The methodology was based on the article by Hadwan MH and Abed HN, [15].

Principle:

Catalase catalyzes the subsequent reaction:

Catalase activity was measured by incubating the enzyme sample in 1.0 mL substrate (20 μmol per ml hydrogen peroxide in 60 mmol/l sodium-potassium phosphate buffer, pH 7.4) at 37^oC for three minutes. The reaction was stopped with titanium sulphate. The method quantifies undecomposed hydrogen peroxide by reacting it with a titanium sulphate reagent to produce pertitanic acid. Absorbance of the yellow pertitanic acid was measured at 405 nm against the blank.

Reagents:

Instrument: Shimadzu 1800 spectrophotometer was used in the study.

Procedure: shown in [Table/Fig-1].

Calculation: Therate constant of a first-order reaction (k) equation is used to determine catalase activity:

t: time.

S°: absorbance of standard tube.

S: absorbance of test tube.

M: absorbance of control test (correction factor).

Vt: total volume of reagents in test tube.

Vs: volume of serum.

*The present method uses a correction factor (Control-test) to eliminate the interference that arises from the presence of a strong yellow background colour in the sample containing catalase enzyme. The absorbance of test tube in procedure belong to two types of compounds, undecomposed hydrogen peroxide and interferences that found in the sample. The absorbance of (Control-test) tube in procedure belongs to interference compounds found in the sample only. By subtracting the absorbance of (control-test) tube from the absorbance of test tube; we exclude the interference of a strong yellow background colour. Consequently; the resulting absorbance was related to un-reacted hydrogen peroxide in enzymatic reaction.

An absorbance spectrum of the colourimetric product was measured from 330 nm to 800 nm and revealed a peak at 405 nm [Table/Fig-2], suggesting the optimum colourimetric wavelength for the yellow reaction product is 405 nm.

[Table/Fig-2]:

Absorption spectra obtained for the colourimetric products of the present assay.

The present method uses a correction factor (Control-test) to remove the interference that arises from the presence of a yellow background colour of serum and/or reducing agents. Sugars and amino acids are the main constituents of the serum. Also, they have the reduction ability to interfere with the reagents that are used for assessment of catalase activity [17]. Possible interferences of compounds that could disturb catalase activity assessment were measured using the methods documented by Hadwan MH and Abed HN, [15]. To determine the effect of interferences that might disturb the catalase measurement, seven volumetric flasks were taken, to each one was added one ml of catalase known activity (500 kU/L) obtained from Himedia (Product Code: TC037). Its activity was calibrated according to Aebi’s method [18] and nine ml of 55.55 μmol/L of one interference only that dissolved in phosphate buffer (50 mM, pH 7.4). The final activity equals to 50 kU/L of catalase with 50 μmol/L of interference. Catalase enzyme activity was measured by the present method (with and without correction factor). [Table/Fig-3] shows the effects of several interferences on the catalase activity. Catalase enzyme activity was not affected significantly by a considerable amount of each interference compound when assayed by the present method. However, interferences affected catalase enzyme activity when method was used without a correction factor. The result established from [Table/Fig-3] proves that the correction factor was necessary to get a precise result.

Results achieved for serum by the present method were compared with that produced by the method of Aebi H [18]. Matching sample and buffer were used in both methods. The results of the present assay showed a good precision [Table/Fig-4] and a good correlation with the Aebi’s method [Table/Fig-5].

Accuracy of the present assay protocol was determined by recovery of hydrogen peroxide added to the reaction solution after the end of enzymatic reaction detailed in the [Table/Fig-6]. The high accuracy of the present assay protocol was belonging to high molar extinction coefficient and correction factor.

The result concluded from [Table/Fig-6] proves that the linearity of the following method reaches to about 300 kU.

This following article elucidates a new method for measuring catalase activity in serum. The method quantifies undecomposed hydrogen peroxide by reacting it with a titanium sulphate reagent to produce pertitanic acid. Absorbance of the yellow pertitanic acid was measured at 405 nm against the blank (as shown in [Table/Fig-2]).

The titanium tetrachloride/sulfuric acid reagent can be represented as a “stop bath” for enzymatic reaction that is regulated by catalase enzyme. When the enzyme solution is mixed with sulfuric acid, its molecules are denatured; any hydrogen peroxide which has not been decomposed by the catalase will react with the titanium to produce a peroxotitanium complex (pertitanic acid), which has a maximum absorbance at 405 nm.

Despite the discovery of the titanium sulfate method for H₂O₂ estimation by Eisenberg [19] several decades ago, it wasn’t appealing to use in the estimation of serum catalase activity because there are two types of interferences. The interferences include a yellow background colour of serum and/or reducing agents that reduce titanium (IV) to titanium (III). The only exception is the work that is done by Baudhuin and his assistants [20,21] to measure catalase activity in some tissues. Baudhuin and his coworkers did not explain how to remove interferences and did not compare their own method with other methods. The present method uses a correction factor (Control-test) to remove the interference that arises from the presence of a yellow background colour of serum and/or reducing agents. Sugars and amino acids are the main constituents of the serum. Also, they have the reduction ability to interfere with the reagents that are used for assessment of catalase activity [17]. [Table/Fig-3] shows the effects of several interferences on the catalase activity. Catalase enzyme activity was not affected significantly by a considerable amount of each interference compound when assayed by the present method. However, interferences affected catalase enzyme activity when method was used without a correction factor. The result established from [Table/Fig-3] proves that the correction factor was necessary to get a precise result.

The present method has introduced several advantages when compared with Aebi’s method. Catalase activity can be considered by measuring the rate of dissociation of hydrogen peroxide at 240 nm (Aebi’s method). There are practical problems with this method, which is related to using very high and un-physiological concentrations of H₂O₂ (5-50 mM) for achieving acceptable initial absorbance (the absorbance of H₂O₂ at 240 nm is only 43.6 M^-1 cm^-1). These high concentrations of hydrogen peroxide cause direct, but variable auto-inactivation of catalase by transformation of the active enzyme - H₂O₂ complex I to the inactive complex II [18]. Additionally, many cellular constituents such as proteins absorb intensely at 240 nm so that low activities of catalase often have to be assessed by the continuous method in contrast to a higher background absorbance.

The present method is more accurate (as shown in [Table/Fig-4]) and sensitive than Aebi’s method (as shown in [Table/Fig-5,6]) because of the high molar extinction coefficient for H₂TiO₄ (ε = 689 M^-1 cm^-1 at 407 nm) [22] compared with the molar extinction coefficient for H₂O₂ at 240 nm (ε = 43.6 M^-1 cm^-1) [16]. Sensitivity is often described in terms of the molar extinction coefficient (ε, M^-1 cm-1) of the measured product. The degree of the sensitivity is very significant in the spectrophotometric determination of an analyte [23].

On the other hand, the current method was preferable when compared with some colourimetric methods such as Hadwan MH and Almashhedy LA, and Ou P and Wolff SP, [7,8]. Hadwan MH and Almashhedy LA, presented simple method to measure catalase activity [7]. They used a certified standard enzyme with identified unit activity as an alternative of necessitation to standardize exact hydrogen peroxide concentration to 30 mM in a difficult process. The disadvantage of this method consists of the difficulty to obtain a certified catalase enzyme with identified activity. Ou P and Wolff SP, introduced a simple method to measure catalase activity. Ou P and Wolff SP, did not clarify how to exclude interferences and did not compare their own method they used with other procedures [8].

The results of the present study were not compared with that obtained by commercial kits.

The study explains a simple discontinuous method for catalase activity assessment which can be completed with few steps, and which allows catalase to be measured in the presence of high concentrations of other molecules as well as at low levels of H₂O₂. The titanium tetrachloride/sulfuric acid reagent is a sensitive assay for H₂O₂ which thus facilitates measurement of catalase at low concentrations of substrate confirming that auto-inactivation of catalase is reduced during the steps of measurement.