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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
UDC 615.327.07
DEVELOPMENT OF METHOD FOR THE MINERAL
WATER CATALASE ACTIVITY DETERMINATION
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.
DOI 10.15276/opu.1.45.2015.29
© O.M. Nikipelova, A.Yu. Kisilevskaya, L.B. Solodova, 2015
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, 2015.
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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 2 2 + 2KMn 4 + 4 2S 4 → 2K S 4 + 2 nS 4 + 8 2 +5 2.
The catalase activity is determined with the amounts of cleaved hydrogen peroxide (mg)
= ( – ) × Q,
(1)
where — catalase activity, mg;
( – ) — difference between results of titrating the control and test samples with 0,1 n-solution of
Potassium permanganate, m3;
Q — amount of hydrogen peroxide (85 mg), corresponding to 1 m3 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.
.
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Table 1
Finding the mineral water
incubation time
Incubation
time, min
0
5
10
15
20
25
30
35
40
Catalase
activity, %
45,17
46,13
45,75
47,68
49,81
50,19
50,58
49,81
49,42
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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.
cat
=
control
−
test
× 100 % ,
control
— catalase activity, %;
— the control (blank) sample optic density;
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 m3 is added
with 2 m3 0,03 % of hydrogen peroxide water solution. The blank sample
contains instead of mineral one, 5 m3 of distilled water. Both samples are
incubated during 30 minutes at 37 ° . To
Table 2 stop the reaction, 1 m3 of 4 % ammonium
molybdate water solution is introduced. Both
Evaluating the MW catalase activity
blank and test samples’ colour intensity is
Sample Catalase RMSD, Relative measured with JEN WAY spectrophotometer;
№ Mineral water
№
activity, %
S
error, Sr wavelength — 410 nm, cuvette — 10 mm.
The MW catalase activity determination
1
74,52
Bottled MW
results, cat, %, are processed following the
2
75,85
“Ploskivs’ka”
formula (1).
1,58
0,91
1
(Zakarpattya re3
77,67
Considering low index of MW catalase
gion)
activity,
estimated is the optimal delay of
75,92
X
mineral
water
incubation (Table 1).
1
82,62
Native MW from
Table 1 does clearly represent that first
2
80,57
wellbore No. 59
30 minutes of incubation running the MW
3
81,80
catalase activity augments and further time
1,03
0,60
2 at Ploske village
increasing we observe its drop. Therefore
(Zakarpattya re81,66
X
the selected incubation time was 30 minutes.
gion)
The results of MW catalase activity
measurement are shown at Table 2. CalcuNative MW
1
11,64
lated are: the mean value, the root-mean
from wellbore
2
11,45
square deviation S and the relative error Sr.
No. 2 Tsary3
11,07
3
0,29
0,17
The standard relative error values (Sr)
chanka village
are 1,58; 1,03; 0,29 and 0,33 % respectively
(Dniprope11,39
X
that make significantly below the bound
trovs’k region)
error assigned for spectrophotometric anal1
9,83
Bottled MW
yses and the given optic density range.
2
9,83
“Tsarychanka”
4
0,33
0,19
Conclusions. After working out the
3
10,40
(Dnipropemethod for catalase activity determining for
trovs’k region)
10,02
X
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.
where
cat
control
.
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ISSN 2223-3814 (online)
, 2015.
. 1(45)
179
Ʌɿɬɟɪɚɬɭɪɚ
1.
, . .
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2.
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, 2002. — . 59 — 68.
/ . .
— 2001: .
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-
,
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, . .
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2001. ― . 159 ― 162.
3.
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. — 2010. —
. 76, № 10. — . 21 — 24.
4.
–
/ . .
[
.]. ―
. 4, . 3. ―
:
, 2005. ― 51 .
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M. Rostamian, B. Yaghmaei, M. Hedayati // Zahedan Journal of Research in Medical Sciences. —
2014. — Vol. 16, No. 2. — . 64—67.
6. Wu, M. Determination of the activity of catalase using a europium(III)–tetracycline-derived fluorescent
substrate / M. Wu, Z. Lin, O.S. Wolfbeis // Analytical Biochemistry. — 2003. — Vol. 320, Issue 1. —
. 129 — 135.
7. Sandeep Kumar Jha. An ultra sensitive method for rapid in vitro catalase assay with software based approach using LabVIEW virtual instrumentation / Sandeep Kumar Jha, S.F. D’Souza // Analytical Methods. — 2011. — Issue 3. — . 1981 — 1987.
8. Erel, O. A novel automated method to measure total antioxidant response against potent free radical reactions / O. Erel // Clinical Biochemistry. — 2004. — Vol. 37, Issue 2. — . 112 — 119.
9. Rapid electrochemical detection and identification of catalase positive micro-organisms / N. Sippy,
R. Luxton, R.J. Lewis, D.C. Cowell // Biosensors and Bioelectronics. — 2003. — Vol. 18, Issues 5–6. —
. 741—749.
10. Majumdar, T. Rapid Electrochemical Quantification of Food Borne Pathogen Staphylococcus Aureus
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method for determination of catalase activity. Zahedan Journal of Research in Medical Sciences, 16(2), 64-67.
6. Wu, M., Lin, Z. and Wolfbeis, O.S. (2003). Determination of the activity of catalase using a europium(III)–tetracycline-derived fluorescent substrate. Analytical Biochemistry, 320(1), 129-135.
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radical reactions. Clinical Biochemistry, 37(2), 112-119.
9. Sippy, N., Luxton, R., Lewis, R.J. and Cowell, D.C. (2003). Rapid electrochemical detection and identification of catalase positive micro-organisms. Biosensors and Bioelectronics, 18(5-6), 741-749.
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10. Majumdar, T., Chakraborty, R. and Raychaudhuri, U. (2013). Rapid electrochemical quantification of
food borne pathogen Staphylococcus Aureus based on hydrogen peroxide degradation by catalase.
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ȺɇɈɌȺɐȱə / ȺɇɇɈɌȺɐɂə / ABSTRACT
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O.M. Nikipelova, A.Yu. Kisilevskaya, L.B. Solodova. Development of method for the mineral water catalase activity
determination. Biological effects of mineral water depend not only on the chemical composition but also on the metabolic
products of microbial cenosis. Among numerous microorganisms constituting the autochthonous microflora of mineral waters, we do evolve the saprophytic organisms producing the catalase, the saprophytes’ physiological and biological role being
proven a long ago. The research aim was to develop a method for determination of mineral water catalase activity. Analysed
are various methods to determine the catalase activity in biological objects. Developed is a spectrophotometric method for
determination of mineral water catalase activity. The method is efficiently tested with series of Ukrainian mineral waters.
Calculated are the relative standard deviations which are significantly below normal errors, admitted at spectroscopic analysis
and at the optic density range. The given method provides sufficient accuracy and convergence when estimating the mineral
waters catalase activity, allowing to introduce a new index to assess the quality and biological value.
Keywords: catalase activity, mineral waters, spectrophotometric method, saprophytic microorganisms.
Received September 30, 2014
.