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Development of method for the mineral water catalase activity determination.

UDC 615.327.07

Introduction. The availability in Ukraine of diversified unique natural mineral water (MW) resources does necessitate a detailed study of that water properties in order of its widespread use in the balneological and extra-resort practice, considering the MW as an important factor of population health preserving and improving [1]. The mineral water rational use is largely determined by its physical and chemical composition, sanitary and microbiological status, the presence of autochthonous microorganisms and those microorganisms' ability to influence the water organoleptic characteristics and biological properties. In MW practical use the key importance is tribute to controlling the water quality parameters for the prediction of water safety degree and the detection of biological exposure.

The MW biological effect depends on the mineralization degree, chemical composition, presence of biologically active components and compounds, as well as on their microbial cenosis' metabolic products. Analyzing the mineral water microorganisms' list we reveal their active biochemical effect [1].

The numerous microorganisms contributing to the mineral water's autochthonous microflora do often include significant concentrations in saprophytic microorganisms producing the catalase as an enzyme of oxidoreductases class, a heme-containing chromoproteid making part of the cell's antioxidant system and acting as an antiperoxidant protector agent [2]. Peroxide mechanisms may occur in the brain and in other tissues during aging, when exposure to poisons and toxins, when cerebral apoplexy, at nervous system injuries, and others processes. The biological role of catalase refers to the complex enzymatic protection of cell membranes against hydrogen peroxide-caused degradation. The catalase does metabolize the hydrogen peroxide, preventing its accumulation in the cell through producing water and oxygen. The most relevant catalase application relates to the treatment of diseases requiring an effective antioxidant protector: cardiovascular, rheumatic diseases, allergies, central and autonomic nervous systems' diseases, gastrointestinal diseases, oncology, plastic surgery, etc. [2].

Given the important physiological role of catalase, it is necessary to determine the mineral water's enzymatic activity. At present the methodology for MW enzymatic activity determination is still absent, so such methods' elaboration problem is top priority one.

Analysis of recent researches and publications. Methods for catalase activity quantitative determination are based on the estimation of enzyme-cleaved hydrogen peroxide quantity when enzymatic activity is assessed by the catalase index.

The reference sources describe several methods of catalase activity assessment, but these methods relate to the biological objects (plasma, serum, etc.). To develop methods for determining the MW catalase activity we proceed to review the available approaches.

Known is the enzymatic activity determining method by Bach and Oparin based onto titration [3], through assessing the quantity of hydrogen peroxide decomposed during incubation with the enzyme. The amount of hydrogen peroxide in the reaction mixture is assessed by titration with a Potassium permanganate solution in an acidic environment. However, this method has such drawbacks as a high labour intensity, results' poor reproducibility and low sensitivity.

Another known approach to catalase activity determining is Warburg's gasometric method and its modifications. This methodology essence relates to capturing and measuring the volume of oxygen liberated after introducing the catalase extract into aqueous solution of hydrogen peroxide [4]. This method's disadvantages refer to the need for calculating and taking into account the vessel's constant volume, the dependence on external conditions (temperature, sea level altitude and atmospheric pressure) and the need in appliance for mechanical shaking device.

There exists also a fluorimetric catalase activity assessment method with further modifications determining the blood serum enzymatic index [5, 6]. Despite the method's high sensitivity, it is very expensive due to the use of a microtabet reader for increasing the sensitivity.

Another highly sensitive but expensive methodology departs from assessing the catalase activity on the base of the Clark oxygen electrode application, and this method modification [7] using the LabVIEW virtual tools.

There exist some polarographic methods, for example, [8], but they require special equipment and laboratory space arrangement.

The potentiometric method is based on measuring the rate of fluoride ions release using the fluorine-selective electrode as a result of 4-fluorophenol enzymatic cleavage with peroxidase in the presence of hydrogen peroxide [9]. In such a way it becomes possible to determine the amount of aerobic (catalase-positive) microorganisms. Still this method requires working out all the parameters for every studied object.

In biomedicine and food industry known is the electrochemical method of catalase-positive microorganisms' detection and identification, developed on the EIA and amperometry basis for highsensitivity enzymes detection [10]. This method uses nitrocellulose membranes as a solid phase for bacteria selective capturing with those microorganisms' antibodies. This is a technologically new approach, of high sensitivity, but the method is also expensive because of the costly equipment use.

Thus, the above spoken catalase assay methods are not available for certain objects' analysis facilities and can never be applied to mineral water analysis.

The Aim of the Research consists in developing a method for the mineral water catalase activity determination with sufficient accuracy and precision, and refers introducing a new parameter to assess the MW quality and biological value.

Main Body. At present, one of the basic analytical methods used at wide range of chemical, clinical, biological etc., laboratories is the spectrophotometry.

As the methodical prototype for catalase activity assessing accepted is Korolyuk's method [11] representing a modification of the biological objects' catalase activity spectrophotometric evaluation; the principle departs from the hydrogen peroxide's ability to interact with molybdenum salts forming a stable coloured complex. The method is based on the titration in an acidic environment with Potassium permanganate of hydrogen peroxide remaining after the catalase action,

5[H.sub.2][O.sub.2] + 2KMn[O.sub.4] + 4[H.sub.2]S[O.sub.4] [right arrow] 2KHS[O.sub.4] + 2MnS[O.sub.4] + 8[H.sub.2]O +5[O.sub.2].

The catalase activity is determined with the amounts of cleaved hydrogen peroxide (mg)

C = (B - A) x Q, (1)

where C--catalase activity, mg;

(B-A)--difference between results of titrating the control and test samples with 0,1 n-solution of Potassium permanganate, [cm.sup.3];

Q--amount of hydrogen peroxide (85 mg), corresponding to 1 [cm.sup.3] of 0,1 n-solution of Potassium permanganate.

Reagents: 4 % ammonium molybdate solution and 0,03 % hydrogen peroxide solution.

This research object was to improve the method for determining the catalase activity by spectrophotometric method of registering the coloured product of reaction when hydrogen peroxide not decomposed after incubation with catalase interacts with ammonium molybdate; the ultimate goal being to elaborate this method application to various types of mineral water.

Results. The formulated problem is solved using for spectrophotometric MW catalase activity estimation the mineral water research sample volume of 5,0 ml, assigning its incubation time as 30 min; the catalase activity is calculated as a percentage.

[] = [A.sub.control]-[A.sub.test][/.sub.Acontrol] x 100%

where []--catalase activity, %;

[A.sub.control]--the control (blank) sample optic density;

[A.sub.test]--the tested sample optic density.

The method essential feature is that the test MW sample volume increased from 0,1 to 5,0 ml, with simultaneous increase of incubation time from 10 to 30 min. Comparing to the prototype model we did change the formula of enzymatic activity calculation, having excluded the incubation time which has the same value. The test MW sample of 5 [cm.sup.3] is added with 2 [cm.sup.3] 0,03 % of hydrogen peroxide water solution. The blank sample contains instead of mineral one, 5 [cm.sup.3] of distilled water. Both samples are incubated during 30 minutes at 37[degrees]C. To stop the reaction, 1 [cm.sup.3] of 4% ammonium molybdate water solution is introduced. Both blank and test samples' colour intensity is measured with JEN WAY spectrophotometer; wavelength--410 nm, cuvette--10 mm. The MW catalase activity determination results, [], %, are processed following the formula (1).

Considering low index of MW catalase activity, estimated is the optimal delay of mineral water incubation (Table 1).

Table 1 does clearly represent that first 30 minutes of incubation running the MW catalase activity augments and further time increasing we observe its drop. Therefore the selected incubation time was 30 minutes.

The results of MW catalase activity measurement are shown at Table 2. Calculated are: the mean value, the root-mean square deviation S and the relative error [S.sub.r].

The standard relative error values ([S.sub.r]) are 1,58; 1,03; 0,29 and 0,33% respectively that make significantly below the bound error assigned for spectrophotometric analyses and the given optic density range.

Conclusions. After working out the method for catalase activity determining for various types mineral water, further the catalase activity index may be used as an additional criterion for evaluating the mineral water quality and biological activity. The elaborated method has been adopted for issuing a respective patent from the State Intellectual Property Office.

DOI 10.15276/opu.1.45.2015.29


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[2.] Nikolenko, S.I., Slavina, N.G., Zaytseva, K.B. and Glukhovskaya, S.N. (2001). Therapeutically active metabolites of mineral waters microflora. Water and Health--2001, 159-162.

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[5.] Naazeri, S., Rostamian, M., Yaghmaei, B. and Hedayati, M. (2014). Fluorimetry as a simple and sensitive method for determination of catalase activity. Zahedan Journal of Research in Medical Sciences, 16(2), 64-67.

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[7.] Sandeep Kumar Jha and D'Souza, S.F. (2011). An ultra sensitive method for rapid in vitro catalase assay with software based approach using LabVIEW virtual instrumentation. Analytical Methods, 3, 1981-1987.

[8.] Erel, O. (2004). A novel automated method to measure total antioxidant response against potent free radical reactions. Clinical Biochemistry, 37(2), 112-119.

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Received September 30, 2014

O.M. Nikipelova, Dr. of Chemistry, A.Yu. Kisilevskaya, PhD, L.B. Solodova, The Ukrainian Research Institute of Medical Rehabilitation and Balneology of Ministry of Health of Ukraine, Odessa
Table 1
Finding the mineral water incubation time

Incubation    Catalase
time, min    activity, %

0               45,17
5               46,13
10              45,75
15              47,68
20              49,81
25              50,19
30              50,58
35              49,81
40              49,42

Table 2
Evaluating the MW catalase activity

No.    Mineral water    Sample     Catalase     RMSD,   Relative
                          No.     activity, %     S      error,

1       Bottled MW         1         74,52      1,58      0,91
       "Ploskivs'ka"       2         75,85
       (Zakarpattya        3         77,67
          region)       [bar.X]      75,92

2     Native MW from       1         82,62      1,03      0,60
      wellbore No. 59      2         80,57
         at Ploske         3         81,80
          village       [bar.X]      81,66

3     Native MW from       1         11,64      0,29      0,17
      wellbore No. 2       2         11,45
       Tsary-chanka        3         11,07
          village       [bar.X]      11,39
      trovs'k region)

4       Bottled MW         1         9,83       0,33      0,19
       "Tsarychanka"       2         9,83
        (Dniprope-         3         10,40
      trovs'k region)   [bar.X]      10,02
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Author:Nikipelova, O.M.; Kisilevskaya, A.Yu.; Solodova, L.B.
Publication:Odes'kyi Politechnichnyi Universytet. Pratsi
Article Type:Report
Date:Mar 1, 2015
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