J. Agric. Food Chem. 2007, 55, 3325−3333
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Electron Spin Resonance Estimation of Hydroxyl Radical Scavenging Capacity for Lipophilic Antioxidants ZHIHONG CHENG,† HUIPING ZHOU,‡ JUNJIE YIN,§
AND
LIANGLI YU*,†
Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742, Department of Microbiology and Immunology, School of Medicine, Virginia Commonwealth University, Richmond, Virginia 23298, and Instrumentation and Biophysics Branch, CFSAN, U.S. Food and Drug Administration, College Park, Maryland 20740
Hydroxyl radical scavenging capacity estimation for lipophilic antioxidants is a challenge due to their poor solubility in aqueous radical generating and measuring systems. In this study, an electron spin resonance (ESR) method was developed and validated for its application in estimating the relative hydroxyl radical (HO•) scavenging capacity for lipophilic antioxidants under physiological pH using a Fenton Fe2+/H2O2 system for radical generation and acetonitrile as a solvent. The Fenton Fe2+/H2O2 system generates a constant flux of pure HO• under the assay conditions. The method was validated by linearity, precision, and reproducibility using selected known lipophilic antioxidants including R-tocopherol, lutein, β-carotene, and BHT. The potential effects of commonly used water-miscible and water-immiscible organic solvents on the Fenton Fe2+/H2O2 HO• generating system as well as their possible interactions with the fluorescent and spectroscopic probes were also reported. In addition, the limitation of the ESR assay was described. KEYWORDS: Hydroxyl radical; lipophilic antioxidant; ESR; Fenton reaction; solvent effect
INTRODUCTION
Antioxidants are well recognized for their potential role in reducing the risk of several aging-associated human diseases such as cardiovascular disorder and cancer (1-3). Free radical medicated oxidative chain reaction is a commonly accepted mechanism of lipid peroxidation in the biological systems, and this mechanism may apply to oxidation of other cellular components such as cholesterol and proteins. Antioxidants may suppress the initiation and propagation of the oxidative chain reaction through a number of mechanisms including but not limited to chelating transition metals, quenching free radicals, and reducing peroxides. Generally, antioxidants are classified into hydrophilic and lipophilic antioxidants according to their solubility in water or nonpolar organic solvents. It is well accepted that hydrophilic or hydrophobic antioxidants may act synergistically and provide stronger protection to biological systems and reduce the damage caused by reactive oxygen species (ROS) including free oxygen centered radicals (4). In contrast to hydrophilic antioxidants, lipophilic antioxidants have a tendency to be distributed in or penetrate into the hydrophobic domain of cell membranes and biological particles such as the nonpolar core of lipoproteins. Tocopherols, the most important representatives of lipophilic antioxidants, may act mainly as oxygen radical scavengers thus to inhibit the initiation and * Author to whom correspondence should be addressed [telephone (301) 405-0761; fax (301) 314-3313; e-mail
[email protected]]. † University of Maryland. ‡ Virginia Commonwealth University. § U.S. Food and Drug Administration.
propagation of the oxidative chain reactions in hydrophobic systems. It was also suggested that tocopherols incorporated in the cellular membrane might interact with and scavenge hydrophilic ROS in the surrounding aqueous phase outside of cells because their chroman head groups might orient near the surface of cell membranes (5). Lipophilic antioxidants may provide stronger protection against lipid peroxidation and other free radical mediated oxidative damage to important biological molecules because of their better cellular availability (4). Rapid, simple, reliable, and physiologically relevant in vitro assays are in high demand for screening and development of novel lipophilic antioxidants. Among all known ROS, hydroxyl radical (HO•) is the most damaging one, which may be produced under the physiological conditions and reacts rapidly with almost any biological molecule and may be involved in pathology of many human diseases (6). The production of HO• in biological systems is mainly attributed to the decomposition of hydrogen peroxide and superoxide catalyzed by transition metals including Fe and Cu. Fenton and Fenton-like reactions generating HO• through a similar chemical mechanism may serve as in vitro models for investigating the HO• scavenging properties of lipophilic antioxidants. In 2006, a novel in vitro fluorometric assay was developed for HO• scavenging capacity (HOSC) estimation using a Fenton-like Fe3+/H2O2 system at the physiological pH (7). This assay may be performed in aqueous acetone and is superior to other existing HO• scavenging capacity assays because pure HO• was generated at a constant concentration under physiologically relevant conditions during the entire
10.1021/jf0634808 CCC: $37.00 © 2007 American Chemical Society Published on Web 03/24/2007
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testing period. However, this assay cannot be used to estimate HO• scavenging capacity of lipophilic antioxidants due to their poor solubility in aqueous acetone. In vitro antioxidant property estimation is still a challenge for lipophilic antioxidants. Antioxidant properties of lipophilic components might be underestimated or could not be measured in aqueous systems because of their poor solubility in these testing systems such as that in the oxygen radical absorbing capacity (ORAC), HOSC, and total peroxyl radical-trapping potential (TRAP) assays (7-9). Efforts have been made to enhance the solubility of lipophilic antioxidants in aqueous phases to be able to examine their antioxidant properties. These included the utilization of amphiphilic solvents such as DMSO (10) and solubilizing agents such as randomly methylated β-cyclodextrin (RMCD) (11, 12). It was reported that RMCD might reduce the total HO• concentration in the assay system when using the Fenton-like Fe3+/H2O2 system for radical generation (7), suggesting its capacity to directly react with and quench HO•, which may lead to overestimation of antioxidant capacity for lipophilic components. These previous studies also indicated that solubilizing agents might alter the radical purity in the testing systems and lead to inaccurate estimation of HO• scavenging capacity for lipophilic antioxidants (7). Several organic solvents including DMSO, methanol, and ethanol were able to generate carbon-centered radicals (7, 13, 14), showing possible solvent interference in HOSC assay, which measures HO• scavenging capacity using the Fenton-like Fe3+/H2O2 system. Therefore, this study was undertaken to develop a practical assay for examining HO• scavenging capacity of lipophilic antioxidants such as lutein and β-carotene under the physiological pH. MATERIALS AND METHODS Chemicals. Disodium ethylenediaminetetraacetate (EDTA), iron(III) chloride, iron(II) sulfate heptahydrate, 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), fluorescein (FL), nitro blue tetrazolium (NBT), 5,5-dimethyl N-oxide pyrroline (DMPO), 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (trolox), butylated hydroxytoluene (BHT), R-, δ-, and γ-tocopherols, lutein, and β-carotene were all purchased from Sigma-Aldrich (St. Louis, MO). 30% hydrogen peroxide (H2O2) was purchased from Fisher Scientific. Randomly methylated β-cyclodextrin (RMCD) was purchased from Cyclolab R&D Ltd. (Budapest, Hungary). Ultrapure water was used for all experiments from an ELGA (Lowell, MA) A Purelab Ultra Genetic polishing system with lutein > trolox > β-carotene > γ-tocopherol > BHT > δ-tocopherol determined at 20 min after the reaction was initiated. The order of trolox, R-tocopherol, and lutein is similar to that of the previous observation (22). The IC50 values of trolox, R-tocopherol, β-carotene, and lutein were also determined and shown in Figure 12B, which demonstrated the similar order of the hydroxyl radical scavenging capacities as shown in Figure 12A. IC50 value is the required concentration of a selected antioxidant to quench 50% of the hydroxyl radicals in the ESR assay system under the testing conditions. A stronger antioxidant has a smaller IC50 value. These data indicate the potential application of this ESR assay in measuring and comparing HO• scavenging capacity of lipophilic antioxidants.
In summary, an ESR protocol was developed and validated for its application in measuring HO• scavenging capacity for lipophilic antioxidants. This study also discussed the effects of commonly used organic solvents on the Fenton Fe2+/H2O2 HO• generating system as well as their possible interactions with the fluorescent and spectroscopic probes. In addition, the limitation of the ESR assay was also described.
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(17) Perricone, N.; Nagy, K.; Horvath, F.; Dajko, G.; Uray, I.; Zs.Nagy, I. The hydroxyl free radical reactions of ascorbyl palmitate as measured in various in Vitro models. Biochem. Biophys. Res. Commun. 1999, 262, 661-665. (18) Zhou, K. Q.; Yin, J. J.; Yu, L. L. ESR determination of the reactions between selected phenolic acids and free radicals or transition metals. Food Chem. 2006, 95, 446-457. (19) Tran, T. T.; Cronin, M. T. D.; Dearden, J. C.; Morris, H. Determination of anti-oxidant activity by electron spin resonance spectroscopy. Pest Manage. Sci. 2000, 56, 818-820. (20) Schaich, K. M.; Borg, D. C. Solvent effects in the spin trapping of lipid oxyl radicals. Free Radical Res. 1990, 9, 267-278. (21) Zhao, H. T.; Joseph, J.; Zhang, H.; Karoui, H.; Kalyanaraman, B. Synthesis and biochemical applications of a solid cyclic nitrone spin trap: a relatively superior trap for detecting superoxide anions and glutathiyl radicals. Free Radical Biol. Med. 2001, 31, 599-606. (22) Naguib, Y. M. A. A fluorometric method for measurement of peroxyl radical scavenging activities of lipophilic antioxidants. Anal. Biochem. 1998, 265, 290-298. (23) Pulido, R.; Hernandez-Garcia, M.; Saura-Calixto, F. Contribution of beverages to the intake of lipophilic and hydrophilic antioxidants in the Spanish diet. Eur. J. Clin. Nutr. 2003, 57, 12751282. Received for review December 1, 2006. Revised manuscript received February 8, 2007. Accepted February 19, 2007. This research was supported by a grant from USDA-CSREES National Research Initiatives with a federal grant number of 20043550314852, and a grant from the Maryland Grain Producers Utilization Board (MGPUB) with a MGPUB grant proposal number of 206198.
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