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    Development and Validation of Oxygen Radical Absorbance PDF

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    AGRICULTURAL AND FOOD CHEMISTRY 2002, 1815-1821 1815 Development and Validation of Oxygen Radical Absorbance Capacity Assay for Lipophilic Antioxidants Using Randomly Methylated 6-Cyclodextrin as the Solubility Enhancer Desi HUANG, BoxiN Qu,* MAUREEN HaMpscii-WoopiLL, Juprri A. FLANAGAN, AND ELIZABETH K. DEEMER, Brunswick Laboratories, 6 Thacher Lane, Wateham, Massschusets 02571 We recently reported the improved oxygen radical absorbance capacity (ORAC) assay using ‘luorescein (FL) as the fluorescent probe, The current ORC assay is limited in hydrophilic antioxidant due to the aqueous environment of the assay, Lipopilc antioxidants mainly include the vitamin E family and carotenoids, which play a critical role in biological defense systems. In this paper, we ‘expanded the current ORACn. assay to lipophilic antioxidants. Randomly methylated f-cyclodextrin (RMCD) was introduced as the water solublity enhancer for lipophilic antioxidants. Seven percent RMCD (wiv) in a 50% acetone—H,0 mixture was found to sufficiently solubilize vitamin E compounds ‘and other lipophilic phenolic antioxidants in 75 mM phosphate buffer (oH 7.4). This newly developed ‘ORAC assay (abbreviated ORAC+. po) was validated through linearity, precision, accuracy, and ruggedness. The validation results demonstrate that the ORACr.-.eo assay is reliable and robust For the fist time, by using 6-hydroxy-2,8,7.8-tetramethyl-2-carboxylic acid as a standard (1.0), the ‘ORAC values of a-tocopherol, (+}--tacopherol, (+-é-tocopherol, a-locopheral acetate, tocotrienols, 2,6-4i-tert-buty-4-methylphenol, and y-oryzanal were determined to be 0.5 ~ 0.02, 0.74 = 0.03, 1.35 + 0.14, 0.00, 0.91 = 0.04, 0.16 = 0.0%, and 3.00 + 0.26, respectively. The structural information of oxidized a-tocopherol obtained by liquid chromatographyimass spectromet’y reveals that the ‘mechanism for the reaction between the vitamin E and the peroxy! radical follows the hydrogen atom transfer mechanism, which is in agreement with the notion that vitamin E is the chain-breaking antioxidant. KEYWORDS: ORAG; cyclodextrin; LC/MS; ipephille antioxidants; vitamin E; chain breaking; hydrogen atom transfer INTRODUCTION Antioxidants ean be physically classified by their solubility into two groups () () hydrophilic antioxidant, such as vitamin C and the majority of polyphenolic compounds, and (Gi) lipophilic antioxidants, mainly including vitamin E and earot- enoids, Similar to hydrophilic antioxidants, lipophilic antioxi- «dans play an important role in a wide speetrum of biochemical and physiological processes. Of primary interest is their optimal antioxidant activity in vitto and in vivo. Unlike hydrophilic antioxidants, which do not accumulate in the body and are exereted in the urine, lipophilic antioxidants penetrate the Lipoprotein cell membrane more easily and therefore reach a higher level of bioavailability (2). Although lipophilic antioxi- ddants are highly bioavailable, itis not a trivial task to accurately measure their antioxidant activity in vito. There are a number of methods available for measuring antioxidant activity such as the oxygen radical absorbance capacity (ORAC) fluorescein * To winom corespondence shouldbe a Esai) bowalbranieklobs som sed Fax (508)295-6615, so-1oz1yort3732 ccc: $22.00, (FL) assay fiom our laboratory; all of these methods are conducted in an aqueous system, as such, none of which are suitable for lipophilic antioxidants. Without knowing the actual cffectiveness of the lipophilic antioxidants, consumers ean be ‘exposed to unsafe concentrations or ineffective dosages. We overcame this obstacle by introducing randomly methylated B-cyelodextrin (RMCD) as @ molecular host to enhar solubility of lipophilic antioxidants in aqueous solution, Speci: ically, cyclodextrins (CDs) are eyelic (4-1,4)-linked oligosac- charides of a-b-gluco-pyranose containing @ relatively hydro- phobic (fatlike) central cavity and hydrophilie (waterlike) outer surface. This property of CD has made it increasingly popular 4a a vehicle for enhancing the solubility of fat soluble ‘compounds in an aqueous environment in pharmaceutical and food industries (3, 4. In this paper, we will report the development and validation of a new ORAC assay specific for lipophilic antioxidant activity. To our knowledge. it is the first time that vitamin E and other common lipophilie phenolic antioxidants were measured using the ORAC assay, © 2002 American Chemical Society se the Published an Web 0208/2002 1816 Vol. 50, No. 7, 2002 MATERIALS AND METHOD ‘Chemicals and Apparatus. RMD was purchased from Cyelola| RAD Lid. (Budapest, Hungary). FL and 6-hydeoxy-2.578-ctrametby- 2-carboxylie acid (Trolox) were purchased from Aliich (Milwaukee, Wh. 2,7-Azobis (2-amidino-propane) dihydrochloride (APH) was obtained fiom Wako Chemicals USA (Richmond, VA). y-Oryzanol was purchased ftom TCI America (Porand, OR). Nutiene (toco- tronols) was obtained from Eastman Chemicals Company (Kingsport, TN), 4Difhuoo-5-(epheny-I-butadieny)-+bora-3a$a-diazt-in- dcene-3-undevanoie acid (BODIPY $81/591 C11) was purchased from Molecular Probes, Inc. (Eugene, OR). ll other standands were lable form Sigma or Aldrich, All ORAC analyses ‘were performed on a COBAS FARA Il analyzer (Roche Diagnostic System In, Branchburg, NJ) using an excitation wavelength of 493, rm and an cmissin filter of $15 nm, The photobleaching experiment fof BODIPY $81/891 CIT was carried out in a Bio-Tek fluorescence microplate reader FL600 (Bio-Tek, Winooski, VT). The identification ‘of oxidized Troox and a-iocopherol induced by AAPH was performed by a HP 1100 high-performance liquid chromatography (HPLC) system (Hewiot-Parkard, Palo Alto, CA) coupled with a Finigan LCQ ion ap ‘mass spectroscope detector (Thermanigan, San Jose, CA. Sample Preparation. Approximately 0.5 g of simple was dissolved in 20 mL of acetone. An aliqut of sample solution was appropriately diluted with 7% RMD solvent (ws) made in a 50% acetone wate ‘mixture (vy) and was shaken for 1 at room ten shaker at 400 rpm. The sample solution was ready for analysis after fuer dilution with 7% RMICD solvent, Automatic ORAC Assay. The automated ORAC assay was carried out on a COBAS FARA I spectrofluorometric centrifugal analyzer. The procedure was based on a previous report of Ou and co-workers (5). With the exception of samples and Trolox standard, which were ‘made in 7% RMCD solvent, al other reagents were peepared at 75 ‘mM phosphate buffer (pH 74). Inthe final assay mixture (044 mL tol volume), FL (63 % 10-® M) was used asa target of free radical attack and AAPH (1.28 x 10°? M) was used as a peroxyl radial generator. Seven percent RMCD was used as the blank, and Trolox (12.5, 28, $0, and 100 pM) was used a the control standard. The analyzer was programmed recor the fuoresence of FL every minute afer the addition of APH. All measurements were expressed relative to the initial reading, Final results were calculated using the differences of areas under the FL decay curves between the blank and a sample ‘These results were expressed as micromoles Trolox equivalent (TE). ‘Competitive Reaction of Trolox and c-Tocopherol with APH. A total of 0.5 mL of 0 mM Trolox and 0.5 mL of OS mM ‘tocopherol (prepared in 7% RMCD) were mixed ina 1.5 mL HPLC sample vial followed by the addition of 20 L of 640 mM APH: the reaction solution was incubated at 37 °C nan autosampler. AL every 10 min itera, $y. of reaction solution was injected nto Zorba C18 column (Hewlett-Packard, Palo Ato, CA) Q.1 mmx 150 mm, 3 um) until the rection was completed. The mobile phase was 70% ‘methanol with low rate of 0.3 mLimin, and the UV detector was set 1280 nm, ‘Characterization of Oxidized Products of Trolox Induced by APH. A misture of 0.2 ml. of 2,0 mM Trolox and 0.8 mL of 640 mM AAPH was incubated at 37 °C for 30 min, The rection products ‘were separated by a Zorbax C18 column (2.1 mm 150 me, 3 4m) A137 °C with a mobile phase of 70% methanol at a Mow rate of 0.3 L/min, andthe UV detector was sot at 280 nm. The oxidized products were characterized using Finnigan LCQ ion trap mass spectrometer ‘suipped with an API chamber and an eloctospray ionization (ESI) source. The ionization was negative fon mode, and Aux gas and Sheath 8s were sett 72 and 14 units, respectively. An ionization reagent of [LS mM ammonium hydroxide was added at «rate of (105 mlimin through a Tee device using & secondary HPLC pump before the API chamber, Trolox was used asa standard for calibrating the system, (Characterization of Oxidized Products of «Tocopherol Induced by APH, A mintre of 05 mL of 20 mM a-tocopherol (prepared in 7% RMCD) and 0.5 mL of 640 mM APH was incubated at 37 °C for 30 min and was extracted with 10 mL. of methylene chloride. The methylene chloride phase was separated and washed with § mL of Huang et a. Relative Fluoresonce Intensity re ee a ee) ‘Time (min) Figure 1. FL fuorescence decay curves induced by APH inthe presence ‘of cctocopheral at ifferentcancenraon. _ — 2s x» a Net AUC © 20 40 © 80 100 120 140 10 160 200 270 Concentration (:M) Figure 2. Regression of nel AUC of e-ocopherol on diferent conconra- tions of tocopherol. The net AUC = AUCsng» ~ AUCapa’ the AUC was cacuaed by the equation previously described by Ou ea, (See). eionized wate 3 times. The oxidized products of a-tocopherol inthe methylene chloride phase were then analyzed by quid chromatography’ mass spectroscopy (LCIMS). With the exception of 100% acetone 4s the mobile phase, the LCIMS conditions are the same as above, Photobleaching Experiment of BODIPY §81/891 C11. BODIPY 581/391 CI was dissolved in 1:9 butyronittle and octane mixture (to givea 7.9 x 10-7 M solution; 200 of the BODIPY solution was then added into a 96 well polyproplene plate, The photostailty ‘of BODIPY was evaluated by monitoring the change of fluorescence imensity over a 1h period. The furescence reading was taken every minute by a Bio-Tek fluorescence microplate reader F600 (Bio-Tek, Winooski, VT) equipped with an excitation filter for $4540 nm and ‘an emission iter for 620/40 nm, RESULTS Method Validation, (a) Linearity. The correlation between the net area under the curve (AUC) forthe antioxidant and its ‘concentration was evaluated using four pure compounds. Figure 1 illustrates the FL fluorescence decay curves in the presence ‘of c-tocopherol and AAPH. Figure 2 depicts the linear response between the concentration and the net AUC for a-tocopherol ‘Table 1 summarizes the net AUCs corresponding to the different ‘concentrations and the linear coefficient (°) for the pure ‘compounds. (b) Limit of Quantitation (LOQ) and Limit of Detection (LOD). The LOQ is the lowest concentration on the calibration ‘curve, while the LOD is the lowest amount of antioxidant that ORAC Assay for Lipophilic Antioxidants be 1. Net AUC vs Conceitation® cam en ut) tea 2 anal B vas 25 ‘sar ose 1625 ast 305 a2 ‘ooapetol 100 mas Py ua osm % 78 docophera 5 sant » na a 1556 09568 5 a3 625 aus cecocopecl 200 wast ‘00 1909 0 045, ose0 B ‘607 = Regression equaton & exressed aS (nel a8) itrcept (eoeenaten) + Table 2. Precision and Accuracy of the ORACrt-uo ASSAY amin act eco acs varinalcanen Ga) o 10 Runt lavaean (a) ae eae 175.40 so 287 20 S67 SRS an 252 an REC: 10425 105.60 10962 N 4 4 4 Rn? lavaean (a) an a0 ima so 25 5 108% RSD 20 557 ou REC 10685 1501 10703 N 4 4 4 Rin ‘amean 902s esas 72 so 56 654 261 RSD 1399 1a 156 SeREC oro worst 10450 N 4 4 4 paced nas btarean (a) ss Bs im 30 368 63 638 RSD 268 524 an soREC 58 10931 10205 N 2 ” 2 7 Slandarédelton* Rave Sanda devon, “Reco can be detected. In our experiment, the LOQ and LOD were determined to be 12.5 and 5.0 uM, respectively (c) Precision and Accuracy. Table 2 summarizes the precision and accuracy of the ORAC assay using a tocopherol as @ candidate compound, The precision, which is expressed as relative standard deviation (%4RSD) for all quality control concentrations, was within +15%, The accuracy of the method varies from 97.60 to 15.01% within individual batches and from 95.39 10 107.05% between all of the batches. (@) Ruggedness. To determine the reproducibility of the method, a ruggedness experiment was performed. Using two COBAS FARA Il analyzers, 12.5 4M y-oryzanol was analyzed for 50 days, Results are shown in Figure 3 ORAC Values for Pure Lipophilic Antioxidants. Table 3 lists the results for common lipophilic phenolic antioxidants, which were referenced as the TEs. y-Oryzanol possesses the Vol. 50, No. 7, 2002 1817 ORAC (Trolox Equivalent) ‘Time (day) Figure 3. Ruggodness of the ORACR-uro assay. An amount of 12.5 {AM y-onyzanol was analyzed using two COBAS FARA Il analyzers for 55 days Table 3, Relative ORAC Values of Pure Compounds ( = 4) comps RAC Tron 70 atacopet 050002 ‘atocoperl cea 00 (}ysoconhe 0742003 Gposocophea 1a6s018 rene (eeotienls) 0912008 22518 periomethyt6-cronana torso 2. bayidnetyphena onz001 aur 0162001 paryzanol 100026, 12 Me tora eto amy) | Time (min) Figure 4. Kite curves fr Trolox and a-tocopherol inthe prosence of ‘AAPH, The reaction mixture coniais 0.25 mM Tiolox, 025 mit ‘clocopherol and 128 mt AAPH in RMICD (7% w) soon, The mitre vas incubated at 37 °C in a HPLC autosampe, and 5 a. mitre was. ‘analyzed by HPLC al every 10 min interval unl the reaction was, completed. highest value of 3.0, while vitamin E acetate shows no antioxidant activity ‘Competitive Reaction between Trolox and c-Tocopherol with APH. The competitive reaction between Trolox and ‘e-tocopherol with AAPH was examined, and the kinetic curves for Trolox and a-tocopherol were illustrated in Figure 4. It is ‘evident that the reaction rate of Trolox with APH is much, faster than that of a-tocopherol with APH. Mechanistic Studies. The mechanisms for peroxidation of | Trolox and a-tocopherol can be clucidated based on their ww18 Vol. 50, No. 7, 2002 Huang et a. pom. — a 1 a B i | Product 2 Product 1 ) ~ | a ae ae | Figue 5. (2) HPLC cromatogram fr the Tilx oiaon pote iduced by AAPH. The mire contained 0.4 mi Trolox and 512 mil AAPA in 75 mnt phosphate bul (pH 74) and was incbaled al 37°C fr 30min. Te reaction pots were separated by a Ztbax C18 column 2.1mm x 150 tm, 3) al 37°C witha mobile phase of 70% methanol at a How rate of 0.3 min and the UV detector was sla 20 rm, The oxidized products wee characterized by sig a Finnigan LCO ion rap mass spectrometer equpped wth an AP chamber and an ESI source. (b) HPLC chromatogram {athe cocoperl oxidation pre induced by AAPH. A mixture of 05 mi of 20 mM tocopherol (repre in 7% RCD) and 0.5 mk of 640 mM -AAPH as incubated at 37 °C for 30 min and was exacted with 10m of methylene chaide. The methylene cli phase was separated and ‘washed wih 5 mL of Dl water 3 tines The xed paul of cacopesl inthe methylene clare phase wee then analyzed by LCS. Wh he exception of 100% acetone asthe mobile phase, the LCIMS conditions are the same as a Net AUC. / - % Concentration of RMCD (wiv) Figure 6. Effect of RMICD concentration (nt) on the net AUC of 50 Mt ‘locopherl oxidized products obtained by LC/MS. As shown in Figure Sab, both Trolox and a-tocopherol were oxidized into two ‘major products with 16 and 32 mass units increase. Figure 8 illustrates the proposed structures for oxidized products and the oxidation mechanism, DISCUSSIONS ‘The ORAC assay was originally developed by Cao and co- workers (6) and was significantly improved by Ou et al. (5). However, the improved ORAC assay does not address the issue of lipophilic antioxidants because the assay is performed in aqueous solution. The application of CD for solubilization of lipophilic compounds has been extensively studied, particularly withthe earotene/fatty acids—CD interaction (3, 4, 7). CDs are °| Seaman] A ge a Qa. 3 ° =o ® mm Concentration (iM) Figure 7. Regression of net AUC of Trlox on diferent concentrations of “Trolox inthe presencelabsence of RMCD (73 wiv). Each data point epresents an average of six separate measurements, 2 group of naturally occurring cage molecules, which are built up from a-b-glucose units. Depending on the number of glucose moieties in the ring (6, 7, or 8), they are named a, >, and -eyclodextrin, CDs are doughnut-shaped and can bind a wide variety of organic “guest” eompounds inside their apolar cavities in aqueous solution. The main driving force for this binding is hydrophobic interactions. Recently, Szente et al. studied the solubilization of carotene and fatty acids in aqueous solution, and their results indicated that the solubilizing power of CD. derivatives are methylated-p-cyclodextrin >> hydroxypropylated= Boyclodextrt -cyelodextrin (4). The use of 10~ 40% methylated-f-cyclodextrin results in the 1000-fold en- hhancements of the aqueous solubility of lipophilic compounds. In light of Szente's work, we employed RMCD in the ORAC ORAC Assay for Lipophilic Antioxidants ean a ‘bet 7 Pique 8 Popssedaaon mechanism br Tbe ard aacophrtn the presence of AAPH, assay for lipophilic antioxidants. As demonstrated in the following sections, RMCD was determined to be an ideal solubility enhancer forthe studied antioxidants, and forthe first time, the antioxidant values for tocopherols, tocotrienols, and ‘other common lipophilic antioxidants were obtained using the ORAC assay. Effect of RMCD Concentration on the Solubility of Vitamin E in Aqueous Solution, Figure 6 shows the net AUC of 50 uM a-tocopherol at different RMCD concentrations (w/ v) in 75 mM phosphate buffer solution (pHT 7.4). The net AUC of a-tocopherol inereases withthe increase of RMCD concen- tration and reaches a plateau after 4% RMCD. The plateau indicates that a-tocopherol is completely soluble in 75 mM phosphate buffer solution. This conclusion was further con- firmed by the HPLC studies (data not shown). Taking into account the concentration variations, 7% RMCD in $0% acetone solution was chosen for sample preparation. Effect of RMCD on the ORAC Value of Trolox. Lipophilic antioxidants, such asthe vitamin E family and oryzanol, consist of along aliphatic tail (> 16 carbons) and a hydrophilic phenol group on the head, The tal ean fit into a CD cavity allowing the hydrophilic phenol group to remain in aqueous solution. Therefore, the reactivity of the headgroup is not inhibited by CD complexation. RMCD itself consists of hydroxyl and ‘methoxyl funetional groups and is doughnutshaped with an ‘open moiety; therefore, RMCD does not possess any antioxidant activity nor does it prevent the complexed antioxidant molecule from functioning as antioxidant. To confirm this, the ORAC values of Trolox were examined in the presence (or absence) of 7% RMCD. Figure 7 shows the linear curves of the net AUC against the Trolox concentrations in phosphate buffer and 7% RMCD solution, respectively. It becomes obvious thatthe two curves are almost superimposed, suggesting that RMCD is inert In the ORAC assay. Difference in ORAC Value between a-Tocopherol and Trolox. Because of the structural similarity, Trolox is expected to possess a similar ORAC value to that of a-tocopherol However, our results reveal that the ORAC value of a-toco- pherol is about 50% less than that of Trolox. The difference in ORAC value prompted us to investigate the reaction kinetics and mechanisms of Trolox/a-tocopherol with AAPH. Figure 4 shows the competitive reaction kinetie curves between Trolox, tnd a-tocopherol in the presence of APH As shown, the rate of Trolox with AAPH tends to be much faster than that of ‘a-tocopherol with APH. This observation is in agreement with their ORAC values. At this point, we suggest thatthe inductive Vol. 50, No. 7, 2002 1819 effect on the phenol group derived from the long (CieHs) aliphatic tal of a-tocopherol, an electron-

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