RU2618447C2 - Method of assessing severity of intoxication - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to laboratory diagnostics in medicine of emergency conditions and experimental medicine, and can be used for assessing severity of any origin in patients and laboratory animals. Method: at first protein concentration is measured in sample of liquid biological material of human or animal, then sample is incubated in boiling water bath for 3 minutes, concentration of protein in heated sample is measured again, and reduction of concentration of protein in sample after heating by more than 25 times shows absence of intoxication, reduction by 25–21 times shows mild intoxication, reduction by 20–16 times shows moderate intoxication, and reduction of concentration of protein by less than 16 times shows severe intoxication.

EFFECT: invention provides simplifying and improving flexibility of intoxication severity evaluation procedure as in patients with suspected intoxication syndrome, so in laboratory animals at stimulating of endogenous or exogenous intoxication.

1 cl, 1 tbl, 7 ex

Description

The invention relates to medicine, namely to laboratory diagnostics in emergency medicine and experimental medicine, and can be used to assess the severity of any etiology in patients and laboratory animals.

Intoxication syndrome is concomitant in most diseases and pathological conditions and determining its degree is of great clinical importance (Grachev S.V., Prokhorenko I.R., Prokhorenko S.V. Role of blood proteins in the initial stage of the pathogenesis of endotoxin shock // Molecular Medicine. - 2004. - No. 1. - P. 20-25; Titov V.N. Exogenous and endogenous pathological factors (pathogens) as the cause of inflammation // Wedge, laboratory diagnostics - 2004. - No. 5. - P. 3-10 ; Afanasyev A.N., Odintsova I.N., Udut V.V. Syndromes of endogenous intoxication and systemic of the response: generality and differences // Anesthesiol. and resuscitation. - 2007, - No. 4. - P. 67-71; Churlyaev Yu.A.; Grigoryev EV; Reinik V.Ya. Method for assessing the severity of endogenous syndrome intoxication in critically ill patients // RF Patent No. 2187810 of 05.22.2001). The timeliness of identifying the severity of endogenous intoxication syndrome determines the set, intensity and effectiveness of methods of active detoxification of the body.

In laboratory practice, for the diagnosis of endotoxemia, plasma studies of medium-weight molecules or peptides (MSM) are widely used. It was established that these compounds have a different structure, and their molecular weight is 500-5000 daltons. The increase in the optical density of the supernatant at a wavelength of 254 nm after the precipitation of serum proteins by trichloroacetic acid (TCA) reflects the level of small pathogens formed in vivo during uncontrolled proteolysis (Gabrielyan N.I., Dmitriev A.A., Sevastyanova O.A. et al. Medium molecules and the level of endogenous intoxication in resuscitation patients // Anesthesiol. And Resuscitation. - 1985. - No. 1. - P. 36-38; Karyakina EV, Belova SV. Medium-weight molecules as an integral indicator of metabolic disorders (literature review ) // Wedge, laboratory. Diagnostics. - 2 004. - No. 3. - S. 3-8; Malakhova M.Ya. Method for registering endogenous intoxication: A manual for doctors. - St. Petersburg, 1995. - 100 p.).

The disadvantages of the known methods for assessing the severity of endogenous intoxication syndrome, based on a study in plasma of molecules or peptides of medium weight (MSM), are:

1) multi-stage and time-consuming, which makes it impossible to use the method for rapid analysis;

2) the technique is not automated and all manipulations are carried out manually, which entails errors in the results associated with the individual professional qualities of the laboratory assistant;

3) the inability to use portable equipment and, as a consequence, the inapplicability for mass and monitoring surveys.

For assessing the degree of intoxication of any etiology for more than 70 years, keen attention of doctors of all specialties has been drawn by calculated indices reflecting the ratio of different classes of leukocytes in the composition of peripheral blood (Kalf-Kalif Ya.Ya. On the leukocyte intoxication index and its practical value // Doctor. - 1941. - No. 1. - S. 31-35). Some of them, such as leukocyte intoxication index (LII) of Kalf-Kalif, LII Ostrovsky, LII Khimich, LII Yabluchansky, nuclear index Dashtayants G.A. (YaID) characterize the degree of intoxication (Abakumov M.M., Borovkova N.V., Aleksandrova I.V., Rei S.I., Khvatov V.B. Method for assessing the severity of endogenous intoxication // RF Patent No. 2357248 of 11.12 .2007; Kidalov V.N .; Lysak V.F .; Makeev B.L. Method for assessing the severity of intoxication of an organism // RF Patent No. 2082971 from 05.13.1993). Others indicate an imbalance in the immune system (Ostrovsky V.K., Mashchenko A.V., Yangolenko D.V., Makarov S.V. Blood counts and leukocyte intoxication index in assessing the severity and determining the prognosis for inflammatory, purulent and purulent destructive diseases // Clin. laboratory. diagnostics. - 2006. - No. 6. - P. 50-53).

The disadvantages of methods based on leukocyte calculated indices are:

1) expensive, since they require a detailed blood test and trained personnel;

2) not applicable for blood diseases;

3) their information content drops sharply in the presence of immune disorders in patients and during immunostimulating therapy;

4) do not reflect the state of the body (and, therefore, intoxication) with exogenous toxicosis;

5) the information content of LII as a hematological index largely depends on the time of the disease and characterizes the presence of an inflammatory process rather than intoxication. Therefore, it must be used in conjunction with other laboratory and instrumental studies, be sure to consider clinical and medical history;

6) with a prolonged inflammatory process and severe intoxication of the body, it can decrease (ie, approach the "norm"), thereby disorienting the doctor.

It is known that with severe intoxication syndrome of endogenous and exogenous etiology, free radical processes intensify and reactive oxygen species (ROS) appear in excess, which leads to peroxidation of substrates and the appearance of various aggressive factors of an endogenous nature in the intercellular environment and then in the blood, initiating oxidative stress (Pankin V.Z., Tikhase A.K., Belenkov Yu.N. Free-radical processes in normal and pathological conditions: A manual for doctors. - M.: RKNPK MZ RF, - 2001. - 78 p.; Zenko NK, Lankin VZ, Menshikov EB Oxidative stress biochemical and pathophysiological aspects -.. M .: IAPC Nauka - 2001 - 343).. On the principle of determining the products of lipid peroxidation (lipid peroxidation), modified ROS proteins, there are methods for assessing intoxication syndrome (Golikov P.P., Nikolaeva N.Yu., Gavrilenko I.A., Matveev SB, Davydov B.V., Marchenko V.V., Smirnov S.V., Lebedev V.V., Golikov A.P. Nitric oxide and lipid peroxidation as factors of endogenous intoxication in emergency conditions // Patol. Physiol. And experimental therapy. - 2000. - No. 2. - S. 6-9).

The disadvantage of these methods is the very complex relationship between the degree of intoxication and the level of oxidative stress, which includes not only the processes of peroxidation, but also the compensatory activation of antioxidant systems that neutralize the physiological excess of the formed ROS (antioxidants, ROS acceptors, glutathione peroxidase enzymes, superoxide dismutase) and do not allow between intoxication syndromes and oxidative stress.

Other disadvantages of the method include the complexity of the analysis, the use of reagents from the list of precursors of the Federal Drug Control Service of the Russian Federation.

The most modern methods for diagnosing the degree of endogenous and exogenous intoxication is a method for determining the binding capacity of albumin in blood serum by adding a fluorescent substance interacting with albumin to the serum, followed by measuring the amount of the obtained product on a fluorimeter and comparing its level with the control serum (Moroz V.V. , Kravchenko-Berezhnaya N.R., Meshcheryakov G.N., Zaks I.O., Radaev S.M. Effective albumin concentration - a marker of endotoxemia in severe mechanical trauma // Anesthesiol reanimatol. - 2000. - No. 6. - P. 54-57; Gryzunov Yu.A., Syromyatnikova E.D., Ilyashenko K.K. Prognostic value of albumin fluorescence test in assessing the outcome of acute poisoning with psychotropic drugs. Clinical laboratory Diagnostics, 2004, No. 11, pp. 39-42). In this method, the total (OKA) and effective albumin concentration (ECA) are determined and the albumin binding capacity reserve (PCCA) is calculated, it is the toxicity index (IT): IT = (OKA / ECA) - 1, which reflects the degree of albumin binding of toxic substances in blood serum (Gryzunov Yu.A., Grinberg A.A., Stupin V.A., Rodoman G.V., Musselius S.G., Fedorovsky N.M., Dobretsov G.E., Chernysh T.I. , Shalaeva T.I., Par V.I., Vasina N.V., Syromyatnikova E.D., Naumov E.K. Informational content of the indicator "effective albumin concentration" with peritonitis e: data from a multicenter study // Anesthesiol. and resuscitation. - 2003. - No. 6. - S. 32-35).

This method is increasingly used in large clinics, however, the need for special expensive equipment (fluorimeters) and fluorescent probes are the main disadvantage and obstacle to the widespread use of this method of assessing intoxication.

Closest to the proposed one is a method for determining the degree of intoxication based on measuring the binding capacity of albumin in blood serum by adding to the centrifuged serum diluted 120-2000 times the fluorescent substance N-carboxyphenylimide dimethylaminonaphthalic acid (K-35), selectively interacting with albumin, followed by measurement the amount of the obtained product on the fluorimeter and comparing its level with the control serum (Miller Yu.I., Dobretsov G.E., Krasovitsky B.M., Kornilova L.I., Erm Olenko I.G. A method for determining the binding capacity of albumin in blood serum // Auth. certificate No. 1681266 of 07.25.1989).

The disadvantages of the prototype are:

1) the need for special expensive equipment and fluorescent probes;

2) the high cost and inaccessibility of fluorimeters necessary for the practical implementation of this method;

3) dependence on suppliers of fluorescent probes;

4) attachment to the equipment of a general clinical laboratory to determine the total concentration of albumin;

5) the need for highly qualified personnel of various specialties;

6) the relatively high cost of analysis;

7) the limitations of the method only to blood serum and the inability to use the method to determine the binding capacity of albumin in whole blood, hemolyzed serum, saliva and other biological fluids.

The proposed method for determining the severity of intoxication is based on measuring the degree of reduction of thermal denaturation of transport proteins adsorbing toxins.

The invention is aimed at simplifying and increasing the universality of the procedure for assessing the severity of intoxication both in patients with suspected intoxication syndrome and in laboratory animals when modeling endogenous or exogenous intoxication.

The specified technical result is achieved by initially measuring the protein concentration in the sample of liquid biological material (hereinafter referred to as the biomaterial) of a human or animal, then the sample is incubated in a boiling water bath for 3 minutes, the protein concentration in the heated sample is re-determined, and when the protein concentration is reduced in after warming the sample more than 25 times, it is estimated as the absence of intoxication, with a decrease of 25-21 times - as mild intoxication, with a decrease of 20-16 times - as moderate intoxication, and with is lowered protein concentration of less than 16 times - as a severe intoxication.

As a liquid biomaterial according to the claimed method, you can use whole blood, blood serum, blood plasma, oral fluid, peritoneal fluid, ejaculate, cerebrospinal fluid.

The proposed method is based on our ability to withstand some tissue and plasma proteins to withstand incubation in a boiling water bath for 3 minutes.

A detailed analysis of the phenomenon of thermal stability of a protein complex with toxins showed that an increase in their resistance to boiling is directly related to intermolecular interactions of low molecular weight ligand substances and medium-weight molecules with macromolecules of whey adsorbent proteins. In addition, preliminary experiments showed that the degree of adsorption of endo- and exotoxins by proteins and the coefficient of thermal stability of whey protein are dependent on the severity of intoxication syndrome (table).

Determination of protein concentration in the sample can be carried out using any available biochemical (Bradford; Lowry) or immunochemical method (radial immunodiffusion according to Mancini's agar, immunoturbidimetry or "rocket" immunoelectrophoresis according to Laurel using commercial antibodies to human serum proteins).

If blood or serum is used as the analyzed biomaterial, preliminary dilution of the sample by 5-10 times is required, which is necessary for the prevention of gelation and for the analysis of the liquid fraction without flakes of denatured protein. In this case, the protein concentration in the diluted blood sample (serum) is measured before and after incubation in a boiling water bath. If oral fluid is used as the biomaterial, exudate or transudate does not require preliminary dilution of the sample.

The thermal treatment of a biomaterial sample in a boiling water bath is explained by the ease of maintaining this temperature parameter in any laboratory (water bath). The use of lower temperatures (for example, thermostatic 56 ° C and 65 ° C) does not lead to massive thermal denaturation of the protein, and the degree of protein denaturation under these conditions does not correlate with the severity of the intoxication syndrome.

Incubation of a biomaterial sample in a boiling water bath for 3 minutes was selected empirically and is minimal and sufficient for the manifestation of the effect of protein thermal stability. When reducing the incubation time in a boiling water bath, the samples do not have time to warm up and the bulk of the protein does not have time to go through the stage of thermal denaturation. On the contrary, increasing the incubation time in a boiling water bath against the declared one increases the percentage of protein denaturation and requires the calculation of new correction factors for the degree of protein denaturation corresponding to different degrees of intoxication severity.

The presence of hemoglobin and red blood cells in blood and serum increases the concentration of protein in the sample before incubation in a boiling water bath and does not distort the coefficients of the degree of protein denaturation corresponding to various degrees of intoxication.

The essence of the invention is illustrated in the table.

The table shows the results of determining the protein concentration (g / l) according to the Lowry method in individual blood sera of patients with various pathologies, women with physiological pregnancy and pregnancy complicated by gestosis (toxicosis), and a healthy male donor before and after incubation in a boiling water bath in within 3 minutes. In addition to serums, the table shows the results of determining the concentration of protein in hemolyzed blood of a donor, blood serum of a rabbit and rat and three samples of saliva. The third column presents the ratio of protein concentrations, which we called the degree of decrease in protein concentration, which is the quotient of dividing the result in the first column by the result in the second column.

For an objective characterization of the degree of intoxication in patients with various pathologies presented in the table, for each of them in the fourth column are two indicators of intoxication - leukocyte intoxication index (LII) and the level of medium-weight molecules (MSM).

The proposed method has been successfully tested at the departments of biological chemistry, pathological physiology, faculty surgery, urology, obstetrics and gynecology, faculty of medicine, anesthesiology and intensive care of the Astrakhan State Medical University during 2004-2015, in the Department of Pregnancy Pathology of the Astrakhan City Clinical Maternity Hospital, Traumatology Department , Department of hemodialysis and the department of resuscitation and intensive care of NUZ "MSCh" in Astrakhan in 75 patients.

Below are the results of testing.

Example 1. For donor No. 6714, the protein concentration in the blood serum sample measured by the Lowry method was 77.8 g / l (table), after incubating a 5-fold diluted sample of this blood serum in a boiling water bath for 3 minutes, the protein concentration in the sample decreased to 0.48 g / l, which, in terms of undiluted donor serum after heating, amounted to 2.4 g / l (table). The decrease in protein concentration in the sample after heating was 77.8 / 2.4 = 32.4 times (table), which characterizes serum as biomaterial from a patient with no intoxication.

In the blood serum of the same donor after precipitation of proteins with trichloroacetic acid (TCA) by measuring the optical density of the supernatant at a wavelength of 254 nm, the concentration of medium-weight molecules (MSM) was determined, which was 0.102 opt. units (table), which also corresponds to the norm and the absence of intoxication (Gabrielyan N.I., Dmitriev A.A., Sevastyanova O.A. et al. Medium molecules and the level of endogenous intoxication in resuscitation patients // Anestesiol. and resuscitation. - 1985. - No. 1. - S. 36-38).

A smear was prepared from the blood of the same donor and a stained blood sample was examined under a microscope with manual counting of the leukocyte formula and differentiated counting of young forms of neutrophils. The leukogram of the same blood sample: leukocytes 4.5 × 10 ⁹ / l, neutrophils 49% (segments 47%, rods 2%, young 0%), lymphocytes 40%, basophils 1%, monocytes 8%, eosinophils 2%, myelocytes and plasmocytes 0%. LII calculated by the Kalf-Caliph formula

составил 0,35.

amounted to 0.35.

In order to compare with the clinical data given in the table, the Kalf-Kalifa LII was also calculated on the basis of the data of the hematological analyzer using the modified formula of A. L. Kostyuchenko. et al. (Nazarenko G.I., Kishkun A.L. Clinical evaluation of the results of laboratory studies. - M .: Medicine, 2000. - 544 p.):

The authors of the modified method found that the rate of LII is from 0.5 to 1.5. The result of LII donor 0.43 (table). The diagnosis of LII: there is no intoxication in the donor.

All three models of intoxication showed the same result: donor toxicity is absent.

Example 2. Patient D., 39 years old, was hospitalized in the burn department of the Non-governmental healthcare institution "Medical Unit" of Gazprom Dobycha Astrakhan LLC on June 13, 2009 with a diagnosis of Flame burn II-III Art. back, back surfaces of both lower extremities, burn area 35%, burn shock. Since June 14, 2009, clinical signs of burn toxemia, oliguria. In the blood of June 17, 2009, leukocytosis (up to 11 × 10 ⁹ / L) was noted, the Kalf-Kalif intoxication index was 16.5 (table), which indicates severe intoxication. In the blood serum, the content of medium-mass molecules was 0.890 opt. units (table), which also corresponds to severe intoxication.

Incubation of a 5-fold diluted blood serum sample of this patient in a boiling water bath and subsequent calculations were carried out as in example 1. Against the background of massive infusion-transfusion therapy, the concentration of protein in the blood serum after incubation in a boiling water bath decreased only 14.7 times ( table), which confirms the diagnosis of a severe degree of endogenous intoxication (burn toxemia).

Example 3. Pregnant S., 27 years old, first one-pregnancy, 32 weeks, was hospitalized in the pathology department of the Alexander Mariinsky Regional Clinical Hospital with gestosis with clinical symptoms of preeclampsia. Incubation of the diluted blood serum of this patient in a boiling water bath and subsequent calculations were carried out, as in example 1. All three indicators of intoxication LII 8.54, MSM 0.620 opt. units and the degree of decrease in protein concentration after incubation in a boiling water bath by 19.8 times (table) consistently indicate moderate intoxication.

Example 4. Patient I., in a state of intoxication of moderate degree, delivered by an ambulance team from a public place to the toxicology department of GBUZ AO GKB No. 3 named after Kirov with a diagnosis of alcohol-toxic coma two hours after admission, all three indicators of intoxication LII 3.58, MSM 0.218 opt. units and the degree of decrease in protein concentration after incubation in a boiling water bath by 20.7 times (table) indicate mild intoxication.

Example 5. In the blood serum of laboratory animals (Chinchilla rabbit, white Wistar rat) contained in vivarium cells of Astrakhan State Medical University and not previously used in scientific experiments, the protein concentration before incubation in a boiling water bath corresponds to physiological norms for this group of laboratory animals, and the degree of decrease in concentration after incubation in a boiling water bath of sera (table) corresponds to the control values for donors - 29.7 times and 29.0 times accordingly (diagnosis: intoxication is absent).

Example 6. In a healthy one-year-old baby B., who has 4 incisors, when examining the gums without signs of teething of the following milk teeth, according to the mother during the last month of healthy, in the collected saliva, the protein concentration after incubation in a boiling water bath decreased by 28 times ( table), which corresponds to the diagnosis: lack of intoxication. Incubation of the oral fluid of the child in a boiling water bath and subsequent calculations were carried out as in example 1, but the oral fluid was not diluted before heating.

Example 7. Patient X., 45 years old, turned to a private dental clinic for right-sided flux for three days. On examination, the general condition of moderate severity, diagnosed with odontogenic purulent periostitis, intoxication syndrome. Before opening the abscess, a study of saliva and blood was performed on intoxication indicators: LII 4.37, MSM 0.420 opt. units and the degree of decrease in protein concentration after incubation in a boiling water bath in saliva by 19.1 times (table), and in the blood - by 17.8 times, which indicates moderate intoxication. Incubation of the oral fluid of this patient in a boiling water bath and subsequent calculations were carried out, as in example 6.

A parallelism was found in the results of the assessment of intoxication syndrome in saliva and blood, despite the lower protein content in the oral fluid compared to blood serum (tenfold).

The proposed method achieves the following positive effect:

- technical simplicity, insignificant labor costs (for the practical execution of the method, 1 specialist is enough);

- there is no need for protein purification, stages of filtration and centrifugation and sample processing with reagents;

- the ability to use for the implementation of the method in addition to blood any other biological fluid (serum, oral fluid, cerebrospinal fluid, extract, exudate, transudate, etc.);

- efficiency (the method does not require exclusive and expensive equipment and reagents, auxiliary equipment and highly qualified medical staff);

- the versatility of the method (determination of protein concentration can be made in any available way);

- the availability of reagents necessary for the practical implementation of this method;

- the possibility of express modification of the test;

- the possibility of non-invasive modification (saliva);

- the possibility of widespread use of the method in animals in experiments on modeling intoxication.

The proposed method achieves simplification and increasing the versatility of the procedure for assessing the severity of intoxication.

The implementation of this method does not require significant costs, will improve the performance of determining the degree of intoxication in humans or experimental animals.

The proposed method can be used both for scientific experiments and in clinical practice as an additional test to diagnose the severity of exogenous and endogenous intoxication syndrome.

Claims (1)

A method for assessing the severity of intoxication, consisting in a biochemical analysis of biological material, characterized in that the protein concentration in the sample of the liquid biological material of a person or animal is initially measured, then the sample is incubated in a boiling water bath for 3 minutes, the protein concentration in the heated sample is re-determined and with a decrease in the concentration of protein in the sample after heating more than 25 times, it is estimated as the absence of intoxication, with a decrease of 25-21 times - as intoxication of a mild degree, with lower enii in 20-16 times - as the average degree of toxicity, while reducing protein concentration less than 16 times - both severe intoxication.