pH of 5.8. The dark appearance of 5CB resulted from pure DOPG without any pretreatment with the Fenton reaction can remain stable for a least one week. Based on these findings, we confirmed that the pH of buffer could influence the oxidation effects of the Fenton reaction on DOPG molecules. High pH triggers iron to precipitate and H2O2 decomposition. Therefore, the oxidation effects on phospholipids were negligible, resulting in the optical response of 5CB at aqueous/LC interface to remain dark. A previous report
FIG. 3. Cross-polarized optical images of 5CB at an aqueous/LC interface after being exposed to (A) DOPG solution oxidized by ferrous-ion catalyzed oxidation, (B) DOPG solution oxidized by Ferric-ion catalyzed oxidation, (C) an aqueous mixture of DOPG only with FeSO4, (D) an aqueous mixture of DOPG only with FeCl3, and (E) observed UV/Vis absorbance of ferrous ion- and ferric ion-catalyzed oxidation reactions at 235 nm as a function of oxidation time. The solid lines serve to guide the eye.
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FIG. 4. Cross-polarized optical images of 5CB at an aqueous/LC interface after being exposed to a series of DOPG solutions oxidized by the Fenton reaction at the following pH values: (A) pH 5.8, (B) pH 7.0, (C) pH 8.0, and (D) after being exposed to 0.5 mM DOPG solution at pH 5.8.
showed that maintaining phospholipid dispersion pH values around 6.5 is beneficial for physicochemical stability (29). Considering these two conditions, a pH of 5.8 was determined to be optimal for experimental use. Detection limit of H2O2 measured by LC-based method To determine the limit of detection of the LC-based technique, the Fenton reagent, 50 mM FeSO4 was mixed with H2O2 at different concentrations and pre-incubated with 500 mM DOPG before the aqueous mixtures were transferred into the 5CB film chamber. The incubation was performed at room temperature for 2 h under continuous shaking. H2O2 concentration at 100 mM or greater yielded a bright optical image of 5CB (Fig. 5AeC). When the concentration of H2O2 was 50 mM, a bright but intermediate optical appearance was observed (Fig. 5D). However, at an H2O2 concentration of 25 mM, only partially bright domains in the optical images of 5CB were observed (Fig. 5E). When the bulk concentration of H2O2 decreased to 2.5 mM or lower, no obvious
optical response of LCs was observed (Fig. 5F), indicating that the LCs adopted a homeotropic state at the aqueous/LC interface. To the best of our knowledge, there has been no previous report on using LC-based aqueous systems to detect oxidation effects of ferrous ion-catalyzed oxidation on phospholipids. LC-based detection systems can detect phospholipid oxidation rapidly using simple analytical devices. The detection limit of H2O2 in this LC-based detection system was w25 mM. In addition, different tilt angles and birefringence of 5CB were represented by the continuous color changes in the optical response. Based on a past study by Brake et al., the quantification of the color changes was a potential way to quantify the interfacial event accompanying LC orientational transitions, which, in our case, corresponds to the oxidation effects of the Fenton reaction on the phospholipid monolayer (30). We also used UV/Vis spectroscopy to confirm the oxidation level of phospholipids (Fig. 6). The accumulation of hydroperoxides in ferrous ion-catalyzed oxidation at pH 5.8 was consistent with the
FIG. 5. Cross-polarized optical images of 5CB at an aqueous/LC interface after being exposed to a series of DOPG solutions oxidized by the Fenton reaction with the following concentrations of H2O2: (A) 250 mM, (B) 200 mM, (C) 100 mM, (D) 50 mM, (E) 25 mM, and (F) 2.5 mM.
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FIG. 6. Observed UV/Vis absorbance of Fenton reaction at 235 nm as a function of different concentrations of H2O2. The solid lines serve to guide the eye.
qualitative observation reported above. H2O2 concentrations of 100 mM or above exhibited strong absorption, whereas the concentration of H2O2 decreased to 50 mM or lower, such that the difference in the absorption between concentrations was trivial. This simple and quick method is comparable or even better to other previously reported methods used to investigate phospholipid oxidation. Compared to the existing assays, which require time-consuming and complicated operations of large-scale instrumentation, the LC-based method reported here is simple and can rapidly detect the presence of analytes. Conclusions We have demonstrated an LC-based approach for the real-time and label-free characterization of phospholipid oxidation by the Fenton reaction. Ferrous ion-catalyzed oxidation was used in this study as an effective catalyst. The optimum pH for phospholipid oxidation in this LC detection system was also investigated. The oxidation of the phospholipid monolayer, which can self-assemble at the aqueous/LC interface, induced orientational changes of LCs. Consequently, the optical signals that represented the oxidation level of phospholipids were observed in a real-time manner. The detection limit of H2O2, a vital component of the Fenton reagent, was also determined. Our work demonstrates the potential for the development of a simple and cost-effective LC-based device for the detection of phospholipid oxidation by the Fenton reaction. Monitoring lipid peroxidation products in phospholipids formed under oxidative stress conditions may provide a new strategy for studying oxidative stress signaling and disease states as well as new insight into the pathogenesis process. ACKNOWLEDGMENTS This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI13C0891) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF2013R1A1A1A05008333). References 1. Striebel, C., Brecht, A., and Gauglitz, G.: Characterization of biomembranes by spectral ellipsometry, surface plasmon resonance and interferometry with regard to biosensor application, Biosens. Bioelectron., 9, 139e146 (1994). 2. Heyse, S., Vogel, H., Sänger, M., and Sigrist, H.: Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes, Protein Sci., 4, 2532e2544 (1995).
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Please cite this article in press as: Zhang, M., and Jang, C.-H., Imaging the oxidation effects of the Fenton reaction on phospholipids at the interface between aqueous phase and thermotropic liquid crystals, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2014.12.016
