RU2798309C1 - Method of assessing the level of oxidative stress in patients with burns - Google Patents

Abstract

FIELD: medicine, combustiology.

SUBSTANCE: invention can be used to assess the level of oxidative stress in patients with burns. The method of induced biochemiluminescence determines in the blood the indicators of free radical oxidation (FRO), which is taken as the S value given by the biochemiluminometer, and the total antioxidant activity (TAA), which is taken as the tg2α value given by the biochemiluminometer, for at least three days in a row. If on each of the three days FRO/OAA is 14.5 or more, the oxidative stress is assessed as critical with a predominance of free radical activity. If a value is 12.2 or less, oxidative stress is assessed as critical with a predominance of antioxidant activity. In other cases, oxidative stress is assessed as adequate.

EFFECT: method provides the possibility of reducing the time for obtaining an assessment of oxidative stress in patients with burns, increasing its efficiency and information content, due to the determination of FRO and TAA in the blood by the method of induced biochemiluminescence.

1 cl, 5 tbl, 5 ex

Description

The invention relates to medicine and can be used in the assessment of oxidative stress in patients with thermal injury.

A high risk of complications in severe burn injuries is now increasingly associated with the development of a systemic inflammatory response syndrome in victims. Thermal injury causes the release of cytokines and prostaglandins, resulting in increased interaction between leukocytes, platelets and endothelial cells. Activation of leukocytes, primarily polymorphonuclear (PMNL), leads to an increase in the formation of reactive oxygen species (ROS) and nitrogen, which, in combination with increased adhesion of PMNL to the endothelium, creates a threat of oxidative damage to its own organs and tissues. On the other hand, a sharp decrease in the radical-producing activity of PMNL can contribute to the development of sepsis. Normally, increased production of ROS is compensated by the activation of protective antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, etc.). An imbalance between the activity of the radical-producing and antioxidant systems leads to an excess of free radicals, which play the role of circulating "pathological signals", and underlies the damage to the lungs and other internal organs (Elena Vladimirovna Mikhalchik. Indicators of oxidative stress in burn injury: thesis... Doctor of Biological Sciences : 03.00.04 Moscow, 2006 234 pp. RSL OD, 71:07-3/54).

A known method for the integral assessment of oxidative stress in emergency conditions (Patent for the invention RU 2226276), based on the determination in the blood serum of indicators of free radical reactions - diene conjugates, malondialdehyde, the degree of lipid oxidation and indicators of the antioxidant system - α-tocopherol, ceruloplasmin, characterized in that that the assessment of oxidative stress is carried out according to the integral indicator K - the coefficient of oxidative stress in blood serum, determined by the formula:

where K is an integral indicator of oxidative stress;

DCi - the content of diene conjugates in the patient's blood serum;

MDAi - the content of malondialdehyde in the patient's blood serum;

COi - the degree of lipid oxidation in the patient's blood serum;

TFi - the content of α-tocopherol in the patient's blood serum;

CPi - the content of ceruloplasmin in the patient's blood serum;

DKn - the content of diene conjugates in the blood serum of a healthy person (norm);

MDAn - the content of malondialdehyde in the blood serum of a healthy person (normal);

COn is the degree of lipid oxidation in the blood serum of a healthy person (normal);

TFn - the content of α-tocopherol in the blood serum of a healthy person (normal);

CPn - the content of ceruloplasmin in the blood serum of a healthy person (normal). An increase in the K coefficient compared to the norm indicates an increase in oxidative stress and the predominance of prooxidant processes over antioxidant ones.

The above method uses a large number of indicators, operating with complex formulas, which requires a lot of time and consumption of reagents. In addition, it is not possible to carry out the assessment necessary for the prognosis of burn disease.

A known method for diagnosing oxidative stress of the human body (Patent for the invention RU 2236008), associated with the determination of the level of thiol groups in the hemolysate. The difference between the average number of thiol groups of the hemolysate of practically healthy people, equal to 0.174±0.004 optical units, and the number of thiol groups of the hemolysate of the examined person determines the number of disulfide groups. If this difference is equal to 0.000±0.008 optical units, the absence of oxidative stress is determined. If this difference is positive, the amount of intermediate and minor products of oxidative modification of biomolecules is additionally determined: proteins, lipids, carbohydrates, nucleic acids reacting with thiobarbituric acid. Additionally, the amount of biomolecule modification products is determined after preliminary chemical induction of Fe2+ peroxidation processes. The coefficient of oxidative modification of erythrocyte biomolecules is determined by the formula:

COMBER.=(TBCher.+TBCher.Ind)⋅(Unit-sh-gr.-Ei-sh-gr.)⋅100, where COMBer. - coefficient of oxidative modification of erythrocyte biomolecules in oxidative units of activity, OEA, TBCher. - quantity in erythrocytes of intermediate and minor products of oxidative modification of biomolecules: proteins, lipids, carbohydrates, nucleic acids reacting with thiobarbituric acid, in optical units, OU, TBCer. Ind - quantity in erythrocytes of intermediate and minor products of oxidative modification of biomolecules: proteins, lipids , carbohydrates, nucleic acids reacting with thiobarbituric acid, induced by Fe2+, in optical units, OE, U-sh-gr. - indicator of the average number of thiol groups of the hemolysate in practically healthy people, expressed in optical units, OU, equal to 0.174±0.004 OU, Ei-sh-gr. - the number of thiol groups of the hemolysate of the examined person, expressed in optical units, OE, 100 - the calculated coefficient. The higher the positive COMBer value, the higher the level of oxidative stress in the body.

The disadvantages of the method include:

1) taking into account the indicators of the prooxidant and antioxidant systems of free radical oxidation only at the cellular level (in erythrocytes) without attention to the plasma link, which reduces the sensitivity of this method and leads to an incorrect reflection of the level of existing oxidative stress, and, consequently, inadequate prescription of therapy aimed at eliminating it ;

2) low information content, since the studied thiol compounds are highly specific, predominantly intracellular antioxidants, and plasma antioxidants, which are essential in protecting against free radicals, are not taken into account, which does not allow us to fully evaluate all the links of the antioxidant system that lead to the development and formation of oxidative stress, therefore, the reliability of this method is reduced;

3) the duration of the assessment, due to the use of a large number of indicators, manual spectrophotometric methods of analysis, multi-stage sample preparation, the need to use additional equipment when using the luminescence method.

Since all patients with extensive burns have some degree of oxidative stress, antioxidant therapy is prescribed upon admission of the patient and the condition is monitored throughout the treatment. In this regard, it is required to obtain objective data for assessing oxidative stress during treatment.

The objective of the proposed invention is the development of a method for a comprehensive assessment of oxidative stress in thermal injury, based on the use of a minimum number of laboratory diagnostic methods for prescribing and monitoring the effectiveness of antioxidant therapy.

The technical result of the invention is to reduce the time to obtain an assessment of oxidative stress in patients with burns, increase its efficiency and information content. The technical result is achieved by the fact that the method of induced biochemiluminescence determines the indicators of free radical oxidation (FRO), for which the S value given by the biochemiluminometer is taken, and the total antioxidant activity (TAA), for which the tg2α value given by the biochemiluminometer is taken for at least three days in a row and if on each of the three days of FRO / TAA 14.5 or more, then oxidative stress is assessed as critical with a predominance of free radical activity, and at a value of 12.2 or less as critical with a predominance of antioxidant activity, in other cases oxidative stress is assessed as adequate .

The method is carried out as follows.

Blood is taken from the patient in a test tube with a 3.8% solution of sodium citrate in a ratio of 9:1 and blood plasma is obtained by the generally accepted method. Further, the activity of free radical oxidation processes and total antioxidant activity is determined in plasma using the method of induced biochemiluminescence on a biochemiluminometer BHL-06 (Nizhny Novgorod) [Piskarev I.M. et al. Study of the level of free radical processes in substrates and biological samples using induced chemiluminescence // Biophysics. - 2015. - T. 60, No. 3. - S. 496-505]. 100 µl of plasma, 400 µl of phosphate buffer (pH=7.5), 400 µl of 0.05 mM ferrous sulfate solution and 200 µl of 2% H2O2 solution are added to the cuvette of the biochemiluminometer. Measure the intensity of the glow for 30 seconds. An information card appears on the computer screen, on which the values characterizing the intensity of FRO and TAA are displayed. The following parameters of the chemiluminogram are evaluated: tg2α is an indicator that indicates the total antioxidant activity and characterizes the rate of decay of free radical oxidation processes, which is taken as FRO; S is an indicator that indicates free radical oxidation of a biological object and is equal to the chemiluminescence light sum in 30 seconds (the number of pulses in 30 seconds), which is taken as TAA.

The ratio of FRO to TAA is calculated, and if on each of three consecutive days the ratio of the free radical oxidation index to the total antioxidant activity index is 14.5 or more, oxidative stress is assessed as critical with a predominance of free radical activity, and at 12.2 or less as critical with predominance of antioxidant activity.

The method of biochemiluminescence induced by H ₂ O ₂ and Fe ²⁺ is based on the catalytic decomposition of H ₂ O ₂ by metal ions with transitional valence according to the Fenton reaction: HOOH + Fe ²⁺ → HO ^• + OH ^- + FeO ₂ ^. leads to the formation of an unstable tetroxide, which decays with the release of a quantum of light [Vladimirov Yu.A., Proskurnina E.V. Free radicals and cellular chemiluminescence // Advances in biological chemistry. - 2009. - T. 49. - S. 341-388]. The intensity of biochemiluminescence is affected by a full range of compounds that have both antioxidant, including enzymes and substances with antioxidant properties, and prooxidant action. Thus, the method makes it possible to comprehensively assess the balance of pro-/antioxidant systems and, therefore, makes it possible to identify the level of compensatory capabilities of the organism.

The causal relationship between the essential features of the invention and the achieved technical result is manifested in the choice of parameters obtained using the biochemiluminescence method FRO and OAA, which are universal indicators of oxidative stress that have an unambiguous interpretation. Only two indicators are used for evaluation, which reduces the number of reagents, reduces the time required for evaluation. Studies are carried out for three consecutive days to improve the accuracy of the assessment by eliminating random changes and measurement errors. The FRO index obtained using the method of induced biochemiluminescence makes it possible to quantify all products of oxidative modification of various nature (lipid, protein, nucleotide, carbohydrate), which are the constituent components of membranes and subcellular structures, which will allow to correctly assess the severity of the formed oxidative stress in a patient with burns . Thus, the study of the total antioxidant activity of plasma and the intensity of FRO, including the amount of oxidized-modified products during the induction of peroxidation by chemical pro-oxidants (Fe2+), will simultaneously allow us to assess the severity of oxidative stress and calculate the amount of antioxidant therapy required.

The method is illustrated with examples.

Example 1

Patient K., 43 years old, was admitted to the burn department; upon admission, the patient's condition was regarded as severe. Diagnosis: Flame burn I-II-III degree on the area of 60% of the body surface. Severe burn shock. The Frank index is 72, which indicates, according to the literature, a questionable outcome for the patient. According to the proposed method, blood was taken and on the BHL-06 biochemiluminometer (Nizhny Novgorod) the FRO and TAA values were obtained on the 1st, 2nd, 3rd days after injury and during treatment. The data obtained are shown in Table 1.

Table 1 Day SRO OAA SRO/OAA 1 12.660 0.450 28.133 2 13.027 0.477 27.310 3 11.034 0.510 21.635 4 10.911 0.547 19.947 5 10.231 0.585 17.489 6 10.321 0.602 17.145 7 10.911 0.641 17.022 8 10.513 0.658 15.977 9 10.324 0.712 14,500 10 10.302 0.740 13.922 eleven 10.315 0.739 13.958 12 10.127 0.713 14.203 13 10.104 0.728 13.879 14 10.008 0.735 13.616

As can be seen from Table 1, for the first three days, the FRO/OAA values exceeded 14.5; therefore, oxidative stress was assessed as critical with a predominance of free radical activity. The patient underwent complex treatment with the inclusion of antioxidant drugs.

According to the proposed method, blood was taken and FRO and TAA were obtained on the 4th, 5th, 6th day in the course of treatment. The ratio of FRO/TAA exceeded 14.5, but was significantly lower than the first three days.

The patient's condition remained severe, but stable, without negative dynamics.

Performed blood sampling and receiving FRO and TAA on the 11th, 12th, 13th day in the course of treatment. The FRO/TAA ratio did not exceed 14.5 and was more than 12.2, so oxidative stress was assessed as adequate. At the same time, the patient's condition is moderate, stable.

As a result of the treatment, the patient was discharged with signs of recovery on the 32nd day.

Example 2

Patient K., 30 years old, was admitted to the burn department. Diagnosis: I-II-III degree flame burn on the area of 51% of the body surface. Burn shock. Thermal injury. Corneal burn. The Frank index is 115, which, according to the literature, indicates an unfavorable prognosis for the patient. According to the proposed method, blood was taken and on the BHL-06 biochemiluminometer (Nizhny Novgorod) the FRO and TAA values were obtained on the 1st, 2nd, 3rd days after injury and during treatment. The data obtained are shown in Table 2.

table 2 Day SRO OAA SRO/OAA 1 10.14 0.547 18.537 2 10.819 0.632 17.119 3 11.032 0.583 18.923 21 10.52 0.728 14,450 22 10.416 0.755 13,800 23 9.823 0.762 12,900

As can be seen from Table 2, the values exceeded 14.5 for the first three days; therefore, oxidative stress was assessed as critical with a predominance of free radical activity.

The patient underwent complex treatment with the inclusion of antioxidant drugs.

By day 21, the patient's condition was of moderate severity with a distinct positive trend. Performed blood sampling and receiving FRO and TAA on the 21st, 22nd, 23rd day in the course of treatment. The FRO/TAA ratio did not exceed 14.5, but was more than 12.2; therefore, oxidative stress was assessed as adequate.

The patient was discharged with signs of recovery on the 67th day.

Example 3

Patient D., 38 years old, was admitted to the burn department. Diagnosis: Burn with boiling water I-II-III degree on a total area of 75% of the body surface. Burn shock. The Frank index is 105, which indicates, according to the literature, an unfavorable outcome for the patient. According to the proposed method, blood was taken and on the BHL-06 biochemiluminometer (Nizhny Novgorod) the FRO and TAA values were obtained on the 1st, 2nd, 3rd days after injury and during treatment. The data obtained are shown in Table 3.

Table 3 Day SRO OAA SRO/OAA 1 8,930 0.355 25.15493 2 35.738 1.090 32.78716 3 53.725 1.645 32.65957 4 53.615 1.674 32.02808 5 53.464 1.680 31.82381 6 53.613 1,730 30.99017 7 53.725 1.764 30.45635 8 53.050 1.235 42.95547 9 43.135 1.753 24.60639 10 42.415 1.784 23.77522 eleven 42.514 1.718 24.74622 12 41.618 1.613 25.80161 13 45.896 1.654 27.74849 14 47.669 1.662 28.68171

As can be seen from Table 3, the values exceeded 14.5 for the first three days; therefore, oxidative stress was assessed as critical with a predominance of free radical activity. In further studies, for any three consecutive days, the values significantly exceeded 14.5, which indicates critical oxidative stress with a predominance of free radical activity. These changes were observed despite the ongoing complex treatment with the inclusion of antioxidant drugs. Clinically, the patient's condition remained severe, without positive dynamics. The patient died 14 days after injury.

Example 4

Patient G., 36 years old, was admitted to the burn department. Diagnosis: I-II-III degree flame burn on the area of 51% of the body surface. Severe burn shock. The Frank index is 121, which indicates, according to the literature, an unfavorable prognosis for the patient. According to the proposed method, blood was taken and on the BHL-06 biochemiluminometer (Nizhny Novgorod) the FRO and TAA values were obtained on the 1st, 2nd, 3rd days after injury and during treatment. The data obtained are shown in Table 4.

Table 4 Day SRO OAA SRO/OAA 1 15.84 1.501 10.553 2 15.53 1.451 10.703 3 15.433 1.265 12,200 4 12.4 0.863 14.368 5 11.62 0.697 16.671 6 10.435 0.584 17.868 7 11.234 0.623 18.032 8 11.117 0.630 17.646 9 10.889 0.652 16.701 10 10.748 0.630 17.060 eleven 11.117 0.632 17,590 12 11.223 0.603 18.612 13 11,546 0.611 18.897 14 11.742 0.582 20.175

As can be seen from Table 4, all the first three days the values were 12.2 or less, therefore, oxidative stress was assessed as critical with a predominance of antioxidant activity. The patient's condition remained severe, without positive dynamics, despite adequate therapy.

The patient underwent complex treatment with the inclusion of antioxidant drugs. In further studies, for any three consecutive days, the values significantly exceeded 14.5, which indicates critical oxidative stress with a predominance of free radical activity. The patient's condition against the background of ongoing complex therapy continued to be severe, without positive dynamics.

The patient died after 1.5 months of treatment from right-sided lower lobe pneumonia.

Example 5

Patient G., aged 43, was admitted to the burn department. Diagnosis: Flame burn I-II-III degree on the area of 52% of the body surface. Thermal injury. Severe burn shock. The Frank index is 128, which indicates, according to the literature, an unfavorable prognosis for the patient. According to the proposed method, blood was taken and on the BHL-06 biochemiluminometer (Nizhny Novgorod) the FRO and TAA values were obtained on the 1st, 2nd, 3rd days after injury and during treatment. The data obtained are shown in Table 5.

Table 5 Day SRO OAA SRO/OAA 1 10.867 0.487 22.314 2 10,480 0.485 21.608 3 10.775 0.49 21,990 4 10.668 0.512 20.836 5 10,534 0.523 20.141 6 10.532 0.534 19.723 7 10,540 0.545 19.339 8 10.520 0.549 19.162 9 9.520 0.584 16.301 10 9.563 0.797 11,999 eleven 9.640 0.740 13.027 12 9.558 0.726 13.165 13 9.548 0.732 13.044 14 9.517 0.740 12.861

As can be seen from Table 5, the values exceeded 14.5 for the first three days; therefore, oxidative stress was assessed as critical with a predominance of free radical activity. Against the background of ongoing complex treatment with the inclusion of antioxidant drugs continued to assess oxidative stress by the proposed method. On the 7th, 8th, 9th day in the course of treatment, the ratio of FRO/TAA exceeded 14.5, but was significantly lower than the first three days. The patient's condition during complex intensive care continued to be severe, but stable, without negative dynamics. Starting from the 10th day, the patient's condition remained severe, stable, with positive dynamics. According to the proposed method, according to measurements for 11-13 days, oxidative stress was assessed as adequate. The patient was discharged for outpatient treatment on the 84th day.

Claims (1)

A method for assessing the level of oxidative stress in patients with burns, characterized in that the method of induced biochemiluminescence determines the free radical oxidation (FRO) in the blood, for which the S value given by the biochemiluminometer is taken, and the total antioxidant activity (TAA), for which the tg2α value is taken, issued by a biochemiluminometer for at least three consecutive days, and if on each of the three days the FRO/OAA is 14.5 or more, then oxidative stress is assessed as critical with a predominance of free radical activity, and at a value of 12.2 or less as critical with a predominance of antioxidant activity , in other cases, oxidative stress is assessed as adequate.