Method of estimating general oxidant status of organism

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to internal diseases, diagnostics. Method is based on determination of general oxidant activity (GOA) and general antioxidant activity (GAA) with further determination of oxidant index (OI), which equals ratio of GOA to GAA. In accordance with the invention oxidant activity is determined by degree of echinochrome A oxidation by components of oxidant system of blood serum or plasma, and general antioxidant activity is determined by degree of echinochrome A oxidation with chloramine B, added to blood serum or plasma. OI index of healthy donor is taken as a unit. OI value higher than a unit testifies to disbalance of general oxidant status of organism. Method ensures up to 20 fold reduction of amount of analysed serum or plasma in comparison with standard methodology, reduction for carrying out analysis from 72 hours to 2 hours. When ranging it is possible to perform analysis of more than 500 samples during one day, using microboards and microplate spectrophotometers.

EFFECT: method is simple in implementation, economical and does not require large volume of thermostatically controlled chambers.

1 tbl, 2 ex

 

The invention relates to medicine, namely to internal diseases, diagnosis.

In the normal process of lipid peroxidation (LPO) occurs in living systems are balanced, is kept at the optimal stationary level, due to the presence of a protective system, representing a hierarchy of antioxidant systems (Zenkov NICHOLAS, Lankin old Testament Oxidative stress. Biochemical and pathophysiological aspects. - M.: Nauka / Interperiodica, 2001. - 490 C.). The first line of defense form antioxidant enzymes, each of which inactivates one of the reactive oxygen species (ROS): O2(SOD), H2O2(catalase), ROOH (glutathione peroxidase). In normal conditions of life, with the functioning of living systems in terms of physiological optimum, there are Pro - and antioxidant balance, which is the most important mechanism of oxidative homeostasis. Balance is a mobile character that is a balanced action of oppositely directed processes and is characterized by oscillatory mode of functioning within the limits compatible with life, and maintaining homeostasis. Any kind of damage to the structures of living systems caused by exogenous and endogenous agents, inevitably accompanied by activation of the FLOOR, shift the Pro - and antioxidant balance.

Intensification of free radical processes in the tissues may be due to hyperproduction ROS and free radicals and/or scarcity of natural antioxidants and reduce the activity of other protective systems of cells, including antioxidant enzymes. Similar to the physiological state of cells, combined with disruption of the normal regulation of free radical reactions in the literature is called "oxidative stress" (Baraboi, VA, etc. // Peroxidation and stress. - SPb.: Science, 1992. - 148 C.; Zenkov NICHOLAS, Lankin old Testament Oxidative stress. Biochemical and pathophysiological aspects. - M.: Nauka / Interperiodica, 2001. - 490 C.). At the present time believe that oxidative stress is a universal mechanism of cell damage, leading to the development of various pathological conditions, including programmed cell death - apoptosis.

Most appropriate for the characteristics of the diseases, the development of which play an important role violations of regulation of free radical processes, it seems the term "free radical diseases" (Lankin old Testament Atherosclerosis as an example the free-radical pathology / bioantioxidants. - Tyumen, 1997. - P.51-53). Examples of free radical pathology is atherosclerosis, coronary heart disease (CHD) and other diseases of the cardiovascular system which we inflammation, diseases of the gastrointestinal tract, ulcerative colitis (UC), Crohn's disease, chronic pancreatitis, pulmonary disease, and many others.

In the pathogenesis of CHD important role of disturbance processes the FLOOR. At the transition of stable angina voltage in a stable form and later in myocardial infarction observed increased severity of violations FLOOR and the reduced function of the antioxidant system. Research shows that patients suffering from angina increased content of lipoperoxides in the blood, whereas the activity utilizing lipoperoxide enzyme GSH-peroxidase, however, significantly reduced compared with healthy people. In patients with unstable angina content increased lipoperoxide and reduced activity of GSH-peroxidase in the blood is more pronounced than in patients with stable angina (A.P. Golikov and other lipid Peroxidation in ischemic heart disease. // Human physiology. - Volume 23, No. 6. - 1997. 49 - 56).

In the literature there are data about the participation of GENDER in the onset and progression of chronic inflammatory bowel disease (UC) (Suprun E.V. Mill obuslovleno-antioxidant homeostasis in hvorih on Granny colt. // Problems iï ï genetics i KLN. iiï. K.-Luhansk-Kharkiv, 2002. - VIP. - Pp.96-100).

An important mechanism of action of circulating immune complexes when UC is their ability to damage cell membranes, which leads to oxidative stress (Patiashvili L.M. Occurrence of secondary immune deficiency and its role in chronic inflammatory bowel disease. Suchasna gastroenterology, №2(8), R. 2002, p.16-17). With positions peroxide homeostasis noted violations, ensuring the progression of endotoxicosis, as well as potentiation of immunopathological process in tissues. Thus, the intensification of free radical oxidation in the background of depression of the antioxidant system supports local pathological process, facilitates the circulation and exchange processes in the tissues of the affected organ, which ultimately can lead to the degradation of the tissues.

There is a standard method of determining the oxidative status of the organism, based on the determination of total oxidant activity (AOA) and total antioxidant activity (OAA) [Lperational and other State peroxidation in patients with gastric ulcer and duodenal ulcer. Clinical laboratory diagnostics, No. 6, 1998, p.10-14].

OOA determined by the accumulation in the model system the end product of peroxidation - malondialdehyde (MDA). As substrate using tween-80, and as the initiator of the blood plasma. Reagents: 1% solution of tween-80; 40% solution of trichloroacetic acid (THU); 0.25% solution thiobarbituric acid (TBA). In a tightly close the container in dark glasses of 250 ml make 2 ml of tween-80. In a test sample is added 0.2 ml of blood plasma and in the control sample is added an appropriate volume of distilled water. Next, the samples incubated at 40°C for 48 hours After each sample add 1 ml of THU and leave at room temperature for 60 minutes and Then centrifuged at 8000 rpm for 15 min. the Supernatant (2 ml) is mixed with 2 ml of TBA and boil for 15 minutes At this TBA reacts with MDA education trimethylboron complex, it has a pink color. The sample is cooled and measure the extinction at 532 nm against distilled water. The calculation is conducted according to the formula: OOA (%) =(EO-EC)/SW*100%, where EO and EC - extinction, respectively, experimental and control samples.

JAA is determined by the degree of inhibition forum-escortinbrisbane oxidation of tween-80 to MDA. Reagents: 1% aqueous solution of tween-80; 1 mm aqueous solution of ferrous sulfate iron (FeSO4); 10 mm aqueous solution of ascorbic acid; 40% THU; 0.25% aqueous solution of TBA. As described above, make 2 ml of tween-80, 0.2 ml of a solution of ferrous sulfate, 0.2 ml ascorbic acid, 0.1 misiorski, in the control sample, the corresponding amount of distilled water. The mixture is incubated for 48 h at 40°C, then add 1 ml of THU and treated samples, as described above. The calculation is conducted according to the formula: JAA (%) =(EC-EO)/EC*100%, where EC and EO - extinction, respectively, of control and test samples.

For judgments about the imbalance of antioxidant and oxidant systems determine the oxidative index (OI). Calculation formula: OI=OOA/JAA.

The disadvantages of this method of assessing the overall oxidative status of the organism is its duration (72 hours), the need of spending significant amounts of the analyzed serum or blood plasma.

Technical result provided by the invention is to reduce up to 20 times the number of test sera or plasma in comparison with the standard method, to reduce analysis time from 72 hours to 2 hours. When zooming, you can analyze more than 500 samples per day using microplates and microplate spectrophotometers. The method is simple, economical and does not require a large amount of cooled cameras.

The technical result is achieved in that in the method of estimating the total oxidant status of the organism by determining the total oxidant activity and total antioxidant activity with subsequent determination of hydroxy is based index (OI), according to the invention a General oxidant activity is determined by the oxidation States of echinochrome And components oxidant system serum or blood plasma, and total antioxidant activity determined by the degree of oxidation of echinochrome And by bleach, added to serum or plasma.

The proposed method estimates the total oxidant status is based on the determination of the total oxidant and total antioxidant activity of the serum or plasma of blood of warm-blooded organisms with lower specific optical density indicators - dye solutions are unstable to oxidation.

When determining the total oxidant activity (AOA) is used as an indicator dye, such as echinochrome And oxidizable components oxidant system of warm-blooded organisms.

Echinochrome And - pigment flat sea urchin Scaphechinus mirabilis, which is used as the active substance drugs series "Histogram" [RU 2352554 C1, 20.04.2009].

Total antioxidant activity (OAA) is determined by the degree of destruction of an oxidant selected for this indicator dye, such as destruction of bleach In, recruited echinochrome A.

Figure OI healthy donors is taken as a unit. The value of RI is greater than one indicates the General imbalance OK adantage status of the organism.

The invention is illustrated by the following examples:

Example 1.

A) Determining the total oxidant activity (AOA).

Reagents: PBS (10 mm phosphate, 150 mm NaCl, pH to 7.6), 50 γ/ml PBS echinochrome A. On the tablet transfer 50 ál of serum or plasma (in a control sample 50 ál PBS), 100 μl of PBS, 50 μl of a solution of echinochrome and measure the extinction at 475 nm. The mixture is then incubated for 30 min at 37°C and repeat spectrophotometrically. Calculated by the formula: OOA (%)=(E1-E2) /*100%, where E1and E2- extinction of the samples in the first and second measurements, K=0,27, while the absorption of the test sample at the first measurement is 0,427.

B) Determination of the total antioxidant activity (OAA).

Reagents: 30 γ/ml dH2O chloramine In PBS (10 mm sodium phosphate, 150 mm NaCl, pH to 7.6), 50 γ/ml PBS echinochrome A. On the tablet transfer 50 ál of serum or plasma (in a control sample of 50 ál PBS), 50 μl of chloramine In (in 2 control sample 50 ál PBS). The mixture is incubated for 30 min at 37°C, then add 50 μl of PBS, 50 μl of echinochrome and measure the extinction at 475 and 700 nm. The mixture is then incubated for 30 min at 37°C and repeat spectrophotometrically. Calculated by the formula: JAA (%)=(Eo-Emin)/(Emax-Emin)*100%, where EmaxEminEo=E2-E1- extinction, respectively, of control and test samples.

Example 2. About what the definition of oxidative status in patients with ischemic heart disease and ulcerative colitis.

We studied 8 patients with angina 2 FC, 2 patients with myocardial infarction (mi) and 10 with ulcerative colitis (UC). The control group consisted of 6 healthy persons.

The estimate of the total oxidant status was performed using 2 methods - known and offer. The results of the study are shown in table. For a more convenient presentation of results the values of AOA and JAA expressed in conventional units, taking values in healthy donors per unit.

In patients with UC and with THEM not observed significant differences of OOA and JAA compared with the control group using the methods of the prototype, whereas in patients with angina 2 FC show increased levels of OOA to 1.43±1,14 and reduction of OAA to 0.76±0,33.

When using the proposed method of assessing the overall oxidative status of the organism there is an increase in the level of OOA and reduction of OAA in the study groups compared with the control group. Moreover, in the group of patients with their level of AOA higher than in the group of patients with angina 2 FC, and the level of the TAA is 2 times lower.

For judgments about the imbalance of antioxidant and oxidant systems were determined oxidant index (OI). Calculation formula: OI=OOA/JAA.

In groups of patients with UC and with angina 2 FC when used the standard method of determination of total oxidant status OI amounted to 1.05±3,71 and a 1.88±1,48, respectively, in the group of patients with THEM significant differences were not found. Using the proposed method, there is an increase of OI in all study groups compared to the control, especially in the group of patients with THEM (or above 3 times and is 3,15). The table presents the results from the prototype method and the proposed method are not fully congruent. This is due to the fact that the standard method to determine the final product (MDA), whereas the proposed method allows to determine the whole complex of high and low molecular weight compounds (malonic dialdehyde, superoxide dismutase, catalase, and so on).

1,00±0,21
Group study patientsAAAJAAJOI
The placeholderThe proposed methodThe placeholderThe proposed methodThe placeholderThe proposed method
Healthy1,00±0,611,00±0,211,00±0,121,00±0,041,00±0,56
UC0,90±0,551,36±0,310,86±0,320,85±0,061,05±3,711,60±0,40
Angina 2 FC1,43±1,141,07±0,110 7b±0,330,93±0,021,88±1,481,16±0,13
THEM0,97±0,241,32±0,060,90±0,210,42±0,011,07±0,533,15±0,22

Method of assessment total oxidant status of the organism by determining the total oxidant activity and total antioxidant activity with subsequent determination of oxidative index, characterized in that the total oxidant activity is determined by the oxidation States of echinochrome And components oxidant system serum or blood plasma, and total antioxidant activity determined by the degree of oxidation of echinochrome And by bleach, added to serum or plasma.



 

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