RU2175445C2 - Incubation medium for determination of plasma blood catalase activity - Google Patents

Abstract

FIELD: medicine, cardiology, therapy, biology, biochemistry. SUBSTANCE: invention relates to development of incubation medium used for assay of catalase activity in plasma blood. Medium has containing phosphate buffer K₂HPO₄/NaH₂PO₄, 60 mM/l; pH = 7; ammonium chloride, 500 mM/l; Triton X-100, 0.769 mM/l; H₂O₂, 11.6 mM/l. Indicated composition of incubation medium provides to determine kinetics of catalase activity in plasma blood by photometric method at λ = 405 nm, t = 37 C for 9 min with high precision. EFFECT: enhanced quality of incubation medium, improved precision of assay. 1 cl, 1 ex

Description

 The invention relates to medicine and can be used in therapy, cardiology, biochemistry, biology.

 Known incubation medium for determining the activity of catalase (CF 1.11.1.6) with potassium permanganate reagent (1) with the reproducibility of the method to 8.7% and to reduce the concentration of hydrogen peroxide in the cell of the spectrophotometer at a wavelength of λ = 240 nm [1].

 Also known is the incubation medium containing phosphate buffer, acetylacetone / methanol, hydrogen peroxide [2].

 The indicated incubation media for determining the activity of catalase in blood plasma do not provide high accuracy in determining the activity of catalase in blood plasma and do not allow a kinetic determination of the activity of catalase.

The closest (prototype) is the incubation medium for determining the activity of catalase in the test system for determining total blood cholesterol [3], containing phosphate buffer 600 mm / l, pH 7.0, acetylacetone / methanol 420 mm / l / 2500 mm / l , hydroxypolyethoxydecane 2.1%, hydrogen peroxide 11.6 mm / L. The final volume of the mixture is 760 μl (optical path length 1 cm). The measurement is carried out at λ = 405 nm and a temperature of 37 ^o C after a 45-minute incubation of the sample. Registration begins after adding 60 μl of methanol / catalase solution. A colored complex of 3,5-diacyl-1,4 dihydrolutidine is recorded with simultaneous determination of cholesterol esterase in an incubation medium.

There is a known incubation medium for determining cholesterol, in which, under the influence of cholesterol oxidase, cholesterol is converted to cholestenone, and the by-product of the reaction is H ₂ O ₂ , which, under the influence of added catalase, is converted to H ₂ O ₂ with the simultaneous oxidation of methanol to formaldehyde. The resulting formaldehyde is readily soluble in water at a temperature of 37 ^o C and transparent. It reacts with acetylacetone in the presence of ammonium phosphate with the formation of yellow-colored 3,5-dihydrolutidine, detected at λ = 405 nm under the condition that the reaction proceeds linearly for 9 minutes.

 As the experimental studies conducted by us showed, the prototype incubation medium does not allow the linear dependence of the reaction to be determined, that is, it does not allow the kinetic determination of catalase activity, since the concentration of ingredients in the prototype incubation medium is optimal for a preliminary study of cholesterol esterase activity, namely the concentration phosphate buffer is high and 10 times different from the optimal molarity of the buffer used in determining the activity of catalase and other oxidation enzymes [2, 4, 5].

 The problem solved by this invention is to improve the quality of the environment due to the possibility of recording the kinetics of catalase activity.

The problem is solved by creating an incubation medium for kinetic determination of catalase activity in blood plasma containing phosphate buffer solution, acetylacetone, methanol, hydrogen peroxide, ammonium chloride and X-100 triton in the following ratio of components, mmol / l:
K ₂ HPO ₄ / NaH ₂ PO ₄ - 60
NH ₄ Cl - 500
Acetylacetone - 500
Methanol - 500
H ₂ O ₂ - 11.6
Triton X-100 - 0.769
In the literature analyzed by the authors, no combination of distinctive features was found and they do not explicitly follow the prior art for a specialist. This incubation medium can be used to record the kinetics of plasma catalase activity.

 Thus, this technical solution meets the criteria of the invention of "novelty" and "inventive step", industrially applicable.

 To determine the activity of catalase in plasma, the incubation medium is obtained in the manner described above.

 Then add 60 μl of blood plasma.

 Registration is carried out within 10 minutes

 Example 1

 Patient V., 40 years old, screened at the NIIKIF MZRF, Tomsk on 05.21.97 for IHD, exertional angina, FC II.

The general blood test did not have abnormalities. ESR 78 mm / h, HB 154 g / l; red blood cells 5.5 • 10 ¹² / l.

 Plasma catalase activity was determined using the proposed incubation medium and amounted to 6.0 μat / L.

 The kinetic activity curve of catalase is shown in graph No. 5 (see Fig. 5).

 The incubation medium for determining the activity of catalase was tested in a number of experiments (see above) and during clinical trials. When kinetic determination of plasma catalase activity in 96 patients with coronary heart disease (CHD) and healthy individuals (table. 5 and schedule No. 5).

 In order to obtain the optimal incubation medium for determining the activity of blood catalase, we conducted experimental studies using the incubation medium of the prototype method.

 Using the incubation medium, it is possible to determine the activity of catalase, which we demonstrated by introducing into the incubation medium instead of catalase blood plasma dry crystalline catalase firm "Fluka" at the concentration specified in the prototype method (2460000 UNITS / l). The kinetics of the reaction and statistical processing of the results are presented in graph No. 1 (see Fig. 1) and in table No. 1. The reaction proceeded nonlinearly.

 Next, we introduced into the incubation mixture a concentration of catalase close to that in blood plasma.

 The results are presented in graph No. 1 and in table No. 1.

 Experimentally, optimal concentrations of reaction ingredients were selected. The results of measuring the concentration of phosphate buffer, pH 7.0, followed by determination of the kinetics of the catalase reaction are presented in graph No. 2 (see Fig. 2). The results of measuring the concentration of acetylacetone with the subsequent determination of the kinetics of the catalase reaction are presented in graph No. 3 (see Fig. 3) and in table No. 3. The results of changes in the concentration of methanol with the study of the kinetics of catalase reaction are presented in graph No. 4 (see Fig. 4) and in table N 4. The results of determining the kinetics of the catalase reaction under optimal conditions are presented in graph No. 1 and table No. 1.

 When the concentration of substances in the incubation medium changes in the direction of their increase or decrease, the reaction slows down, which indicates the authors selected the optimal concentration of ingredients for the photometric determination of the kinetics of catalase activity. At other concentrations of the ingredients of the incubation medium, a similar effect was not achieved.

Thus, the proposed incubation medium for determining the activity of catalase in blood plasma allowed to determine the activity of catalase in blood plasma with a sufficiently high specificity. This fact was verified with the blocking of the enzyme NaN ₃ • 10 m / l with a sensitivity of up to 0.01 units. optical density (the FP-901 device allows you to measure it with an accuracy of 0.001) and with reproducibility up to 4.06%.

 Used the same incubation medium of the prototype method allowed to obtain the reproducibility of the method, equal to 16.7%.

These advantages of using the proposed incubation medium for determining plasma catalase activity were achieved by the additional introduction of NH ₄ Cl into the incubation medium, a 10-fold decrease in the concentration of phosphate buffer [4-6], and a change in the concentration of acetylacetone / methanol.

The introduction of NH ₄ Cl and the change in the concentration of the components of the incubation medium allowed us to determine the kinetics of catalase activity in blood plasma.

 The proposed incubation medium made it possible to create optimal conditions for the kinetic determination of catalase activity in blood plasma within 9 minutes, which allows us to recommend the developed incubation medium for conducting clinical laboratory studies. Currently, there is no doubt the need to simultaneously determine the intensity of lipid peroxidation processes (determination of malondialdehyde concentration, myeloperoxidase activity, superoxide dismutase, glutathione peroxidase), determination of plasma catalase activity to assess the hereditary predisposition to atherosclerosis, the possibility of early diagnosis of coronary heart disease and acute infarction myocardium, as well as a number of autoimmune diseases [6, 7].

 Currently, in clinical practice, there is no optimal incubation medium for determining the activity of catalase in blood plasma, since until recently it was believed that the activity of this enzyme in the blood and urine changes only with iron deficiency anemia, thalassemia, pernicious anemia, hematuria, protenuria, pyuria. However, at present, in connection with the solution of the most urgent tasks of preventive medicine, it has become necessary to determine the activity of catalase simultaneously with the determination of the activity of creatine phosphokinase, transaminases, antioxidant defense enzymes at different stages of coronary atherosclerosis and in the development of methods for its prevention.

 Therefore, the development of an incubation medium for determining the activity of catalase in blood plasma is urgently needed in clinical laboratory practice, since this incubation medium allows the clinical determination of catalase activity in biological objects.

LITERATURE
1. Arkhipova O. G. in the book: "Research Methods in Occupational Cardiology", 1988, 127-157.

 2. Burs R.F. SIZER L.W. BIOL. CHEM. 1952. N 195. P. 133-140.

 3. ROSCHLAN R. et al. Z. KLIN. CHEM. KLIN. BIOCHEM. 1974. N 12. P. 403.

 4. Lankin V.Z. and other Cardiology, 1980, N 7, p. 97-99.

 5. Lankin V.Z. Ter. Archive, 1985, N 5, p. 58-62.

 6. Davidenkova E.F., Shafran M.G., Veksler B.M. Wedge. Med., 1990, N 2, p. 34-38.

 7. Davidenkova E.F. et al. Clinical Medicine, 1990, N 2, p. 34-38.

 8. Prior. Free radicals in biology. - M .: Mir, 1979, volume 1, 317 p.

 9. Osipov A.N., Azizova O.A., Vladimirov V.A. Advances in biological chemistry. - M .: Nauka, 1990, Volume 31, p. 180-208.

Claims (1)

The incubation medium for determining the catalase activity in blood plasma, containing phosphate buffer solution of acetylacetone, methanol, hydrogen peroxide, detergent, characterized in that the incubation medium additionally contains ammonium chloride, and as a detergent, Triton X-100 in the following ratio of components, mmol / l :
K ₂ HPO ₄ / NaH ₂ PO ₄ - 60
Acetylacetone - 500
Methanol - 500
H ₂ O ₂ - 11.6
Triton X-100 - 0.769
NH ₄ Cl - 500s

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