A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation

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1 Clinical Biochemistry 37 (2004) A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation Ozcan Erel* Clinical Biochemistry Department, Medical Faculty, Research Hospital, Harran University, Sßanlıurfa TR63200, Turkey Received 20 May 2003; received in revised form 27 November 2003; accepted 28 November 2003 Abstract Objectives: To develop a novel colorimetric and automated direct measurement method for total antioxidant capacity (TAC). Design and Methods: A new generation, more stable, colored 2,2V-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS *+ ) was employed. The ABTS *+ is decolorized by antioxidants according to their concentrations and antioxidant capacities. This change in color is measured as a change in absorbance at 660 nm. This process is applied to an automated analyzer and the assay is calibrated with Trolox. Results: The novel assay is linear up to 6 mmol Trolox equivalent/l, its precision values are lower than 3%, and there is no interference from hemoglobin, bilirubin, EDTA, or citrate. The method developed is significantly correlated with the Randox- total antioxidant status (TAS) assay (r = 0.897, P < ; n = 91) and with the ferric reducing ability of plasma (FRAP) assay (r = 0.863, P < ; n = 110). Serum TAC level was lower in patients with major depression (1.69 F 0.11 mmol Trolox equivalent/l) than in healthy subjects (1.75 F 0.08 mmol Trolox equivalent/l, P = 0.041). Conclusions: This easy, stable, reliable, sensitive, inexpensive, and fully automated method described can be used to measure total antioxidant capacity. D 2004 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: ABTS; ABTS radical cation; Antioxidant; Antioxidant capacity; Automated measurement; FRAP; Free radicals; Oxidative stress; Total antioxidant capacity Introduction Reactive oxygen species (ROS) is produced in metabolic and physiological processes, and harmful oxidative reactions may occur in organisms that remove them via enzymatic and non-enzymatic antioxidative mechanisms. Under certain conditions, the increase in oxidants and decrease in antioxidants cannot be prevented, and the oxidative or antioxidative balance shifts towards the oxidative status. Consequently, oxidative stress, which has been implicated in over 100 disorders, develops [1]. Antioxidant molecules prevent or inhibit these harmful reactions [2]. Serum (or plasma) concentrations of different antioxidants can be measured in laboratories separately, but the measurements are time-consuming, labor-intensive, costly, and require complicated techniques. Because the mea- * Fax: address: erelozcan@hotmail.com. surement of different antioxidant molecules separately is not practical and their antioxidant effects are additive, the total antioxidant capacity of a sample is measured, and this is called total antioxidant capacity (TAC) [3], total antioxidant activity (TAA) [4], total antioxidant power (TAOP) [5,6], total antioxidant status (TAS) [7], total antioxidant response, or other synonyms. Various methods have been developed to measure TAC [3 13]. In these methods, generally, a type of radical is generated in the assay and the antioxidant activity of the sample against the radical is measured. The most widely used colorimetric methods are 2,2V-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS S+ )-based methods, in which a colorless molecule, reduced ABTS, is oxidized to a characteristic blue-green ABTS S+. When the colored ABTS S+ is mixed with any substance that can be oxidized, it is reduced to its original colorless ABTS form again; in contrast, the reacted substance is oxidized. This feature is the basic principle of the methods that use ABTS /$ - see front matter D 2004 The Canadian Society of Clinical Chemists. All rights reserved. doi: /j.clinbiochem

2 278 O. Erel / Clinical Biochemistry 37 (2004) According to the reported methods, reduced ABTS molecules have been oxidized by various agents. In some studies, various oxidants have been used to oxidize ABTS molecules such as potassium persulfate [8], MnO 2 [14], and 2,2V-azo-bis(2-aminopropane) (ABAP) radicals [15]. In some studies, couples including hydrogen peroxide and a peroxidase enzyme [16] or a non-enzymatic peroxidase agent such as metmyoglobin [3] have been used to oxidize ABTS molecules. To the best of my knowledge, no peroxidase agent besides hydrogen peroxide has been used alone to oxidize ABTS molecules. In this study, I produced a more stable ABTS S+ with a very long lifetime without using any peroxidase agents besides hydrogen peroxide. According to the literature, this is the first time that this has been achieved. In the first ABTS-based colorimetric method developed, metmyoglobin reacts with hydrogen peroxide to produce ferrylmetmyoglobin radical. This radical oxidizes the ABTS molecule to ABTS S+ and the characteristic color develops. Antioxidants suppress the color development [3]. However, its working reagent lifetime is short (48 h at 4jC) and the commercially available kit is expensive. In addition, the dilution of serum samples leads to false-positive results [12]. Faster-reacting antioxidants may contribute to the reduction of ferrylmyoglobin radical and so they may lead to positive interference [17]. Furthermore, hemoglobin and peroxides produce additional color and lead to negative interference with the assay. The new versions of ABTS-based methods are postadditional. In these methods, ABTS S+ is produced before the addition of the sample [8]. Although there are many reports of the use of post-additional methods to measure the TAC of beverages, fruit juices, and pure solutions, there are an insufficient number of reports concerning the use of these methods to measure the TAC of serum (plasma). This study aimed to develop an easy, reliable, sensitive, automated, and inexpensive direct measurement method, with reagents having a long lifetime, for total antioxidant capacity. Materials and methods Chemicals Ferric chloride, ferrous ammonium sulfate, vitamin C (L(+) ascorbic acid), ascorbate oxidase, bilirubin, uric acid, reduced glutathione (GSH), (F)-catechin, 2,4,6-tripyridyl-striazine (TPTZ), ethylenediaminetetraacetic acid (EDTA), 2,2V-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS), hydrogen peroxide, potassium persulfate, and sodium citrate were purchased from Sigma and Merck Co. The water-soluble analogue of vitamin E (Trolox; 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was from the Sigma-Aldrich Chemical Co. All chemicals were ultra pure grade, and type I reagent-grade deionized water was used. Samples This novel measurement method can be performed on a wide range of complex biological fluids, including serum, plasma, semen plasma, cerebrospinal, pleural and ascites fluids, urine, simple and heterogeneous solutions of pure antioxidants, beverages, and fruit juices. Blood samples Blood samples were obtained from healthy subjects who were admitted to our check-up polyclinic and blood bank, and from patients with major depression who were diagnosed by specialist psychiatrists in our hospital. All patients had not been receiving psychotropic medications for at least 2 months. Patients with a history of drug abuse or dependence, serious medical illness, severe head injury, or seizure disorders were excluded from the study. The healthy subjects had not been taking any medication for at least 2 months before blood sampling. Both groups were made up of nonsmokers. Subjects fasted for 12 h before sample collection. They were informed of the purpose of the study. Venous blood samples obtained were centrifuged at 1500 g for 10 min. The samples were run immediately or stored at 80jC. Antioxidants Stock solutions (1.0 mmol/l) of ascorbic acid, glutathione, and (F)-catechin were prepared separately in deionized water. Uric acid and solid bilirubin were dissolved in 10 mmol/l NaOH solution. Trolox was dissolved in phosphate buffer (30 mmol/l ph 7.4). Apparatus A Cecil 3000 spectrophotometer with a temperaturecontrolled cuvette holder (Cecil) and an Aeroset automated analyzer (Abbott) were used. Assays The novel assay Assay principle of the novel measurement method The reduced ABTS molecule is oxidized to ABTS S+ using hydrogen peroxide alone in acidic medium (the acetate buffer 30 mmol/l ph 3.6). In the acetate buffer solution, the concentrate (deep green) ABTS S+ molecules stay more stable for a long time. While it is diluted with a more concentrated acetate buffer solution at high ph values (the acetate buffer 0.4 mol/l ph 5.8), the color is spontaneously and slowly bleached. Antioxidants present in the sample

3 O. Erel / Clinical Biochemistry 37 (2004) accelerate the bleaching rate to a degree proportional to their concentrations. This reaction can be monitored spectrophotometrically and the bleaching rate is inversely related with the TAC of the sample. The reaction rate is calibrated with Trolox, which is widely used as a traditional standard for TAC measurement assays, and the assay results are expressed in mmol Trolox equivalent/l. Assay reagents Reagent 1 The 0.4 mol/l acetate buffer solution (ph 5.8) was obtained as follows: 32.8 g of CH 3 COONa was dissolved in 1000 ml of deionized water (final concentration: 0.4 mol/ l). Reagent-grade glacial acetic acid (22.8 ml) was diluted to 1000 ml with deionized water (final concentration: 0.4 mol/ l). The sodium acetate solution (940 ml) was mixed with 60 ml of the acetic acid solution under a ph meter; the ph of the acetic acid sodium acetate buffer was 5.8. The buffer solution was stable for at least 6 months at 4jC. Reagent 2 The 30 mmol/l acetate buffer solution, ph 3.6, was prepared as follows: 2.46 g of CH 3 COONa was dissolved in 1000 ml of deionized water (final concentration: 30 mmol/ l). Reagent-grade glacial acetic acid (1.705 ml) was diluted to 1000 ml with deionized water (final concentration: 30 mmol/l). The sodium acetate solution (75 ml) was mixed with 925 ml of the acetic acid solution under a ph meter; the ph of the acetic acid sodium acetate buffer was 3.6. Then 278 Al of commercial H 2 O 2 solution (35%, Merck) was diluted to 1000 ml with the buffer solution (final concentration, 2 mmol/l). Then g ABTS was dissolved in 100 ml of prepared solution (final concentration: 10 mmol/l). After 1 h of incubation at room temperature, the characteristic color of ABTS S+ appeared. The colored reagent was stable for at least 6 months at 4jC. Automated measurement After manual spectrophotometric optimization processes, the method was applied to an automated analyzer (Aeroset). The assay format of the test is shown below. Reagent 1 volume 200 Al (Reagent 1: acetate buffer 0.4 mol/l ph 5.8). Sample volume 5 Al (serum or other fluids, pure or complex antioxidant solutions). Reagent 2 volume 20 Al (Reagent 2: the ABTS S+ in acetate buffer 30 mmol/l ph 3.6). Wavelength 660 nm (or other peak wavelength 420 and 740 nm, as seen in Fig. 1). Reading point The first absorbance of the assay is taken before the mixing of R1 and R2 (as sample blank) and the last absorbance is taken at the end of the incubation period (5 min after the mixing). Calibration type Linear Total antioxidant capacity measurement methods Measurement methods based on the decolorization of ABTS radical cation Trolox equivalent antioxidant capacity (Randox-TEAC; Randox-TAS) assay. The automated TEAC assay was carried out on an Aeroset (Abbott) automated analyzer with commercially available kits (Total Antioxidant Status, Randox Laboratories). It was thus sometimes called the Randox- TAS assay in this study. In this assay, 2,2V-azinobis(3- ethylbenzothiazoline-6-sulfonate) (ABTS) is incubated with metmyoglobin and hydrogen peroxide to produce ABTS S+. This species is blue-green. Antioxidants present in the sample cause a reduction in absorption proportional to their concentration. The TAC value of the samples tested is expressed as an equivalent of the millimolar concentration of Trolox solution [3,12]. The improved ABTS radical cation decolorization assay (a post-additional assay). ABTS was dissolved in water to make a concentration of 7 mmol/l. ABTS S+ was produced by reacting the ABTS stock solution with 2.45 mmol/l potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for h before use. For the study of samples, the ABTS S+ stock solution was diluted with phosphate-buffered saline 5 mmol/l, ph 7.4 to an absorbance of 0.70 at 734 nm. After the addition of 1.0 ml of diluted ABTS S+ to 10 Al of sample, the absorbance reading was taken 5 min after the initial mixing [8]. Decolorization of the assay was linear with increasing Trolox concentrations. The ferric reducing ability of plasma (ferric reducing/ antioxidant power) (FRAP) assay. The FRAP assay developed by Benzie and Strain [5,6] was performed as previously described in detail. In short, 1.5 ml of working, prewarmed 37jC FRAP reagent (10 volumes of 300 mmol/l acetate buffer, ph vol of 10 mmol/l 2,4,6-tripyridyl-Striazine in 40 mmol/l HCl + 1 vol of 20 mmol/l FeCl 3 ) was mixed with 50 Al of test sample and standards. This was vortex mixed and absorbance at 593 was read against a reagent blank at a predetermined time after sample reagent mixing. The test was performed at 37jC and the 0 4-min reaction time window was used. The final results are expressed as mmol Trolox equivalent/l. Measurements of albumin, total protein, uric acid, and bilirubin concentrations of serum samples Serum albumin, total protein, uric acid, and total bilirubin concentrations were measured by commercially available kits (Abbott) using an automated analyzer (Aeroset, Abbott). Measurement of reduced ascorbate concentration of plasma samples Plasma-reduced ascorbate concentration was measured by FRASC assay using ascorbate oxidase [6].

4 280 O. Erel / Clinical Biochemistry 37 (2004) Statistical analyses Student s t test, correlation analyses, and linear regression analyses were performed using SPSS for Windows Release 9.5 (SPSS Inc.). Results Optimization studies Type, molarity, and ph of buffer solutions used in the assay ABTS molecules could be oxidized to ABTS S+ only in the acetate buffer at low ph values and when a sufficient amount of hydrogen peroxide is present. At high ph values, oxidation did not occur. In the acetate buffer with the lowest ph values, the lifetime of the ABTS S+ produced was the longest (at least 6 months at 4jC). The reduction (decolorization; bleaching) of ABTS S+ could be performed using the acetate buffer at high ph values. The maximal reduction rate was observed when the acetate buffer at the highest ph value was used. The optimal ph value of acetate buffer to produce ABTS S+ was 3.6 and it was 5.8 for the reduction processes. On the other hand, the acetate buffer concentration for the oxidation process was 30 mmol/l and it was 400 mmol/l for the reduction. The volumes ratio of the acetate buffer (0.4 mol/l)/ the acetate buffer (30 mmol/l) was 200/20 in the assay medium. Optimizations of ABTS and hydrogen peroxide concentrations The second reagent was diluted 21-fold by reagent 1 in the reaction medium; for this reason, ABTS was used as 10 mmol/l to obtain sufficient absorbance and the 1/5 ratio was optimal for [hydrogen peroxide]/[abts]. At this ratio, there was no interference due to hemolysis or ferrous ions. Spectral analyses The spectral absorbances of the ABTS S+ and its re-reduced form (ABTS) are given in Fig. 1. As seen in the figure, maximal absorbances are at 420, 740, and 666 nm. Reaction kinetics of blank and various serum samples The reaction kinetics, which were performed in the automated analyzer, of blank and the serum samples from a healthy person, a healthy pregnant woman, and an infant with neonatal physiological icterus are given in Fig. 2. Reaction kinetics and dose-response characteristics of pure antioxidants Fig. 1. Absorbance spectrums of the ABTS radical cation and its rereduced form. The reaction kinetics and characteristics of some endogenous and exogenous antioxidants, namely Trolox (a watersoluble analogue of vitamin E), vitamin C, reduced glutathione (GSH), bilirubin, uric acid, and (F)-catechin, were determined. The dose-response characteristics of pure antioxidant solutions are shown in Fig. 3. The antioxidative effects of vitamin C, Trolox, and (F)-catechin were fast and were completed at the beginning of the reaction. The antioxidative effect of GSH was completed later than those of Trolox and vitamin C. The antioxidative effects of bilirubin and uric acid were completed later than that of GSH (Fig. 4). Linearity Serial dilutions of the Trolox solution were performed. The upper limit of the linearity in the assay was 6.0 mmol Trolox equivalent/l. In the regression analyses, the r value was ( P < Sy/x = 0.023), the slope was ( P < Sy/x = 0.019), and the intercept was ( P < Sy/x = 0.021). In addition, by decreasing the sample volume ratio the upper limit of the linearity can also be increased. Analytical recovery The percentage recovery of the novel method was determined by adding 1 mmol/l vitamin C to serum samples. The mean percentage recovery of added vitamin C was %. Analytical sensitivity Analytical sensitivity, which is the slope of the calibration line, was Absorbance / [Amount], [AX (mmol/l) 1 ]. Lower detection limit The detection limit of the method was determined by evaluating the zero calibrator 10 times. The detection limit,

5 O. Erel / Clinical Biochemistry 37 (2004) Fig. 2. Reaction kinetics of various serum samples. defined as the mean TAC value of the zero calibrator + 3 SD, was 0.04 mmol Trolox equivalent/l. Precision To determine the precision of the novel method, I assayed three levels of sera. A serum pool that had high TAC was obtained from samples of infants with neonatal icterus. The serum pool with medium TAC was obtained from healthy persons. The serum pool with low TAC was provided from normal pregnant women. Within- and between-batch precision calculated for each of the three sera pools is shown in Table 1. Serum TAC was not affected by storage at 4jC for 1 day or at 80jC for 3 months. Interference Hemolysis and bilirubin did not interfere with the assay; neither did heparin, EDTA, citrate, nor oxalate. TAC values Fig. 4. Rates of decreases in absorbance at 660 nm of individual antioxidants solutions (1.0 mmol/l) and serum sample (after the subtraction of the reagent blank). of plasma samples were 12% higher than those of serum samples in the same individuals. The difference between plasma and serum TAC values was significantly correlated with the difference between total protein levels of plasma and serum samples (r = 0.63, P = 0.02; n = 21). It is known that fibrinogen, which is a protein, is only present in plasma and not in serum, and it has been shown that fibrinogen has an antioxidant activity [18]. The difference between serum and plasma TAC values may have originated from fibrinogen. The effect of lipemia was examined by mixing a specimen with increased triglyceride and TAC levels proportionally with a specimen with low triglyceride and TAC concentrations. Triglyceride concentrations up to 15.8 mmol/l (1400 mg/dl) had no effect on recovery. Uremic plasma samples did not interfere with the assay. Dilution The dilution of serum did not affect the novel assay results. It has been reported that the dilution of serum Table 1 Within- and between-batch precision data of serum obtained by the novel method Number Mean (mmol Trolox equivalent/l) SD CV (%) Within-batch measurement High Medium Low Fig. 3. Dose-response characteristics of pure antioxidant solutions of some endogenous and exogenous antioxidants. Between-batch days High Medium Low

6 282 O. Erel / Clinical Biochemistry 37 (2004) Table 2 Relative antioxidant activities of individual antioxidants and their estimated contributions to total antioxidant capacity (TAC) of fresh fasting serum Antioxidant component of serum Relative activity Serum concentration (Amol/l) Estimated contribution to TAC of serum (mmol Trolox equivalent/l) -SH Uric acid Vitamin C Bilirubin total Vitamin E Others Total Estimated contribution to TAC of serum (%) samples produced up to a 15% increase by Randox-TAS assay [12]. Relative antioxidant activities of the antioxidant components of serum and their estimated contributions to TAC Relative antioxidant activities of individual antioxidants and their estimated contributions to the TAC of fresh fasting serum are shown in Table 2. As seen in the table, the major antioxidant components of serum were total protein and uric acid. The relationships among the novel method, the other TAC measurement methods, and antioxidant components of serum (and plasma) samples As seen in Table 3, serum TAC measured by the novel method was significantly correlated with serum uric acid and total protein levels in the healthy group (n = 25). The novel assay was also significantly correlated with the Randox-TAS assay, the improved ABTS S+ decolorization assay, and the FRAP assay. The novel assay was positively correlated with bilirubin and reduced ascorbate levels but not significantly. Fig. 5. The relationship between serum TAC values measured by the novel method and the first ABTS S+ -based (Randox-TAS) assay. On the other hand, to determine the relationships between the novel method and the Randox-TAS assay, the improved ABTS S+ decolorization assay, the FRAP assay, and total protein and uric acid levels in serum samples obtained from patients selected so as to include a wide variety of pathologic conditions were studied (Figs. 5 7). The correlations among the measured parameters in the mixed patients group were similar to those in the healthy group. Reference interval To determine the reference interval for serum TAC, serum specimens from 113 healthy individuals (56 women, 57 men, years old) were assayed. The reference range was mmol Trolox equivalent/l. Table 3 The relationships among the novel method, the other total antioxidant capacity measurement methods used, and antioxidant components of serum (and plasma) samples of healthy subjects Randox-TAS assay Improved ABTS S+ decolorization assay FRAP assay Total protein Uric acid Bilirubin total Vitamin C The novel method r = 0.902, r = 0.773, r = 0.853, r = 0.511, r = 0.871, r = 0.05, r = 0.091, P < ; P < ; P < ; P < 0.011; P < ; P = 0.812; P = 0.665; n =25 n =25 n =25 n =25 n =25 n =25 n =25 Randox-TAS assay r = 0.621, r = 0.847, r = r = 0.901, r = 0.038, r = 0.128, P = 0.001; P < ; P = 0.088; P < ; P = 0.857; P = 0.541; n =25 n =25 n =25 n =25 n =25 n =25 Improved ABTS S+ r = 0.575, r = 0.774, r = 0.559, r = 0.012, r = 0.017, decolorization assay P = 0.003; P < ; P = 0.004; P = 0.959; P = 0.939; n =25 n =25 n =25 n =25 n =25 FRAP assay r = 0.167, r = r = 0.228, r = 0.036, P = 0.424; P < ; P = 0.273; P = 0.863; n =25 n =25 n =25 n =25 r = correlation coefficient, P = significancy, n = number of subjects.

7 O. Erel / Clinical Biochemistry 37 (2004) Fig. 6. The relationship between the novel method and the improved ABTS radical cation decolorization assay. Results from healthy subjects and patients with major depression There were no significant differences in age, body mass index (BMI), or female/male ratio between patients with major depression and healthy subjects. Serum TAC levels were lower in patients with major depression than those in healthy subjects (1.69 F 0.11 mmol Trolox equivalent/l n = 30, 1.75 F 0.08 mmol Trolox equivalent/l n = 30; P = 0.041, respectively). There was an inverse relationship between duration of the disease and TAC (r = 0.387), but the correlation was not statistically significant ( P = 0.154). version has been widely used during the last decade. In this method, there are two reagents. The first reagent contains ABTS and metmyoglobin and the second contains hydrogen peroxide. A sample is added to reagent 1, and then reagent 2 is added. Ferrylmyoglobin radical is produced and this radical oxidizes the colorless reduced ABTS molecule and thus leads to the development of the characteristic blue-green color. Antioxidants suppress the color formation because they reduce ABTS S+. This method is currently being produced as a commercial kit by Randox. For this reason, some researchers refer to it as the Randox-TAS or Randox-TEAC (Trolox equivalent antioxidant capacity) assay [12]. The latter technique is an improved (post-additional) technique in which the ABTS radical cation is generated directly in a stable form before reaction with putative antioxidants. Although it has been reported that the results obtained with the improved method might not always be directly comparable with those of the Randox-TAS assay [8], in this study it was shown that the results of both methods were significantly correlated with the novel method in healthy subjects and the mixed group (Table 3, Figs. 5 and 6). The FRAP assay is another widely used colorimetric TAC measurement method [5,6]. In this method, colorless Fe 3+ - TPTZ complex is reduced to a blue Fe 2+ -TPTZ complex by antioxidants. The antioxidative effects of uric acid, bilirubin, vitamin C, Trolox, and polyphenols can be seen but the effects of proteins are weak; hence, the TAC levels of serum samples measured by the FRAP assay are lower than those of the Randox-TAS assay. The novel method also exhibited a significant correlation with the FRAP assay (Fig. 7, Table 3). This is an advantage because it has been reported that no Discussion Various measurement methods have been developed for total antioxidant status, but there is not yet an accepted reference method. The final decisions concerning the standardizations, and the terms and units used have not been made yet. This implies that this topic needs to be studied further. Indeed, research efforts have been accelerating in this area over the last two decades. The most widely used methods for TAC measurement are colorimetric, fluorescence, and chemiluminescence [10 12]. The fluorescence and chemiluminescence methods need sophisticated techniques and, in most routine clinical biochemistry laboratories, these improved systems are not present. ABTS S+ -based methods are the most commonly used colorimetric TAC measurement methods. There are two main versions of ABTS S+ -based methods [3,8]. The first Fig. 7. The relationship between serum TAC values measured by the novel method and FRAP assay (FRAP: the ferric reducing ability of plasma).

8 284 O. Erel / Clinical Biochemistry 37 (2004) significant correlation has been found between the FRAP and Randox-TAS assays [12]. The reference range of serum TAC changes from method to method because there is no decided target molecule or assay standard. In various studies, Trolox [3,8,12], uric acid [4], vitamin C [19], and ferrous ion solutions [5,6] have been used for the calibration of the assays. However, Trolox is the most widely used traditional standard and hence Trolox was used as the assay standard in the novel method. In the FRAP assay, the reference range of serum TAC is lowest because this assay practically measures nonprotein total antioxidant capacity. However, proteins constitute the main antioxidant component of serum (plasma). As seen in Table 3, the correlation between serum total protein and FRAP levels was not significant. The Randox-TAS assay can determine the antioxidative effects of bilirubin, vitamin C, uric acid, polyphenols, and proteins; hence, the reference range for serum TAC is higher than that for the FRAP assay because the antioxidative effect of proteins is accounted for. Although the correlation between serum total protein level and TAC value measured by Randox-TAS assay is higher than that of the FRAP assay, it is not statistically significant (Table 3). In the post-addition ABTS S+ decolorization method, the antioxidant effects of proteins are pronounced and the correlation between total protein levels and TAC levels measured by the post-addition method of serum samples is significant. The novel method is more sensitive for determining the antioxidative effects of bilirubin, uric acid, vitamin C, polyphenols, and proteins. The antioxidative effect of reduced glutathione is higher than that of Trolox in the novel assay (Fig. 3, Table 2), and significant correlations between serum TAC and total protein levels were found in the healthy and mixed groups. Hence, serum TAC measured by the novel method is higher than those of the Randox-TAS and FRAP assays. The reaction time of the Randox-TAS assay is shorter than that of the novel method. If we shorten the reaction time of the novel method, the results obtained approach those of the Randox-TAS assay, but, in this condition, an amount of antioxidant capacity will not relate to a true TAC value. Antioxidative activity rates of proteins are not as fast as that of Trolox. However, Trolox has been used as a traditional standard to measure TAC. In the Randox-TAS assay, total reaction time is about half that of the novel method. Serum contains large amounts of proteins and they constitute the major antioxidant component of serum. This may explain why in the novel method serum TAC is higher than those of Randox-TAS and FRAP assays. On the other hand, the reported results of the recently developed methods [4,9] are higher than those of Randox- TAS and FRAP assays. Serum contains different antioxidant molecules. Proteins constitute the main antioxidant component of serum. Free sulfhydryl groups of proteins are mainly responsible for their antioxidative effects. Free sulfhydryl groups of serum belong to proteins in practice because the serum concentration of linoleic acid, which also has -SH groups, is very low and the contribution to serum total free sulfhydryl level can be ignored. It was calculated that total protein contributes 52.9% to the measured serum TAC in healthy subjects (Table 2). The antioxidative role of vitamin C is well known. In this study, it was determined that vitamin C constituted about 4.7% of the measured serum TAC in healthy subjects (Table 2). For many years, the bile pigment bilirubin was considered a toxic waste product formed during heme catabolism. However, more recent studies have shown that it is a strong physiological antioxidant that may provide important protection against atherosclerosis, coronary artery disease, and inflammation [20]. It has also been suggested that bilirubin might play a particularly crucial role in protecting the neonate from oxidative damage [21]. In this study, it was shown that bilirubin is a strong antioxidant molecule (Fig. 3, Table 2) and constituted about 2.4% of the measured serum TAC in healthy subjects (Table 2). Uric acid serves as a potent antioxidant by radical scavenging and reducing activities. Its concentration in plasma is almost 10-fold higher than that of other antioxidants, such as vitamin C or vitamin E [13,22]. As seen in Fig. 3 and Table 2, the antioxidant activity of uric acid is higher than those of vitamin C and Trolox. Uric acid constitutes about 33.1% of serum TAC measured by the novel method in healthy subjects (Table 2). The protective roles of tea, fruits, and vegetables, which are rich in antioxidant polyphenolic compounds, against cancer and atherosclerosis have been shown [23,24]. The number of studies in this area has been increasing at an accelerating rate. It has been shown that a single dose of tea increased plasma total antioxidant capacity [25]. In this study, the antioxidative activity of (F)-catechin, which is abundantly present in tea, was determined and compared with those of other known antioxidants. It has been shown that the antioxidant activity of (F)-catechin was higher than those of vitamin C, Trolox, bilirubin, uric acid, and GSH (Fig. 3). As seen in Fig. 4, the antioxidant activity velocities of vitamin C, Trolox, and catechin were fast and their antioxidant effects were completed within the first minute. The antioxidant activity rate of GSH was medium and its antioxidant effect was completed within 3 min. The antioxidant activity rates of uric acid and bilirubin are not slow but their antioxidant effects were completed within 5 min. There are few studies about the oxidative or antioxidative status of patients with depression. To date, only three articles appear to have been published in this area [26 28]. Maes et al. [26] found that the level of vitamin E, a minor component TAC of plasma, was low; Srivastava et al. [27] found that the specific activity of glutathione peroxidase, an antioxidative enzyme, decreased; and Bilici et al. [28] found that the level of erythrocyte malondialdehyde (MDA), an erythrocyte oxidative stress indicator, was high in patients with major

9 O. Erel / Clinical Biochemistry 37 (2004) depression. To the best of our knowledge, there is no report showing the TAC of serum (plasma) in patients with major depression. Our results are the first to be published on this subject. Our findings support previous reports. The mentioned reports explain that some components of antioxidative status are deficient and oxidative injury occurs in patients with depression. We determined that the TAC of the patients decreased. It may be inferred that the patients are exposed to oxidative stress and this may play a role in the etiopathogenesis of the disease. Supplementation of antioxidant vitamins, such as vitamin C and vitamin E, may be considered in the treatment of the disease. The novel assay is rapid, easy, stable, reliable, sensitive, inexpensive, and fully automated. The developed method has high linearity and the results are highly reproducible. The reagents are easy to prepare and their lifetimes are long. 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