A validated method to assess glutathione peroxidase enzyme activity

This article presents a reliable, effective and easy procedure for measuring glutathione peroxidase (Gpx) activity. Enzyme samples were incubated with phosphate buffer, which included the appropriate concentrations of glutathione (GSH) and peroxide as substrates, to determine Gpx activity. In the CUPRAC method, the CUPRAC reagent Cu(Nc)22+ was added to stop the enzymatic reaction after a sufficient period of incubation. The unreacted substrates acted to reduce the Cu(II)–neocuproine complex into the strongly coloured Cu(I)–neocuproine complex that could be measured spectrophotometrically at 450 nm. GPx activity was linked to a decrease in the absorbance of the coloured Cu(I)–neocuproine complex. The Box–Behnken design was used to optimise the formation of the Cu(I)–neocuproine complex. Response surface methodology was applied to determine the accuracy of the method. This new protocol was confirmed by performing the Bland–Altman plot analysis of Gpx activity in matched samples through the Gpx–DTNB assay. The correlation coefficient between the two protocols was 0.9967. This result indicated that the new protocol was very accurate and on par with the comparison method.


Introduction
Glutathione peroxidases (Gpx) act to reduce hydroperoxides (ROOH) by glutathione (GSH): ROOH + 2GSH→ROH + H2O + GSSG R may be an aliphatic, aromatic, or hydrogen-containing organic group. H2O, alcohol (ROH) (or a second H2O when H2O2 is the substrate), and glutathione disulphide (GSSG) are the products. The enzyme glutathione reductase is responsible for regenerating GSH from GSSG in the cell [1,2]. The glutathione peroxidase family (GPx1-8) catalyse the reduction of organic and inorganic peroxides by using reduced GSH. Proteins that contain selenocysteine make up five of the eight glutathione peroxidases (GPx1-4 and GPx6) [3]. The tendency of various GPxs to catalyse the degradation of hydroperoxides by thiols is their common denominator [4]. Gpx is a significant system to protect against endogenously and exogenously mediated lipid peroxidation that is present in many animal tissues. The enzyme is stoichiometric in selenium, and it reacts with several organic hydroperoxides as well as hydrogen peroxide [5].
Even though several protocols for assessing glutathione peroxidase activity have been established, only a few are still useful. To assess glutathione peroxidase activity in biological tissues, only two different test systems have been used. The first system [1,6] was based on measuring ROOH or GSH consumption at regular interval. The second system monitors GSSG production by coupling to the glutathione reductase-catalysed reaction [1,7]. The decrease in NADPH concentration is continuously measured spectrophotometrically or fluorometrically [6,7].
Ellman's reagent (DTNB) is most commonly used in the first system to colourimetrically evaluate glutathione consumption as a function of glutathione peroxidase activity [6]. Compared to other tests, the GPx-DTNB assay is insensitive [8] and Ellman's reagent is relatively unstable [9]. Moreover, the process is timeconsuming [10]. Flohé and Günzler [11], on the other hand, showed another polarographic method. It employed strong acid to stop enzyme-catalysed or spontaneous GSH-hydrogen peroxide reactions at a specified time (t). Polarography is then used to determine the GSH content.
Ugar et al. [12] recommended a microplate-based method that reduced the Cu(II)neocuproine complex to highly coloured Cu(I)-neocuproine complex by using unreacted GSH. Catalase enzyme with high activity was used to stop the Gpx reaction.
The absorbance decrement was correlated with Gpx activity. The method was suitable to assess Gpx activity in pure samples but not for assessing its activity in biological tissues because this does not take into account the interference arising from the presence of the catalase enzyme. Glutathione peroxidase and catalase act on hydrogen peroxide as a common substrate. All previous methods work to block interference with the catalase enzyme by adding sodium azide, which inhibits the catalase enzyme selectively.
Fluorescent methods occupy an important part of the second system of methods used to estimate the Gpx activity. Weiss et al. [13] documented a fluorometric method to measure Gpx activity in less than 100 µg of tissue. That assay depends on the fluorometric behaviour of NADP + that arises from the oxidised glutathione created by the Gpx reaction. Martinez et al. [14] developed a fluorometric procedure with high sensitivity that used the assay of oxidised glutathione with o-phthalaldehyde. Kamata et al. [15] developed a sensitive method to assess Gpx activity using the fluorometric reaction of oxidised glutathione with N-(9-acridinyl) maleimide. The procedure was used to evaluate human plasma samples and liver homogenates.
Paglia and Valentino [7] were developed the most widely used protocol for measuring Gpx activity. It was based on the change of absorbance at 340 nm when NADPH is consumed by oxidised glutathione (GSSG). The protocol was modified by Lawrence and Burk [16] to study the activity of the Gpx enzyme in the liver supernatant of rats fed with a Se-deficient diet. The protocol was simple and selective, but the sensitivity was poor, and the enzyme and NADPH are costly [12]. Furthermore, NADPH + H + seems to be a potent Gpx inhibitor [10]. Since proteins and DNA absorb UV light, the method cannot have precise results when calculating Gpx activity in biological tissues.
A simple method for determining Gpx enzyme activity is identified in this paper.
Phosphate buffer was used to incubate the enzyme samples, which had appropriate concentrations of glutathione and peroxide as substrates. After appropriate incubation, the CUPRAC reagent (Cu(Nc)2 2+ ) was added to stop the enzyme's reaction. Unreacted substrates reduced the Cu(II)-neocuproine (Cu(Nc)2 2+ ) complex to highly coloured Cu(I)-neocuproine (Cu(Nc)2 + ) complex, which has a maximum absorbance at 450 nm (CUPRAC method). The Gpx activity was correlated inversely with the decrease of absorbance of coloured Cu(I)-neocuproine (Cu(Nc)2 + ) complex.
The current protocol is precise, efficient, and trustworthy. The method is interferencefree, simple to implement in laboratory experiments, and appropriate for clinical diagnosis.

Animals:
The albino rats were obtained from Animal House, University of Babylon, Babylon governorate, Iraq. They were kept in well-ventilated cages with monitored light and humidity, as well as free access to regular food and water. The current study was conducted in accordance with the WSAVA Animal Welfare Recommendations [17].  6. Neocuproine (7.5 * 10 -3 M OF 2,9-dimethyl-1,10-phenanthroline) (Nc) was composed 0.039 g Nc that was dissolved in 25 ml of 96% ethanol. 7. Fresh working reagent (CUPRAC reagent) was used for the experiment. The reagent was composed of Cu(II):Nc: NH4Ac at a ratio of 1:1:

Instrument
A Shimadzu 1800 spectrophotometer was used to take the measurements in the current study.

Glutathione peroxidase purification
Rat's liver Gpx was purified, as described by Chafik et al. [18]. Gpx activity was measured using the GPx-DTNB assay [6]. The specific activity of Gpx was estimated to be about 1.2 U.mg -1 protein.

Tissue preparation
After the animals were sacrificed, the livers were washed thoroughly and rinsed with ice. They were weighted in an objective equilibrium after being carefully blotted between folds of filter paper. A polytron homogeniser was used to prepare 10% of the homogenate in 0.1 M phosphate buffer (pH 7.0). Unbroken cells, cell debris, nuclei, mitochondria, and erythrocytes were removed from the homogenate by centrifugation at 10,000 rpm for 20 minutes. The Gpx level was measured in the supernatant.

Detailed of procedure
The details of the procedure are shown in table 1.
The reaction was initiated by adding peroxide: Mix by vortex and incubate for 10 minutes at 37°C, after that, the reaction was terminated with 0.5 ml of 8% TCA Mix well and centrifuge for 15 minutes at 3000 xg, then remove 1 ml of supernatant in a clean tube , and add: Working reagent 3ml 3ml 3ml 3ml Absorbance was read against blank at 450 nm after 30 min.

Calculation
Unit definition: one unit of GPx was defined as the amount of enzyme capable of )×D.f.

Standard curve preparation
To create a standard curve for the assay, the stock solution of the standards (glutathione and peroxide) were diluted with the phosphate buffer (0.1 M, pH 7.0) according to the layout in table 2. Once each standard tube was created and mixed, 1000 µL of each was added to the three ml of working solution according to the protocol listed in table 1.
Absorbance was read against blank at 450 nm after 30 min (as shown in fig. 1).

Performance of the method
Performance of the current method was achieved according to the guideline on bioanalytical method validation from the Committee for Medicinal Products for Human Use [19].

Optimising the protocol
To optimise the Gpx-CUPRAC protocol, the BBD was used to apply RSM. To design the Gpx assay experiment and optimise the method, the statistical parameters were calculated using Chemoface software, Version 1.5 [20]. The enzymatic reaction was performed with Gpx solution (500 U.L -1 ), which was prepared fresh before the experiment by dissolving 417 mg of Gpx standard in 100 ml of 100 mM phosphate buffer solution (pH 7.0). The Gpx-DTNB procedure [6,11] was used to adjust the final Gpx activity to 500 U.L -1 . The independent variables were glutathione, peroxide, and neocuproine concentrations (Table 3), and the dependent variable was the obtained Gpx activity compared to the current spectrophotometric protocol.
The mathematical modelling of a second-order polynomial equation was used to determine the interaction between the dependent and independent variables.
where Y refers to the response variable, XiXj refers to the independent variables, and β0, βi, βii, and βij refer to the intercept, linear, quadratic and interaction coefficients, respectively. Random error is represented by the symbol ε. Table 3. shows the results of using the Box-Behnken design to optimise the glutathione peroxidase activity assay. The independent variables were glutathione, peroxide, and neocuproine concentrations, and the dependent variable was glutathione peroxidase activity based on current spectrophotometric sensitivity.

Accuracy, selectivity, and reproducibility
Three kinds of interfering biochemicals were dissolved in four separate flasks to test the accuracy of the new Gpx procedure. The first included 100 mM phosphate buffer solution (pH 7.4), the second 5 mM sucrose, glucose, mannose, galactose, and ribose, the third 5 mM isoleucine, leucine, aspartic acid, methionine, and valine, and the fourth 3% casein and 3% bovine albumin. The phosphate buffer was used to dissolve all the interfering biomolecules. One ml of 3000 U/l Gpx enzyme was combined with 9 ml of the solutions containing possibly interfering biochemicals in an enzymatic reaction.
Using the Gpx-DTNB method, the activity of Gpx was calibrated to 300 U/l. The Gpx-CUPRAC protocol recovery was calculated for each possible interfering biomolecule.
The association between relative percentage errors and interfering biological substances is shown in Table 4. Several biological samples were used to test the current method's inter-and intra-day reproducibility, and the results were shown using RSD.

Signal stability
A Gpx solution (150 U.L -1 ) was used to study the stabilisation of the coloured chelate complex. After adding the working solution, the absorbance reading at 450 nm was measured after 15 minutes, 30 minutes, 45 minutes, 60 minutes, 5 hours, one day, three days and one week.

Sensitivity and linearity
The linearity and sensitivity of the protocol were tested using a range of Gpx activities (0, 5, 10, 25, 50, 100, 200, 400, 600, 700 and 1000 U.L -1 ). The linearity was assessed by comparing it to the Gpx-DTNB method [6,11] using a web-based program for bias assessment and comparison of analytical methods [21]. Limits of quantitation (LOW) and detection (LOD) were used to calculate the sensitivity of the Gpx-CUPRAC assay [22].

Validation
The Passing-Bablok regression [23] and Bland-Altman analysis [24] were used to compare the Gpx-CUPRAC method to the Gpx-DTNB method. The QiMacros program linked to Microsoft Excel 2016 was used for mathematical analyses (QiMacros, Know Ware International, Denver, USA).

CUPRAC reagent as a suitable probe to measure Gpx activity [the Gpx-CUPRAC method]
By using the cupric neocuproine complex (Cu(Nc)2 2+ ) as a suitable chromogenic oxidising probe, the present work explains a basic procedure to assay Gpx activity in biological samples (CUPRAC method). Apak et al. [25] introduced

Optimising the Gpx-CUPRAC assay
To achieve the optimum conditions, statistical methods were adapted with the Box-Behnken design (BBD) [26]. To optimise the Gpx-CUPRAC assay, BBD is an important measuring method with three central points to optimise glutathione, peroxide, and neocuproine concentrations to achieve optimal Gpx activity (see Table 3). The regression model for the Gpx-CUPRAC assay was determined using the analysis of variance (ANOVA) of the response surface methodology (RSM), as shown in Table 4.
The model's F-value (12.82) showed that it was significant, while the lack-of-fitness Fvalue (2.8405) showed that it was not significant as compared to the related p-value.
The significance of model terms was proved by obtained p-value (p= 0.0019).
The adjusted response (Adjusted R 2 = 0.9918) was in acceptable agreement with the coefficient predicted response (Predicted R 2 = 0.9482). As a result, the Gpx-CUPRAC assay's ANOVA showed that the specific correlation between the independent variables of the proposed model was appropriate for description and highly significant.
To investigate the graphical results of the independent variables, contour diagrams and three dimensional (3D) of the BBD were used. When the third factor was constant, the creation of 2D and 3D graphs at the midpoint stage was based on a combination of two variables. In the response plot in Fig. 3a-

Signal stability
The coloured complex in the current study was remarkably stable at room temperature.
At 25 °C, the CUPRAC complex's 450 nm absorbance remained remarkably stable for more than a week.

Linearity and sensitivity
According to the results shown in Fig 4, Figure 4 The Gpx activity of diluted tissue homogenates that were obtained by using the Gpx-CUPRAC method compared to the values achieved using the GPx-DTNB method.

Selectivity, reproducibility, and accuracy of the Gpx-CUPRAC method
The findings in Table 5 showed that the analysed biomolecules cannot interact with the  The Gpx-CUPRAC assay was used to determine the Gpx activity of homogenates of liver tissue. The findings revealed that Gpx activity was elevated as predicted in liver tissue homogenates (Fig. 5). The Gpx-CUPRAC method demonstrated reasonable inter-day (RSD% = 2.2.8%-3.2%) and intra-day (RSD% = 2.7%-3.8%).

Fig. 5.
Comparison of Gpx activity of diluted tissue homogenates that were obtained by the Gpx-CUPRAC method and the GPx-DTNB method.
Gpx activity assessment is a useful parameter for evaluating the liver's ability to reduce the susceptibility to oxidative stress. Furthermore, several scientific experiments have focused on Gpx activity in the livers of some types of lab animals to assess the oxidative stress inclination [27,28].

Validation
Using matched enzymatic samples, Bland-Altman plot analyses (QI Macros, 2016) were used to compare the Gpx activity assessed by the present method with the Gpx activity assessed by the GPx-DTNB process [6]. Bland-Altman plot shows the relative differences between Gpx-CUPRAC and GPx-DTNB methods, as well as the mean relative bias (Fig. 6). The correlation coefficient between the two protocols was 0.9994.
This means that the new protocol is almost as accurate as the reference protocol. The comparison between the Gpx-CUPRAC method and the GPx-DTNB method using the Passing-Bablok similarity analysis showed a good agreement correlation (Fig. 7). Fig. 6. Bland-Altman plot shows the relative differences between the Gpx-CUPRAC and GPx-DTNB protocols, as well as the mean relative bias. Fig. 7. Gpx activities were measured using the Gpx-CUPRAC and GPx-DTNB methods over a series of Gpx dilutions.

Conclusion
In this research, a simple and accurate protocol for calculating Gpx activity with a single reagent solution was developed. The BBD calculated the optimum glutathione, peroxide, and neocuproine concentrations. The new method is free of the interference that can arise when proteins, amino acids, and sugars are present. The CUPRAC assay's working solution allows for calculating Gpx activity at low concentrations of substrate.