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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2012 Nov 30;94(1):25–33. doi: 10.1111/iep.12001

Developing SyrinOX total antioxidant capacity assay for measuring antioxidants in humans

Endry N Prasetyo *, Otto Knes †, Gibson S Nyanhongo *, Georg M Guebitz *
PMCID: PMC3575870  PMID: 23198957

Abstract

Accurate monitoring of the antioxidant status or of oxidative stress in patients is still a big challenge in clinical laboratories. This study investigates the possibility of applying a newly developed total antioxidant capacity assay method based on laccase or peroxidase oxidized syringaldazine [Tetramethoxy azobismethylene quinone (TMAMQ)] which is referred to here as SyrinOX, as a diagnostic tool for monitoring both oxidative stress and antioxidant status in patients. Attempts to adapt the Randox total antioxidant procedure [simultaneous incubation of the radical generating system (metmyoglobin and H2O2) and antioxidant sample] for SyrinOX were abandoned after it was discovered that the H2O2 reacted with enzymatically generated TMAMQ and ABTS radicals at a rate of 6.4 × 10−2/μM/s and 5.7 × 10−3/μM/s respectively. Thus this study for the first time demonstrates the negative effects of H2O2 in the Randox system. This leads to erroneous results because the total antioxidant values obtained are the sum of radicals reduced by antioxidants plus those reacting with the radical generating system. Therefore they should be avoided not only for this particular method but also when using other similar methods. Consequently, SyrinOX is best applied using a three-step approach involving, production of TMAMQ, recovery and purification (free from enzyme and other impurities) and then using TMAMQ for measuring the total antioxidant capacity of samples. Using this approach, the reaction conditions for application of SyrinOX when measuring the total antioxidant capacity of plasma sample were determined to be 50% (v/v) ethanol/50 mM sodium succinate buffer pH 5.5, between 20 and 25 °C for at least 1 h.

Keywords: 2,2′-azinobis (3-ethylbenzothiazoline 6-sulphonate); free radicals; oxidative stress; plasma antioxidants; syringaldazine; tetramethoxy azobismethylene quinone; total antioxidant capacity assay

Oxidative stress caused by an imbalance between reactive oxygen species and the ability of the body's system to readily detoxify these species is implicated in many pathophysiological diseases including cancer, neurodegenerative, cardiovascular diseases etc. These oxidative stress-related diseases are notorious for being diagonized too late for successful intervention. It is believed that successful intervention aimed at preventing or curing oxidative stress-related diseases depends on being able to establish the presence of stress or its footprints early. It can also be further inferred that accurate measurement of the total antioxidant capacity may also assist in evaluating physiological and nutritional factors that promote the development of oxidative stress. Several methods including those based on peroxyl radical scavenging [Oxygen radical absorbance capacity (ORAC), Total reactive antioxidant potential (TRAP), total oxidant scavenging capacity (TOSC)], metal reducing power [Ferric reducing antioxidant power (FRAP), Cupric reducing antioxidant capacity (CUPRAC)], hydroxyl radical scavenging (deoxyribose assay), cellular antioxidant activity assay (CAA) and organic radical scavenging [the Randox Total antioxidant status (TAS), (2,2-diphenyl-1-picrylhydrazyl (DPPH)] (Huang et al. 2005; Moreira et al. 2012) have been developed. However, none of these methods are internationally standardized by Huang et al. (2005).

Among these methods, the Randox TAS assay is operationally simple and is used in food, clinical analytical laboratories as well as in many research laboratories as described in the study by Karadag et al. (2009). This assay first reported by Miller et al. (1993) is based on simultaneously incubating a radical generating system (metmyoglobin + H2O2) which oxidizes ABTS substrate producing ABTS cation radicals (intense green-coloured product monitored spectrophotometrically in the range of 600–750 nm) with the antioxidant sample. The metmyoglobin reacts with H2O2 to produce ferrylmetmyoglobin radical, a powerful oxidizing agent which oxidizes the ABTS molecule to ABTS cation radicals. The Randox TAS assay, therefore, measures the ability of the antioxidant test sample to suppress the formation of ABTS cation radical as described in the study by Karadag et al. (2009), Roginsky and Lissi (2005). However, studies have shown that this approach only measures hydrophilic antioxidants (Karadag et al. 2009), and although frequently used at pH 7.4, the stability of ABTS radical at this pH has been reported to be problematic by Karadag et al. (2009). Further, many studies have also reported no relationship between the obtained values and the number of electrons that an antioxidant molecule can donate (MacDonald-Wicks et al. 2006; Ozgen et al. 2006; Karadag et al. 2009). Recently, our group developed an antioxidant capacity assay method based on laccase or peroxidases oxidized syringaldazine [Tetramethoxy azobismethylene quinone (TMAMQ)], a deeply purple quinone with absorbance maxima at 530 nm as described in the study by Nugroho Prasetyo et al. (2009; Nugroho Prasetyo et al. 2010a,b, 2012). This method unlike other existing methods is the only one with alternative ways of validating obtained data by adopting a dual UV–VIS spectroscopy. Data obtained by monitoring the decrease in TMAMQ can be verified by comparing with the formation of syringaldazine (parent compound). As part of ongoing studies aimed at investigating the suitability of TMAMQ assay (referred here as SyrinOX) as an alternative method for measuring total antioxidant capacity of clinical samples, this study aims at adapting the operational simplicity of the Randox TAS assay procedure (simultaneously mixing radical generating system, ABTS and antioxidant sample) by substituting ABTS for syringaldazine. Further, this study also investigates the optimal solvent reaction conditions needed for the routine assay of the total antioxidant capacity of plasma samples using SyrinOX. This is because solvent effects are crucial to the chemical behaviour of antioxidant compounds and have been shown to affect all existing total antioxidant capacity assay methods (Prior & Cao 1999; Perez-Jimenez & Saura-Calixto 2006; Karadag et al. 2009; Çelik et al. 2010). Significant differences were found between the values obtained using the same method (ABTS, FRAP, DPPH and ORAC) in different solvents (Prior & Cao 1999; Perez-Jimenez & Saura-Calixto 2006; Moreira et al. 2012). The total antioxidant capacity values using ABTS were higher in water than in acetone/water solutions and were also consistently higher in protic solvents than in aprotic solvents (Perez-Jimenez & Saura-Calixto 2006). Perez-Jimenez and Saura-Calixto (2006), even ranked the effects of solvents on antioxidant capacity assays in the order; ORAC > ABTS > DPPH > FRAP. Thus, solvents effects are also partly to blame for hampering efforts to compare results and develop internationally standardized methods (Prior et al. 2005). Surprisingly, there is limited information regarding the effects of solvents even on commercially available methods (Çelik et al. 2010). This, therefore, also emphasizes the need to investigate the effects of solvents on any antioxidant capacity assay in order to obtain any meaningful and accurate results that can be interpreted between different studies.

Materials and methods

Enzyme and chemicals

Analytical grade chemicals were used. Syringaldzine, trolox and 2,2′-azinobis (3-ethylbenzothiazoline 6-sulphonate; ABTS) were purchased from Sigma-Aldrich, Steinheim, Germany. The solvents [acetonitrile, methanol, dimethylsulphoxide (DMSO) and ethanol] of HPLC grade were also purchased from Sigma-Aldrich. All the other chemicals were purchased from Merck, Darmstadt, Germany. Trametes modesta laccase was produced and purified as previously described by Nyanhongo et al. (2006). The Randox Kit [Randox Total Antioxidant Status (TAS)] and human plasma samples were a kind gift from the ‘Institute für angewandte Biochemie AG’, Switzerland. The blood was collected from the subjects by venipuncture, anticoagulated with heparin (20 U/ml) and further prepared by centrifuging at 10,000 × g at 4 °C for 10 min. The supernatant was recovered and used as it is for the measuring of the total antioxidant capacity assay.

Ethical approval

No ethical approval was required as the samples used were residual from other studies and were anonymised.

Production of TMAMQ and ABTS radicals

Tetramethoxy azobismethylene quinone was produced as previously described by Nugroho Prasetyo et al. (#b100). Briefly, Syringaldazine (0.17 mM) was incubated with 50 μl of laccase (20 nkat/ml) in 50 mM sodium citrate buffer at pH 4.5 for 10 min at 30 °C. Tetramethoxy azobismethylene quinone was then extracted by adding ethyl acetate and recovered in solid form under vacuum. Stock solutions of ABTS cation radicals for investigating the effect of H2O2 were prepared as described by Re et al. (1999) with slight modifications. Briefly, a 10 mM ABTS aqueous solution was incubated with 3 mM potassium persulfate (final concentration) and allowed to stand in the dark at room temperature for 24 h before use.

Adapting TAS procedure for TMAMQ

The Randox TAS procedure as described by the manufacture was followed and in another experiment, ABTS was substituted for syringaldazine. The Randox TAS assay, commercialized by Randox Laboratories Ltd. (Finsbury House, London, UK), is based on the suppression of the absorbance of ABTS cation radicals by antioxidants in the test sample when ABTS is incubated with a radical generating system (metmyoglobin + H2O2). A reaction mixture containing 100 μM H2O2, metmyoglobin, 610 μM ABTS or 100 μM syringaldazine was incubated with 5 μM trolox at 37 °C. Spectra were recorded in time scan mode until the reaction reached a plateau.

Effect of H2O2 on TMAMQ and ABTS cation radicals

A stock solution of TMAMQ (10 mM) was made in acetonitrile from solid TMAMQ prepared as described before. An ABTS radical stock solution (10 mM) was prepared chemically using potassium persulfate as also described before. To investigate the effect of H2O2 on TMAMQ and ABTS radicals, 65 μM TMAMQ or 80 μM ABTS radicals were incubated with 30 μM H2O2 in 50% (v/v) ethanol pH 5.5 at 37 °C. The reactions were then monitored in time scan mode at 530 nm for TMAMQ and 600 nm for ABTS cation radicals. In another set-up, small amounts of substrates (syringaldazine or ABTS) were added stepwise to the reaction mixture containing the radical generating system (metmyoglobin + H2O2) in 50% (v/v) ethanol pH 5.5 at 37 °C. This allowed investigating the effect of excess H2O2 on the formed TMAMQ or ABTS radicals. Once the reaction reached plateau, 5 μM trolox was also added sequentially resulting in the reduction of the formed TMAMQ and ABTS radicals. Several stepwise additions of the same amount of substrate (syringaldazine or ABTS) and trolox was carried out to investigate the reproducibility of the data.

Effect of solvent on total antioxidant capacity activity measured using TMAMQ

As noted in previous studies, plasma precipitates when mixed with pure organic solvents like methanol, ethanol and acetonitrile which impact negatively on UV–VIS spectroscopy measurements. Preliminary dilution studies showed that at 50% (v/v) of organic solvent/PBS buffer, no precipitation was observed. This concentration was adopted for further studies. The effect of 50% (v/v) solvent (methanol, ethanol, acetonitrile and DMSO) on the total antioxidant capacity TMAMQ was investigated by incubating the samples between 20 °C and 37 °C in buffer solutions adjusted to pH 5.0, 5.5, 6, 7 and 7.4 and measuring the residual absorbance at defined times.

Effect of solvents on TMAMQ reduction by trolox and plasma samples

A stock solution of TMAMQ (2 mg in 50 ml HPLC grade acetonitrile) was prepared. From this, stock solution of 200 μl TMAMQ was incubated in the different solvents with Trolox. Tetramethoxy azobismethylene quinone profiles were followed in time scan mode at 530 nm until steady state. The concentrations of the reduced TMAMQ were calculated from their respective standards. For the measurement of the effect of solvents on TMAMQ reduction by plasma samples, similar reactions and monitoring conditions were set-up as described for trolox.

Monitoring the kinetic reduction in TMAMQ

Spectrophotometric data were acquired by incubating various TMAMQ concentrations (in excess with respect to antioxidants) while varying the concentrations of the individual antioxidants. The rate of reduction in TMAMQ was monitored at 530 nm using either a stopped-flow spectrometer (PBP Spectra Kinetic 05-109 monochromator from Applied Photophysics, London, UK) or the above-mentioned UV–Vis spectrometer (Hitachi U-2001). The temperature of all the reagents was first adjusted by means of a thermoset bath. A typical procedure consisted of adding a freshly prepared solution of the antioxidant to a freshly prepared solution of TMAMQ in appropriate solvent and placing it in the spectrometer cell. Spectra were recorded until the reaction reached a plateau.

Data analysis

The exact concentration of TMAMQ was calculated from a calibration curve recorded for each solvent. For each antioxidant concentration tested, data were fitted by using the software program Origin Pro 8.5 to obtain the kinetic parameters and confirmed by mathematical derivation. The kinetic parameters were estimated using curve fittings achieved through least-square regression analysis and yielded optimized values for the parameters.

Results

Adapting Randox TAS procedure for SyrinOX

The Randox TAS procedure is an operationally simple automated system which makes it very attractive for high-throughput analysis of samples. In this study, this procedure was adapted for SyrinOX by substituting the ABTS with syringaldazine. Comparing the adapted TAS procedure (syringaldazine + metmyoglobin and H2O2 + trolox as antioxidant) and the normal TAS procedure (ABTS + metmyoglobin and H2O2 + trolox as antioxidant) showed that both reaction systems reached steady state in less than 100 s. Addition of 15.00 μM trolox into the Randox TAS system suppressed the formation of 55.68 μM ABTS cation radicals of the 125.00 μM ABTS cation radicals formed in the control (Figure 1). Strangely, doubling the concentration of trolox from 15.00 to 30.00 μM resulted in the suppression of the formation of 95.21 μM ABTS cation radicals (Figure 1). The addition of 2.50 μM trolox into the adapted Randox TAS for SyrinOX resulted in the suppression of the formation of 3.33 μM TMAMQ, and doubling the trolox concentration to 5.00 μM resulted in the suppression of the formation of 8.26 μM TMAMQ (Figure 1).

Figure 1.

Figure 1

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Reduction of TMAMQ and ABTS radical using the Randox TAS approach. The radical generating system [metmyoglobin, H2O2, radical substrates (ABTS or syingaldazine)] and antioxidant sample (trolox) were added at the same time.

To further investigate the unexpected results in both systems (Randox TAS and SyrinOX system), another experiment aimed at investigating the effect of H2O2 on preformed ABTS cation radicals and TMAMQ was conducted. As shown in Figure 2, both TMAMQ and ABTS radicals were not stable in the presence of H2O2. The rates of reaction between the generated ABTS radicals or TMAMQ and H2O2 were estimated to be 5.7 × 10−3 and 6.4 × 10−2/μM/s respectively.

Figure 2.

Figure 2

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Effect of H2O2 on TMAMQ and ABTS cation radicals. The H2O2 was added to previously generated TMAMQ and ABTS radicals.

The effects of H2O2 on TMAMQ and ABTS cation radicals were even more pronounced when small amounts of the substrates (syringaldazine or ABTS) were added stepwise in a reaction mixture containing excess the radical generating system (metmyoglobin + H2O2; Figure 3).

Figure 3.

Figure 3

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Effect of H2O2 on the production of TMAMQ and ABTS radicals.

The formed ABTS radicals (Figure 3) were continuously disappearing at each level attributed to their reaction with excess H2O2. Although TMAMQ was also disappearing, it was not as pronounced as in the ABTS system (Figure 3). However, it is also interesting to note that although same amounts of substrates (syringaldazine or ABTS) were added stepwise, none of the values were exactly the same at each level. This was also true when trolox was later added stepwise.

To demonstrate this point, the total antioxidant capacity of the same plasma samples measured using the adapted TAS method for SyrinOX (syringaldazine + metmyoglobin + H2O2 + plasma samples) and the usual procedure (plasma samples + preformed TMAMQ) were measured and were shown to be very different (Figure 4).

Figure 4.

Figure 4

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Reaction profile of the adapted TAS assay for SyrinOX (right) and standard SyrinOX (left) with same plasma samples sample a and b.

The plasma samples A and B in the adapted TAS method using syringaldazine as substrate suppressed the formation of 10.6 and 13.4 μM TMAMQ, and the same plasma samples quenched 5 and 7.6 μM TMAMQ using the normal SyrinOX procedure respectively. These results represent over 45% difference. Again using the original Randox system, the plasma samples A and B suppressed the formation of 10.3 and 18.2 μM ABTS radicals, and using the chemically produced ABTS radicals quenched 7.9 and 10.7 μM ABTS radicals respectively. These results again vary from each other by over 40%. The observed differences can only be attributed to the radical generating system. This means that in the adapted TAS assay using SyrinOX, the gradual increase in the concentration of TMAMQ is due to depletion of both radical generating system (H2O2) and antioxidants. Consequently, the already established two-step procedure for SyrinOX involving incubating laccase or peroxidase with syringaldazine resulting in the production of TMAMQ, recovery and purification of TMAMQ (free of the enzyme and other impurities) and then in the second step incubating the TMAMQ with antioxidants was chosen as the best approach as it is the only way which guarantees that TMAMQ reacts with the antioxidant sample only.

Operational reaction conditions for the SyrinOX

Effect of solvent on absorption intensity of TMAMQ

In previous studies, it was observed that TMAMQ was stable in acetonitrile for over 2 years when stored in the dark at −4 °C (Nugroho Prasetyo et al. 2009; Nugroho Prasetyo et al. 2010a,b, 2012). However, since plasma precipitates in absolute organic solvents, a 50% of aqueous solution did not result in precipitation. Building on these earlier studies, the optimal operational reaction conditions for SyrinOX were investigated. In the first study, the effect of different solvents on the absorbance intensity of TMAMQ was investigated by dispensing the same amounts of TMAMQ into different solutions of 50% (v/v) solvents (methanol, acetonitrile, ethanol and DMSO). As shown in Figure 5, the absorbance values were different depending on the solvent. The absorbance in ethanol was highest, while the absorbance was lowest in DMSO. This result has direct implications on the total antioxidant value, extinction coefficient and calibration curve used for estimating the total antioxidant capacity of any given sample. For example, the measured extinction coefficients in the different solvents were 1.26 × 104, 1.38 × 104, 1.6 × 104 and 1.29 × 104/M/cm for TMAMQ in 50% (v/v) ethanol, methanol, DMSO and acetonitrile respectively.

Figure 5.

Figure 5

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Effect of solvents on the absorbance intensity of TMAMQ.

Effect of solvent on the rate of reduction and total antioxidant capacity assay of TMAMQ using trolox

The rate of reduction in TMAMQ by trolox incubated in different solvents [50% (v/v) acetonitrile, ethanol and methanol] was investigated (Figure 6). The rate of reduction in TMAMQ depended on the type of solvent, with methanol and ethanol showing high rates of 9.42 × 10−3 and 8.14 × 10−3/μM/s respectively. The rate of reduction in TMAMQ in acetonitrile was 3.44 × 10−4/μM/s, three orders of magnitude lower than that of methanol and ethanol.

Figure 6.

Figure 6

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Effect of solvents on the total antioxidant measurement using TMAMQ. Trolox applied at a concentration of 16.5 µM while solvents were used at 50% (v/v) ethanol/50 mM sodium succinate buffer pH 5.5 incubated at 25 °C.

The estimated total amount of TMAMQ reduced by 16.5 μM Trolox was 11.0, 8.8 and 8.4 μM measured in 50% (v/v) methanol, ethanol and acetonitrile respectively. The total antioxidant activity obtained with acetonitrile and ethanol was closer to the expected ratio 2:1 of Trolox:TMAMQ. However, the total antioxidant capacity assay values obtained in methanol were higher.

Effect of solvents on the reduction in TMAMQ by plasma samples

Further investigation of the effect of solvent on the rate of reduction in TMAMQ by plasma samples showed a similar trend as above (Figure 7). The rate of reduction in TMAMQ by plasma samples in methanol, ethanol and acetonitrile was 2.1 × 10−4, 1.9 × 10−4 and 9 × 10−6/μM/s respectively. The total incubation time needed for the complete consumption of all antioxidants in the sample was 1 h for ethanol, 45 min for methanol and 1 h 45 min for acetonitrile (Figure 7). The total amount of reduced TMAMQ in samples incubated in 50% ethanol, acetonitrile and methanol is summarized in Table 1.

Figure 7.

Figure 7

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Effect of solvent on the total antioxidant capacity of plasma samples (5 µM) obtained in different solvents 50% (v/v) ethanol/50 mM sodium succinate buffer pH 5.5 incubated at 25 °C.

Table 1.

Effects of different solvents on the total antioxidant value using similar antioxidant samples

Total amount of reduced TMAMQ (μM)
Plasma samples Methanol Ethanol Acetonitrile
A 10.2 ± 0.3 7.6 ± 0.3 7.2 ± 0.2
B 5.8 ± 0.3 3.6 ± 0.1 3.5 ± 0.2
C 11.4 ± 0.2 7.7 ± 0.1 7.4 ± 0.1
D 9.7 ± 0.3 6.5 ± 0.2 6.6 ± 0.2
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The total antioxidant capacity of plasma samples A and C in ethanol was almost similar to those obtained in acetonitrile although the antioxidants values obtained in methanol were consistently higher (Table 1).

Effect of pH and temperature in 50% (v/v) ethanol

Because the total antioxidant activity capacity values obtained when using 50% (v/v) acetonitrile and ethanol were closer to the expected ratio 2:1 of Trolox:TMAMQ (Figures 6 and 7, Table 1), 50% (v/v) ethanol was chosen as the best solvent because the reaction proceeds faster than in acetonitrile. Subsequent studies were, therefore, carried out to investigate the effect of temperature and pH on the stability of TMAMQ in 50% (v/v) ethanol/50 mM sodium succinate buffer. As illustrated in Figure 8, TMAMQ was not stable at pH 7.4 (physiological pH) at all incubation temperatures. Tetramethoxy azobismethylene quinone was stable at pH 5.5 between 20 and 25 °C for over 6 h, and a 10% of TMAMQ was lost after 6 h (Figure 8).

Figure 8.

Figure 8

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Effect of pH and temperature on the stability of 10 µM TMAMQ in 50% (v/v) ethanol/buffer solution.

Discussion

Adopting the Randox TAS system for SyrinOX revealed quite interesting information which may serve to explain the challenges encountered in using this assay partly. The study showed that while the radical generating system (metmyoglobin + H2O2) is producing the ABTS cation radicals, some of the H2O2 is also reacting with the formed reactive species (TMAMQ or ABTS radicals) leading to their disappearance. This means that the suppression of the formation of ABTS cation radicals or TMAMQ is due to multiple effects which include reaction of antioxidants with the reactive species and reaction of H2O2 with formed reactive species. As observed in this study, this leads to an overestimation of antioxidant capacity of given samples as clearly demonstrated by over 50% reduction in ABTS radicals (Figure 1). This effect of H2O2 on TMAMQ and ABTS cation radicals taken together with the long-established ability of H2O2 to oxidize ascorbic acid and vitamin E (an important non-enzymatic antioxidant in humans: Vatassery 1989; Deutsch 1998), the ability of metmyoglobin to use ascorbic acid for redox cycling (Galaris et al. 1989; Vatassery 1989) and the relatively high redox potential of metmyoglobin (Schlereth & Maentele 1992) which enables it to oxidize many phenolic based antioxidants results in meaningless and contradictory values. This has already been observed by many previous researchers [Karadag et al. (2009), MacDonald-Wicks et al. (2006)] although the reason behind these problems was not yet clear. This maybe a possible explanation why efforts to develop internationally standardized total antioxidant capacity assay methods are proving difficult with systems which incorporate the radical generating system together with the antioxidant sample. From these observations, it is therefore extremely important if not imperative to develop a two-step approach, separating the radical generating step from the antioxidant activity measurement. Consequently, the already established three-step approach for SyrinOX-involving (i) producing TMAMQ by incubating laccase or peroxidase with syringaldazine (ii) recovery and purification of TMAMQ (free of the enzyme and other impurities); and (iii) in the third step using the purified TMAMQ for measuring the antioxidant capacity of antioxidants samples-remains the method of choice. This is the only way which guarantees that TMAMQ reacts only with the antioxidant sample thereby making the decrease in TMAMQ absorbance directly proportional to the antioxidant concentration.

Further studies investigating the optimal operational reaction conditions for the SyrinOX showed that other reactive species used in antioxidant assay (ABTS cation radicals, DPPH etc.) are affected by the solvent, pH and temperature. The observed different absorption coefficients of TMAMQ in different solvents shows that it is extremely important to make a calibration curve under similar conditions as the sample antioxidant activity measuring conditions. In previous studies, Ozcelik et al. (2003) observed similar effects using DPPH, while El-Daly et al. (2003) and Diaz et al. (2009) noted that solvent polarity affected the absorption maxima. The solvents also affected the reactivity and total antioxidant capacity values obtained using TMAMQ. For example, reaction in methanol reached steady state earlier and also produced abnormally higher values than in acetonitrile. The long incubation period required for reactions incubated in acetonitrile as compared to ethanol and methanol, maybe attributed to its properties as an aprotic solvent. In similarly previous studies, short incubation times and higher values were also consistently observed in protic solvents including methanol (BłauŜ et al. 2008; Moreira et al. 2012) than in aprotic solvents. Litwinienko and Ingold (2003) contented that antioxidant activities in alcohol solvents especially methanol generally lead to an overestimation of their activities and attributed this fact to its well-known properties as the alcohol that supports best ionization (Litwinienko & Ingold 2003). Using DPPH, antioxidants reduced two molar equivalents of DPPH radicals in aprotic solvents, yet in protic solvents an equivalent of five molar radicals was reduced (Saito & Kawabata 2004). Oxygen radical absorbance capacity values in water were also observed to be lower than in acetone/water, while antioxidant values obtained using ABTS in water were higher than the values obtained in acetone/water (Litwinienko 2007). The antioxidant activities of α-tocopherol in different solvents decreased in the order of acetonitrile = hexane > ethanol = methanol, which indicates that the antioxidant activity of α-tocopherol is smaller in protic solvent than in aprotic solvent (Iwatsuki et al. 1994). This further illustrates that the effects of solvents on antioxidants capacity assay methods are complex. It should be also noted that preliminary studies using DMSO as a solvent for TMAMQ gave ridiculously high total antioxidant capacity (i.e. 16.5 μM Trolox reduced 14.4 μM TMAMQ), way out of the expected 2:1 ratio. The observed higher antioxidant values observed in DMSO may be attributed to its reported antioxidant properties (Sanmartin-Suarez et al. 2011). Although Perez-Jimenez and Saura-Calixto (2006) noted that increased antioxidant values in ethanol using ABTS, strangely the reduction in TMAMQ in ethanol as compared to methanol was closer to the expected value. These results are in agreement with previous observations by Serpen et al. (2012), who recently noted and recommended 50% (v/v) ethanol as the most appropriate solution for standardizing antioxidant assay with TAC. Similarly, Çelik et al. (2010) also noted that trolox equivalent antioxidant capacities of hydrophilic antioxidants in ethanol did not differ significantly from those reported in using CUPRAC method, demonstrating that 50% ethanol is a good solvent medium for antioxidant activity measurement. Tetramethoxy azobismethylene quinone antioxidant activity measurement was also less influenced by pH in agreement with previous studies which also showed that the change in pH did not affect DPPH and ORAC assay (Perez-Jimenez & Saura-Calixto 2006).

Consequently, because antioxidant values obtained in methanol were consistently higher than those obtained in ethanol and because the total antioxidant values obtained in ethanol were similar to those obtained in acetonitrile during a shorter incubation time, ethanol was, therefore, chosen as the best solvent for total plasma antioxidant capacity assay using SyrinOX. From the combination of reaction conditions investigated, this effectively means that the total antioxidant capacity plasma samples using TMAMQ are preferably measured in 50% (v/v) ethanol at pH 5.5 and between 20 and 25 °C for 2 h with a correction factor of less than 4% (background loss of TMAMQ in the absence of antioxidants). In all cases, it is recommended to incubate a control containing TMAMQ alone under similar conditions alongside the sample.

Concluding remarks

The TAS procedure cannot be adapted for SyrinOX for the measurement of total antioxidant capacity of plasma samples because the radical generating system (metymyoglobin + H2O2) introduces many problems in the assay. These studies provide the first evidence for the negative effects introduced by the radical generating system, especially H2O2 even in the Randox TAS assay. It is not surprising to realize that efforts to develop internationally recognized total antioxidant capacity assay methods based on TAS have not been successful. This may also be true with other similar methods which incorporate the radical generating systems with the antioxidant measuring phase. This study strongly recommends the separation of the radical generating system from the antioxidant measuring phase even for the Randox TAS assay. A three-step approach involving laccase or peroxidase production of TMAMQ, recovery and purification followed by incubating TMAMQ (free of enzyme and other impurities) with antioxidant sample is recommended for SyrinOX. This study also shows that SyrinOX like other similar methods is affected by solvents which need careful studying. Thus, the optimum conditions for measuring total antioxidant capacity of plasma samples using SyrinOX were determined to be 50% (v/v) ethanol/50 mM sodium succinate buffer pH 5.5 and temperature between 20 and 25 °C for at least 1 h. A control containing TMAMQ minus the antioxidants must always be incubated under similar conditions alongside the sample for correcting background loss of TMAMQ in the absence of antioxidants. It can also be concluded that, in any antioxidant capacity assay activity, an appropriate calibration curve must be prepared under similar conditions using the same solvent composition.

Acknowledgments

The authors are grateful for the financial support offered by EU NOVO project and the Austrian Centre of Industrial Biotechnology (ACIB).

Conflict of Interest

There is no conflict of interest All authors have agreed to publication.

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