Analytical Biochemistry 423 (2012) 36–38

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Analytical Biochemistry

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Notes & Tips

Differences in responsivity of original cupric reducing antioxidant capacity andcupric–bathocuproine sulfonate assays to antioxidant compounds

S. Esin Çelik, Mustafa Özyürek, Kubilay Güçlü, Res�at Apak ⇑Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, 34320 Istanbul, Turkey

a r t i c l e i n f o a b s t r a c t

Article history:Received 26 August 2011Received in revised form 10 January 2012Accepted 15 January 2012Available online 26 January 2012

Keywords:CUPRAC antioxidant assayNeocuproine (Nc)Bathocuproine sulfonate (BCS)

0003-2697/$ - see front matter � 2012 Elsevier Inc. Adoi:10.1016/j.ab.2012.01.011

⇑ Corresponding author. Fax: +90 212 473 7180.E-mail address: rapak@istanbul.edu.tr (R. Apak).

1 Abbreviations used: CUPRAC, cupric ion reducinneocuproine; BCS, bathocuproine disulfonic acid disodicapacity; NH4Ac, ammonium acetate; EDTA, ethyleneddichloromethane; TEAC, trolox equivalent antioxidant cantioxidant power; TE, trolox equivalent.

The cupric reducing antioxidant capacity (CUPRAC) method using bathocuproine sulfonate (BCS) ascuprous-chelating ligand claims to be an efficient substitute for the conventional CUPRAC assay usingneocuproine; however, differences in their physical chemistry give rise to significantly varying responsiv-ities to antioxidant compounds. The antioxidant capacity due to b-carotene (in acetone or dichlorometh-ane) was fully measurable by conventional CUPRAC but not at all by cupric–BCS assay. Furthermore,CUPRAC exhibits distinctly stronger responses than cupric–BCS to naringenin, a-tocopherol, andbilirubin.

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1

The cupric ion reducing antioxidant capacity (CUPRAC) assay isbased on the 450-nm absorbance measurement of the CUPRAC chro-mophore, Cu(I)–neocuproine (Nc) chelate, formed as a result of theredox reaction of antioxidants with the CUPRAC reagent, Cu(II)–Nc[1]. The CUPRAC method was successfully applied to both lipophilicand hydrophilic antioxidants using methyl-b-cyclodextrin [2] and torat plasma [3]. In this respect, Campos and coworkers [4] developeda similar spectrophotometric method using bathocuproine disulfonicacid disodium salt (BCS) as the chelating agent for Cu(I) emergingfrom the reaction of Cu(II) with antioxidants (i.e., the cupric ionreducing capacity–BCS assay, which is based on the commercial anti-oxidant power [AOP] assay method of Da Cruz [5]). In this study,total antioxidant capacity (TAC) assays based on copper(II) ionreducing ability of antioxidants using Nc and BCS chelators werecompared and evaluated on the basis of reaction kinetics and re-sponse to lipophilic plasma antioxidants.

For the TAC test of CUPRAC, 10 mM CuCl2 aqueous solution,7.5 mM Nc (2,9-dimethyl-1–10-phenanthroline) ethanolic solu-tion, and 1 M ammonium acetate (NH4Ac) aqueous solution wereprepared. For the TAC test of Cu(II)–BCS, 0.25 mM BCS solution in10 mM phosphate buffer (pH 7.4), 0.5 mM CuSO4 aqueous solution,and 0.01 M ethylenediaminetetraacetic acid (EDTA) solution wereprepared. Trolox, uric acid, ascorbic acid, glutathione, butylated

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g antioxidant capacity; Nc,um salt; TAC, total antioxidantiaminetetraacetic acid; DCM,apacity; FRAP, ferric reducing

hydroxy anisole, p-coumaric acid, gallic acid, naringenin, bilirubin,a-tocopherol, and b-carotene were used as antioxidants at 10 mMconcentration. Uric acid and bilirubin were dissolved in 0.5 MNaOH and diluted with water. b-Carotene was prepared in dichlo-romethane (DCM) and acetone separately. Ascorbic acid and gluta-thione were dissolved in water. The remaining antioxidants wereprepared in absolute ethanol. The absorption measurements weremade using a Varian CARY Bio 100 ultraviolet–visible (UV–vis)spectrophotometer (Mulgrave, VIC, Australia).

For the CUPRAC assay as described by Apak and coworkers [1],to a test tube were added 1 ml each of Cu(II), Nc, and NH4Ac solu-tions. Antioxidant standard solution or food/biological samplesolution (x) and H2O or MeOH (1.1 – x) ml were added to the initialmixture so as to make the final volume 4.1 ml. The tubes werestoppered, and after 30 min the absorbance at 450 nm (A450) wasrecorded against a reagent blank. For incubated sample measure-ment, the tubes were stoppered and incubated for 20 min in awater bath at a temperature of 50 �C. The tubes were cooled toroom temperature, and their A450 values were measured. For lipo-philic antioxidants and extracts, the CUPRAC–DCM method [6] wasapplied and the A450 of the formed Cu(I)–Nc was recorded in theorganic phase against a reagent blank extract after 30 min. Theexperimental procedure described by Campos and coworkers [4]was applied by changing volumes of solutions. To a test tube,2 ml of synthetic antioxidant solution or real sample solutiondiluted at a 1:40 (v/v) ratio with 0.25 mM BCS in 10 mM phosphatebuffer was added, and A490 was recorded. After the addition of0.5 ml of 0.5 mM CuSO4, the reaction mixture was vortexed andincubated for 3 min. The reaction was stopped by adding 0.5 mlof 0.01 M EDTA, and A490 was recorded again. The difference inabsorbance (DA490) was used in calculations.

Table 2TACs of food extracts and biological samples as trolox equivalents measured withconventional CUPRAC and cupric–BCS assays (n = 3).

Sample TACCUPRAC TACcupric–BCS

Grapefruit (mM TE) 2.55 ± 0.094 1.99 ± 0.122Grapefruit hydrolysate (mM TE) 3.78 ± 0.186 2.43 ± 0.103Grapefruit hydrolysate (incubated) (mM TE) 6.76 ± 0.276 3.57 ± 0.203Carrot extract (MeOH/H2O, 4:1, v/v)

(lmol TE/g)9.89 ± 0.341 1.91 ± 0.122

Carrot extract (acetone, 100%) (lmol TE/g) 5.55 ± 0.136 NDOlive oil extract (methanolic extract)

(lmol TE/g)0.61 ± 0.014 0.17 ± 0.012

Human serum extract (hydrophilic) (mM TE) 0.32 ± 0.012 0.15 ± 0.010Human serum extract (lipophilic) (mM TE) 0.07 ± 0.002 NDUrine sample (mM TE) 11.29 ± 0.312 5.77 ± 0.100

Note: Data presented as (mean ± SD); TE, trolox equivalent; ND, not detected.

Notes & Tips / Anal. Biochem. 423 (2012) 36–38 37

Reproducibility studies were carried out in five replicate sam-ples of ascorbic acid at 24.4 lM. The intraday precision values ofthe CUPRAC and cupric–BCS assays, expressed as the relative stan-dard deviation (RSD) for the analyte, were 0.97 and 2.07% (n = 5),respectively. The interday precision values of the two methodswere 1.17 and 2.30% (n = 5), respectively. The trolox equivalentantioxidant capacity (TEAC) coefficients of the tested antioxidants(Table 1) and the TAC values of food extracts and biological sam-ples (Table 2) were determined by these two assays. These coeffi-cients were simply calculated by dividing the indirect (i.e., due toCu(I)–chelate chromophore) molar absorptivity of the species un-der investigation by that of trolox under identical conditions.Although the CUPRAC–DCM method [6] developed for the mea-surement of lipophilic antioxidant capacity of synthetic or realsamples was responsive toward carotenoids, the TAC value dueto the important plasma antioxidant, b-carotene (dissolved in bothDCM and acetone separately), could not be measured with the cup-ric–BCS assay (Table 1) because no color formed in the DCM or ace-tone phase. Most likely, the Cu(II)–BCS reagent is not effective asan electron transfer agent at the interface, and the reaction prod-uct, Cu(I)–BCS complex, could not be extracted to the organicDCM phase due to its high charge. Likewise, the Folin and FRAP(ferric reducing antioxidant power) chromophores were shownto exhibit a higher affinity to hydrophilic antioxidants due to theirhigh charges, meaning increased ion–dipole interactions withwater molecules [7]. Moreover, the reactive part of carotenes, suchas lycopene and a- and b-carotene, is the conjugated polyene chainin the center of the molecule [8]. This fact makes it difficult for ste-ric-demanding oxidants to interact with the carotenoid, especiallywith the bicyclic structures of b-carotene [9]. As a result, b-caro-tene did not respond to the FRAP assay [10], probably due to thecircumstance that the oxidizing agent, ferric ion, is incorporatedinto the steric-demanding di-tripyridyltriazine (TPTZ) complex.In our case, cupric–BCS, the sterically more demanding oxidant,gave no response to b-carotene in either DCM or acetone, whereascupric–neocuproine (of conventional CUPRAC) rated TEAC valuesof 1.75 and 3.35 in acetone and DCM, respectively, towardb-carotene, close to that of the ABTS [2,20-azinobis-(3-ethylbenzo-thiazoline-6-sulfonic acid)] assay [9].

The TAC values (as trolox equivalents, TEs) of several foodextracts and biological samples measured with both assays werecompared (Table 2). In the assay of human serum antioxidants,hydrophilic antioxidants were measured after precipitation of pro-teins with HClO4, whereas lipophilic ones such as a-tocopherol andb-carotene were determined by n-hexane extraction, evaporation,

Table 1TEAC coefficients of some antioxidant compounds with conventional CUPRAC andcupric–BCS assays.

Antioxidant TEACcupric–BCSa TEACCUPRAC [1,2,6,8]

Uric acid 0.94–1.08 0.96Ascorbic acid 1.02–1.09 0.96Glutathione 0.61–0.64 0.57Butylated hydroxyanisole 0.94–1.22 1.40p-Coumaric acid 0.45 0.55Gallic acid 2.14–2.25 2.62Naringenin 0.05–0.93b 0.05–2.28b

Bilirubin 0.92 3.18a-Tocopherol 0.83 1.10b-Carotene –c 3.35d–1.75e

a TEACcupric–BCS coefficients shown in bold were obtained from Campos andcoworkers [4].

b The incubation procedure was done at 50 �C for 20 min.c b-Carotene (dissolved in both DCM and acetone) did not respond to cupric–BCS

assay.d Solvent medium: DCM solution.e Solvent medium: pure acetone solution.

and subsequent color development in DCM of the Cu(I)–Nc chelateformed from their CUPRAC reaction [6]. The TAC values of lipo-philic extract could not be measured by cupric–BCS assay. TheTAC values of hydrophilic serum extract were 0.32 and 0.15 mMTE, as measured by CUPRAC and cupric–BCS assays, respectively.These methods were also applied to methanolic (for hydrophilics:MeOH/H2O, 4:1, v/v) and acetonated (for lipophilics: 100% acetone)carrot extracts. The TAC values of lipophilic extract of carrot couldnot be measured with cupric–BCS assay. Besides, the hydrophilicTAC value of carrot sample measured by cupric–BCS assay wasfound to be 5 times lower than the corresponding CUPRAC value.The extraction of olive oil samples was performed according tothe procedure described by Pirisi and coworkers [11]. The TACvalues of methanolic olive oil extracts (MeOH/H2O, 60:40, v/v)were measured to be approximately 4 times higher with the CU-PRAC assay. The TAC values of a urine sample—diluted with bidis-tilled water—provided from a healthy adult volunteer were foundto be 11.29 and 5.77 mM TE using CUPRAC and cupric–BCS assays,respectively.

The main difference between CUPRAC and cupric–BCS assaysshowing similar chemistry is expected to arise for those antioxi-dants exhibiting slow kinetics with the assay reagent (oxidantprobe) depending on the solvent. Conventional CUPRAC scoredhigher TEAC values for naringenin, bilirubin, a-tocopherol, and b-carotene (Table 1). The method yielding the higher TAC values astrolox equivalents (with trolox as the reference compound being� 100% oxidized by both assays) for a given real sample of complexantioxidants is understood to oxidize that antioxidant mixture to agreater extent under the assay protocol conditions. These dataprove that the conventional CUPRAC method using Cu(II)–neo-cuproine reagent as the oxidant probe is much more diverselyapplicable to a wide range of biological and food extracts thanthe cupric–BCS assay.

Among the antioxidant compounds listed in Table 1, naringeninshows the lowest antioxidant capacity because it is a singly hydro-xy-substituted flavanone having a high redox potential. The rise ofabsorbance with time at room temperature shows a similar profilefor both the CUPRAC and cupric–BCS methods (Fig. 1A). In theoriginal CUPRAC method [1], it was reported that slow-reactingantioxidants such as naringenin required an incubation at 50 �Cfor 20 min for maximal color development, reflecting the com-pleteness of oxidation. After incubation, the TEAC value of naringe-nin increased from 0.05 to 0.93 in the cupric–BCS assay, whereas itincreased to 2.28 in the CUPRAC assay. The rate of oxidation ofnaringenin is obviously greater with the Cu(II)–Nc reagent(Fig. 1B), and considering the fact that the molar absorptivity ofthe reference compound, TR, is not temperature dependent, thefinal TEAC value of naringenin with conventional CUPRAC showsa net increase of 145% compared to that with Cu(II)–BCS under

Fig.1. Kinetic curves for the reduction of Cu(II)–Nc and Cu(II)–BCS with naringenin: Normal A and incubated B measurements.

38 Notes & Tips / Anal. Biochem. 423 (2012) 36–38

identical conditions of incubation (Fig. 1B). Naringin turns to itsaglycone, naringenin, by acidic hydrolysis (reflux in 1.2 M HCl-con-taining 50% MeOH at 80 �C for 2 h). Grapefruit hydrolyzate (incu-bated) showed the higher TAC value with CUPRAC, probablybecause the TAC contribution of naringenin could not be com-pletely assayed with the cupric–BCS method.

In conclusion, Cu(I)–Nc is distinctly more hydrophobic thanCu(I)–BCS (with the latter being cell membrane impermeable [12]due to its higher charge responsible for ion–dipole interactionswith polar water molecules), and therefore Cu(I)–Nc as the CUPRACchromophore can be more useful to the TAC assay of tissue homog-enates. Electron transfer-based antioxidant assays may show sig-nificant solvent dependencies and differences in proton-coupledelectron transfer rate [13], and it has been shown here that the cup-ric–BCS assay is not competent with conventional CUPRAC usingcupric–Nc reagent in regard to reaction kinetics and response tolipophilic plasma antioxidants (e.g., b-carotene, a-tocopherol). Asa result, it may be suggested that the cupric–bathocuproine assayshould not be further abbreviated as ‘‘CUPRAC–BCS’’ [4] to avoidconfusion but rather should be suitably referred to by the originalname as ‘‘copper(II) reduction assay using bathocuproinedisulfonicacid disodium salt’’ given by Campos and coworkers [4].

Acknowledgment

Authors would like to thank to T.R. Ministry of Development forthe Advanced Research Project of Istanbul University(2011K120320).

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