Measurement of Glutamate-Oxalacetate Transaminase Activity in Human Blood Serum Using Differential Pulse Anodic Stripping Voltammetry

A new electrochemical method for measuring the activity of glutamate-oxalacetate transaminase (GOT) in human blood serum was developed based on the appearance of a voltammetric peak current of oxalacetate at -0.4 V vs. (Ag/AgCl, 3 mol/L KCl) as a reference electrode using phosphate buffer (pH 7.4) as a supporting electrolyte. It is successfully applied for measuring GOT activity in certain diseases such as: Liver cirrhosis and Myocardial infarction, as well as normal cases. The results of our proposed method were compared with those obtained from colorimetric method for 38 specimens of human blood serum. A good relationship between the two methods were obtained with a correlation coefficient (r = 0.9976). ــــــــــــــــــــــــــــــــــــــــــــــــــــ تيماتولك ميزنا ةيلاعف سايق مادختساب ناسنلإا مد لصم يف زينيما سنارت تيتيساولازكوا يلضافتلا يضبنلا يدونلاا عازتنلاا يرتماتلوف


INTRODUCTION
Glutamate-oxalacetate transaminase GOT also known as aspartate aminotransferase (AST) has the ability to catalyze the following enzymatic reaction as shown in equation ( 1) :

Oxogltarate Glutaminate
Transamination reactions play significant role in intermediary metabolism.They require pyridoxal phosphate as a coenzyme for their catalytic activity (Nelson and Cox, 2005).Aspartate aminotransferase found in many animals and plants tissues and its activity is especially high in mammalian heart and liver (Zilva and Pannal, 1984).The enzyme exists in two distinct isoenzyme forms in mammalian tissues, the mitochondrial form and the cytoplasmic form.However, GOT levels in human blood serum are used in clinical diagnosis of liver and heart diseases (Burtis and Ashwood, 1994).Serum GOT levels in healthy cases are low, but the levels are significantly elevated in a number of clinical conditions such as acute and chronic hepatitis, obstructive jaundice, carcinoma of liver, myocardial infarction and muscular dystrophy (Sherwin and Sohenes, 1996).Therefore, determination of serum GOT level has great clinical and diagnostic significant.It has applications in couple enzyme reactions for the measurement of metabolite levels in biological fluids (Kim et al., 2001;Maitra et al., 2005).
Several methods were available for measuring GOT activity, the colorimetric method based on monitoring the concentration of oxalacetate hydrazone formed with 2,4dinitrophenylhydrazine (Reitman and Frankel, 1957).Stripping voltammetry is a very sensitive electroanalytical technique that used for trace analysis.It has a lowest determination limits in comparison with other commonly used electroanalytical techniques.The three most commonly used variations of stripping voltammetry used for quantitative determination of electroactive compounds are: anodic stripping voltammetry (ASV), cathodic stripping voltammetry (CSV), and adsorptive voltammetry (AdSV) (Wang, 1990).Due to the high sensitivity and selectivity of stripping voltammetry, it has the ability to use it with another voltammetric technique such as square-voltammetry (SWV) or differential pulse voltammetry (DPV).(Brett and Bret, 1993) In the present work, differential pulse anodic stripping voltammetry (DPASV) was used for measuring GOT activity in the real specimens of human blood serum as a best voltammetric technique for this purpose.

Apparatus
The electrochemical studies were carried on EG&G 384 B Polarographic Analyzer with hanging mercury dropping electrode (HMDE) as a working electrode, (Ag/AgCl, 3M KCl) as a reference electrode, and Pt wire as an auxiliary electrode.The colorimetric studies were performed on Cecil UV-Vis CE 10211 digital single beam spectrophotometer.
Temperature control was performed using thermostatic water bath type radiometer VTS 13 for electrochemical measurement, and thermostatic water bath model SB 10 from British Grants Instruments Limited for colorimetric measurements.

Chemicals and reagents
The GOT contains phosphate buffer (100 mmol/L, pH 7.4), L-aspartate (100 mmol/L), α-oxoglutarate (2.0 mmol/L) in a total volume of 100 ml Sodium Hydroxide (4.0 N) in a total volume of 100 ml, and 2,4-dinitrophenylhydrazine (2.0 mmol/L) in a total volume of 100 ml were supplied from Randox laboratories Ltd.Company (Randox, Cat.No. AS 147).(0.2 mol/L) Phosphate buffer pH 7.4 was prepared by dissolving 3.4836 g of dipotassium hydrogen phosphate K 2 HPO 4 and 2.7218 g of potassium dihydrogen phosphate KH 2 PO 4 in a total volume of 100 ml deionized distilled water.(10 -3 mol/L) oxalacetate stock solution was prepared freshly by dissolving 0.0013 g of oxalacetic acid in a total volume of 100 ml deionized distilled water.(0.4 N) NaOH solution was prepared freshly from NaOH stock solution.For quality control tests, three human assayed serum levels were prepared freshly by dissolving each vial of lypophilized serum with exactly 5 ml of deionized distilled water, then waiting for 30 min to complete dissolving process by keeping all of them out of light before use.All real specimens of human blood serum were collected from The Republic Hospital in Mosul, and then they are prepared and assayed within an hour, otherwise serum samples should be kept frozen.

Procedure
For voltammetric measurements, three-electrode cell has contained 5 ml of 0.2 mol/L phosphate buffer pH 7.4 and 0.1 ml of stock solution GOT buffer.The enzymatic reaction was initiated by addition 0.1 ml of human blood serum, the solution was de-aerated for 30 s using N 2 gas and the voltammogram was recorded after affixing the potential range from -0.1 to-0.6 V vs. (Ag/AgCl).The activity of GOT was determined depending on the increment in the current value of the reduction wave of oxalacetate that produced through the enzymatic reaction which appeared at -0.4 V vs. (Ag/AgCl).Moreover, the electrochemical cell was thermostated at 37 o C.
For colorimetric method, the sample cuvette contained 0.5 ml of GOT buffer and 0.1 ml of a real sample of human serum thermostated at 37 o C for 30 min, then 0.5 ml of 2,4-dinitrophyenylhydrazine was added and left for 20 min at 20 o C for complete reaction, and 5.0 ml of 0.4N NaOH was added finally to complete the colorimetric reaction.The absorbance of sample (A sample ) was measured at 546 nm against a blank solution.

Voltammetric behavior of oxalacetate produced during GOT enzymatic reaction
The optimum conditions were examined using DPASV as shown in Tables (1-4) as follows: deposition time 0 s, conditioning time 10 s, equilibrium time 5 s, and scan rate 400 mV/s.The voltammetric cell was thermostated at 37 o C, the solution de-aerated by passing N 2 gas through it for 240 s.The calibration curve was constructed using serial additions of 10 -3 mol/l oxalacetic acid solution, the linearity appeared in the range from 0 to 0.2 ml.The linear equation is: y = 122.2x-0.0005;where y is the reduction peak current of oxalacetate, Ip (nA) and x is the volume of oxalacetic solution (ml), as shown in Fig. (1).The GOT activity in human blood serum was calculated using equation ( 2) in order to detect the activity of GOT in the real specimens of human blood serum using DPASV.However, one unit of GOT activity (U/l) is equal to the rate of production of one micromole of oxalacetate per one minute under the assay conditions.
GOT Activity (U/l) = According to the enzymatic reaction of GOT, the oxalacetate which has been produced during this reaction by using our proposed method DPASV gives us the differential pulse anodic stripping voltammogram at -0.4 V vs. (Ag/AgCl) in phosphate buffer pH 7.4 after human blood serum addition as shown in Fig. (2B).Moreover, Fig. 2A represents the differential pulse anodic stripping voltammogram for phosphate buffer pH 7.4 before human serum addition; this indicates the major role of oxalacetate produced through the enzymatic reaction.Effect of human blood serum amount 0.1 ml of a real specimen of human blood serum was selected as the optimum value in our proposed method DPASV.A set of additions ranging from 0.02 to 0.1 ml of human serum were added into the voltammetric cell.The later contains 5 ml of phosphate buffer pH 7.4 and 0.1 ml GOT buffer, respectively.The optimum value gives a maximum value of the reduction wave of oxalacetate at -0.4 V vs. (Ag/AgCl) as shown in Table 5.

Comparison study between DPASV and colorimetric methods
The comparison between our proposed method DPASV and colorimetric method were carried out using 38 samples which were divided into normal cases and patients suffering from different diseases such as: Liver cirrhosis and Myocardial infarction.Table 6 shows the data which showed a consistent difference in the GOT activity between our proposed method and colorimetric method in normal and patient cases.Figure 3 displays the linear relationship for measuring GOT activity in human blood serum using DPASV and colorimetric methods with correlation coefficient (r = 0.9976).The latter indicates a good agreement between these methods.The relationship between two methods could be represented by equation ( 3

Quality control study
To examine the accuracy and reproducibility control from the DPASV and colorimetric methods, quality controls Levels 1, 2 and 3 were assayed respectively.Table 7 shows that the results are displayed a good agreement between our proposed method DPASV and colorimetric method.

Fig. 1 :
Fig.1: Calibration curve between the reduction wave current value of oxalacetate(Ip) and the series volume additions of 10 -3 mol/L oxalacetate solution.

Fig. 2 :
Fig. 2: Differential pulse anodic stripping voltammograms of oxaloacetate in phosphate buffer pH 7.4 as: (a) before human blood serum addition, (b) after human blood serum addition.

Fig. 3 :
Fig.3: The linear relationship between DPASV and colorimetric methods that used for determination of serum GOT activity in normal and patient cases.

Table 1 :
Effect of deposition time on the reduction current of oxalacetate

Table 2 :
Effect of conditioning time on the reduction current of oxalacetate

Table 3 :
Effect of equilibrium time on the reduction current of oxalacetate

Table 4 :
Effect of scan rate on the reduction current of oxalacetate

Table ( 5
): Effect of human blood serum amount on the reduction wave of oxalacetate.

Table 6 :
GOT activity measured by DPASV and colorimetric methods in normal and patient cases.

Table 7 :
Quality control of GOT activity in human serum measured by DPASV and colorimetric methods.