RU2324943C2 - Method of determination of endogenous intoxication by content of substances with low and moderate molecular mass in blood - Google Patents

Abstract

FIELD: medicine; clinical biochemistry.

SUBSTANCE: high-molecular proteins are separated from serum by boiling and following centrifuging; then the spectrophotometry of the supernatant water solution is performed at wave length from 200 to 330 nm; after that, evaluating index of content of substances with low and high molecular mass is calculated. Basing on obtained spectral values, the evaluating index of serum content of substances with low and high molecular mass (Δ) is calculated and if Δ is more than 50%, the endogenous intoxication is determined.

EFFECT: increased precision of diagnostics of endogenous intoxication and available express-diagnostics for dynamic control of therapeutic efficiency are provided.

1 dwg, 2 tbl, 4 ex

Description

The invention relates to medicine, in particular to clinical biochemistry, and is intended to determine endogenous intoxication in various diseases, accompanied by pathological protein catabolism and the development of toxic conditions. The invention can be used in laboratory diagnostics of endogenous intoxication by the content of low and medium molecular weight substances in the blood.

единиц оптической плотности, а для больных D_больн.(254 нм)≥0,136 единиц оптической плотности (патент RU №2193780, МКИ G01N 33/68, 33/48 от 27.11.2002).A known method for determining endogenous intoxication, including the dilution of 0.1 ml of blood serum 0.9 ml of physiological saline before deproteinization, protein precipitation by adding 0.5 ml of a 10% trichloroacetic acid solution to the resulting solution, followed by centrifugation at 3000 rpm for 10 minutes, diluting the obtained supernatant ten times with distilled water and measuring the optical density of the resulting solution at a wavelength of 254 nm. The presence of endogenous intoxication is judged by the value of optical density, assuming that for practically healthy people

units of optical density, and for patients D _sick. (254 nm) ≥0.136 units of optical density (patent RU No. 2193780, MKI G01N 33/68, 33/48 of 11/27/2002).

The disadvantage of this method is that the research results are affected by the dependence of the optical density of the solutions on the physicochemical properties of trichloroacetic acid preparations and its concentration that are difficult to take into account (A. Kovalevsky, O. Nifantiev, O.Y. / Laboratory work. - 1989. - No. 10. - S.35-39). In addition, the difference between the maximum value of optical density for practically healthy people and the minimum value of optical density, at which the presence of endogenous intoxication in patients is recorded, is only 0.012 units of optical density. From the practice of measuring optical density using a two-beam technique, it is known that in the ultraviolet region of the spectrum (200-300 nm), the difference in the spectral properties of the cuvettes installed in the reference and measuring channels can lead to systematic errors of up to 0.05-0.06 optical density units (Zaydel A.N., Ostrovskaya G.V., Ostrovsky Yu.I. Technique and practice of spectroscopy. // M .: Nauka, 2nd ed. - 1976. - P.88-94). As a result, the reliability of determining the presence of endogenous intoxication in this method is low.

Closest to the proposed method is a screening method for determining endogenous intoxication, which consists in determining the content of substances of low and medium molecular weight in biological fluids (Medical laboratory technology // edited by A.I. Karpishchenko, St. Petersburg, "Intermedica", 1999, v.2, C.29-31).

This method involves the precipitation of high molecular weight proteins from the test fluid with a 10% trichloroacetic acid solution, followed by centrifugation (3000 rpm, 20 minutes) and determining the light absorption coefficient of 10 times diluted supernatant. The measurement of optical density is carried out on a spectrophotometer at a wavelength of light of 254 and 280 nm against distilled water. The content of low and medium molecular weight substances in the studied liquid is characterized by the optical density value, since according to the Bouguer-Lambert-Baire law, the optical density of the solution of absorbing centers is proportional to their concentration. By the values of optical density at wavelengths of 254 and 280 nm, and the presence of endogenous intoxication is judged. This method suffers from several disadvantages:

1) The presence in the supernatant of an uncontrolled residual amount of trichloroacetic acid introduces an error in determining the concentration of substances of low and medium molecular weight when measured at a wavelength of 254 nm against distilled water.

2) The condition of proportionality between the optical density and the concentration of absorbing centers (Bouguer-Lambert-Baer law) is fulfilled only for a single-component solution. In a multicomponent system, such as blood serum, the proportionality between the optical density and the concentration of substances of low and medium molecular weight depends not only on their total content, but also on the chromophore composition of substances of low and medium molecular weight. Therefore, in some diseases, the presence of endogenous intoxication does not lead to a noticeable change in the optical density of the supernatant at wavelengths of 254 or 280 nm.

3) Substances of low and medium molecular weight consist of oligopeptides with a molecular weight of 500-6000 daltons and small molecules with a mass of 150-300 daltons. Therefore, the chromophore fragments of substances of low and medium molecular weight are mainly amino acid residues. Therefore, the absorption spectrum of the supernatant in the ultraviolet region will be determined by the sum of the absorption spectra of amino acid residues. Of the 20 amino acids involved in the formation of proteins and peptides, only three contain an aromatic radical - tryptophan, tyrosine, phenylalanine, so most of the chromophores that make up low and medium molecular weight substances intensively absorb light in the range from 200 to 230 nm (region peptide bond absorption) and weakly in the range of 250-300 nm (Dawson R., Elliot V., Elliot W., Jones K. // Biochemist Handbook, M., Mir, 1991, pp. 15-65). Since trichloroacetic acid intensively absorbs light precisely in the range of 200-250 nm, the known method does not allow the most informative region of the spectrum from 200 to 230 nm to be used for diagnostics.

4) A significant drawback of the known method is the fact that as an estimated indicator of the content of substances of low and medium molecular weight use the value of optical density. The value of optical density depends not only on the concentration of substances of low and medium molecular weight, but also on the measurement conditions (thickness and quality of the used cuvettes, spectral resolution, error and dynamic range of the used spectrophotometer), which affects the results of determination of endogenous intoxication.

The objective of the invention is to obtain a new technical result, which consists in increasing the sensitivity and reliability of the method for determining endogenous intoxication by eliminating the main disadvantages of the known method, namely by creating conditions for expanding the range of spectrophotometry towards short wavelengths up to 200 nm with the inclusion of the most informative region from 200 to 230 nm, characteristic for peptide communication.

The problem is solved in that the blood serum is freed from high molecular weight proteins by boiling, followed by centrifugation, then spectrophotometry of the aqueous solution of the supernatant is performed in the wavelength range from 200 to 330 nm, after which the estimated content of low and medium molecular weight substances is calculated by the formula:

where D _x (λ) is the optical density spectrum of the supernatant solution obtained from a blood serum sample;

- средний спектр оптической плотности, полученный путем усреднения индивидуальных спектров поглощения растворов супернатанта из сыворотки крови здоровых доноров;

- the average spectrum of optical density obtained by averaging individual absorption spectra of supernatant solutions from the blood serum of healthy donors;

λ is the wavelength of light.

The value of the estimated indicator Δ determines the presence of endogenous intoxication in the patient.

In particular, when determining endogenous intoxication in patients with acute destructive pancreatitis, blood serum is boiled for 3 minutes at a temperature of 100 ° C and centrifuged for 5 minutes at 6000 rpm, then the supernatant is diluted with distilled water and spectrophotometric in the wavelength range from 200 to 230 nm, after which an estimated indicator of the content of substances of low and medium molecular weight is calculated by the formula:

and when the value of the estimated indicator Δ more than 50% determine the presence of endogenous intoxication in a patient with acute destructive pancreatitis.

In contrast to the known method, in the inventive method, the precipitation of high molecular weight proteins is carried out by boiling, followed by centrifugation, and not by treating blood serum with trichloroacetic acid, followed by centrifugation.

The absence of trace amounts of trichloroacetic acid in the supernatant allows us to expand the spectrophotometry range from 250-330 to 200-330 nm.

Replacing the method of deproteinization of blood serum eliminates the influence of the concentration and physico-chemical properties of trichloroacetic acid preparations that are difficult to take into account on the optical density of supernatant solutions.

As a result, the accuracy of determining the content of low and medium molecular weight substances in blood serum increases.

In order to determine the efficiency of high molecular weight protein precipitation by boiling blood serum, we studied the supernatant from the blood serum of a donor and a patient with destructive pancreatitis using high performance liquid chromatography. From the chromatogram, where the molecular mass is indicated along the X axis and the peak intensity (see the drawing) along the Y axis, it can be seen that both in the supernatant from the blood serum of the donor (1) and in the supernatant from the blood serum of the patient with destructive pancreatitis (2) , the content of the fraction of substances with a molecular weight of less than 10,000 daltons is from 60 to 80%. The results obtained indicate a high efficiency of the applied protein deposition technique.

An important difference of the method is the introduction of a new estimated indicator Δ of the content of substances of low and medium molecular weight in the blood of the patient. As an estimated indicator in the claimed method, a relative excess of the concentration of low and medium molecular weight substances in the supernatant solution from the blood serum sample under study is used over the average concentration of low and medium molecular weight substances in the supernatant solution from the blood serum of healthy donors. To calculate Δ by the present method, the integral value of the optical density in the spectral range corresponding to the absorption of the main chromophores of substances of low and medium molecular weight is used.

The wavelength integration interval is selected based on the optical density of the supernatant in available spectrophotometry and the most informative range for a particular disease. In the known method, the content of low and medium molecular weight substances in the blood is characterized by the optical density at a fixed wavelength in the absorption spectrum of the supernatant solution from a blood serum sample. In accordance with the Bouguer-Lambert-Baer law and the property of additivity of the absorption spectra, the optical density of the solution will depend on the measurement conditions (thickness and spectral properties of the cuvettes) and the chromophore composition of the main components of the low and medium molecular weight substances included in the pool:

where ε _j (λ) is the molar extinction coefficient of the jth chromophore;

C _j is the molar concentration of the jth chromophore;

l is the thickness of the cell.

From the above expression it follows that in the known method, the conclusion about the presence of endogenous intoxication is influenced by the measurement conditions and the type of disease.

In the inventive method, the use of the estimated indicator Δ, which is calculated using the integral values of optical density, eliminates the influence of the measurement conditions on the conclusion about the presence of endogenous intoxication. In addition, the inventive method of calculating the estimated indicator Δ is less dependent on the type of disease.

The validity of this statement follows from the fact that the expression for calculating the estimated indicator of the content of substances of low and medium molecular weight can be converted to a form that is completely independent of the properties of the applied cuvettes and the wavelength of the measurement of optical density:

- интегральный коэффициент экстинкции j-го хромофора в исследуемом образце;Where

- integral extinction coefficient of the jth chromophore in the test sample;

- интегральный коэффициент экстинкции j-го хромофора в составе веществ низкой и средней молекулярной массы у здоровых доноров;

- integral extinction coefficient of the jth chromophore in the composition of substances of low and medium molecular weight in healthy donors;

- концентрация веществ низкой и средней молекулярной массы в исследуемом образце;

- the concentration of substances of low and medium molecular weight in the test sample;

- средняя концентрация веществ низкой и средней молекулярной массы в супернатанте из крови здоровых доноров.

- the average concentration of low and medium molecular weight substances in the supernatant from the blood of healthy donors.

и

Thus, the use of integral values of optical density allows us to determine the percentage of excess of the content of substances of low and medium molecular weight up to an insignificant difference in the chromophore composition of the pool of substances of low and medium molecular weight (if the chromophore composition varies slightly, then

and

The claimed method is not obvious to specialists working in this field. The method for determining endogenous intoxication by the content of low and medium molecular weight substances in the blood has been known for a long time, but none of the specialists in this field used the thermal method to precipitate large molecular weight proteins of blood serum. The apparent simplicity of such a solution actually required lengthy experiments due to the high responsibility of obtaining significant results for diagnosis and treatment.

The non-obviousness of the method is associated with the point of view that it is necessary to precipitate large molecular proteins of blood serum exclusively inorganic acids. We first applied the method of deproteinization, which allowed us to eliminate errors associated with the content of inorganic acids in the experimental samples. The value of this new approach is convincingly justified by high performance liquid chromatography. The absence of the remainder of inorganic acids in the experimental samples allows the most informative region of the spectrum of 200-230 nm, typical for peptide bonds of low molecular weight substances, to be used in the diagnosis of endogenous intoxication.

For the first time in the claimed method, we applied a new quantitative indicator Δ, which we calculated taking into account the integral values of the optical density of the blood serum supernatant. For the first time, the influence of measurement conditions on the conclusion about the presence of endogenous intoxication was eliminated. In addition, the inventive method of calculating the estimated indicator Δ is less dependent on the type of disease.

The proposed method is characterized by increased diagnostic accuracy, as well as stability and comparability of the research results, which is extremely important for the treatment of this group of patients. Endogenous intoxication is the main criterion for determining the prognosis, severity and outcome of the disease, as well as the choice of extracorporeal detoxification methods.

The found technical solution was the result of lengthy searches and experiments and, to some extent, was unexpected for us.

Currently, the claimed method is a method of choice in determining substances of low and medium molecular weight for the diagnosis of endogenous intoxication, in particular in patients with a surgical profile.

The invention can be illustrated by the following examples.

Example 1

15 healthy donors were examined, the results were compared with the survey data of 15 patients with various surgical diseases associated with the presence of endogenous intoxication syndrome (appendicitis, acute pancreatitis, purulent infection of soft tissues).

The determination of the degree of endogenous intoxication was determined by the claimed method No. 1 as follows.

Tubes containing 1.0 ml of blood serum of 15 patients and 15 healthy donors were placed in a water bath and kept for 3 minutes at a temperature of 100 ° C. Then 4 ml of distilled water was added to the tubes, the samples were thoroughly mixed and centrifuged for 5 minutes at 6000 rpm. 0.5 ml of the obtained supernatant was added to clean tubes containing 7 ml of distilled water and mixed thoroughly. The supernatant solutions were poured into quartz cuvettes and the absorption spectra were measured in the range from 200 to 330 nm against distilled water, with a measurement step of 4 nm. For each sample, the integrated absorption in the wavelength range from 200 to 330 nm and the estimated content of substances of low and medium molecular weight were calculated by the formula

где

Where

A comparative analysis of the student’s criteria in table 1 indicates that the ratio of the difference between the average values of the estimated indicators of endogenous intoxication Δ, determined by the claimed method for a group of patients and donors, to the standard error of the difference of these indicators exceeds 12.2, which indicates a high degree of reliability differences (99.99%) of the average estimated indicators of endogenous intoxication Δ between the group of patients and the group of donors with a significance level of p <0.0001.

A similar comparison for the same blood samples in terms of endogenous intoxication, determined by a known method at a wavelength of 254 and 280 nm, showed that the ratio of the difference between the average values of optical densities for groups of patients and donors determined at a wavelength of 254 and 280 nm by a known method, the standard error of the difference between these indicators was 1.2 and 1.4, respectively. The results obtained indicate that the significance of the difference between the optical densities for the group of patients and healthy donors determined by the known method does not exceed 76 and 83% with a significance level of p 0.24 and 0.17, respectively.

Thus, a comparison of student criteria for the two methods showed that the determination of endogenous intoxication by the present method is superior to the known method in the significance of differences between healthy and sick groups. The probability of error in determining endogenous intoxication in a patient by the present method is 0.01%. The known method does not provide such reliability.

Example 2

Patient B-ev N.A., 50 years old. He entered the surgical department of the City Clinical Hospital No. 13 with complaints of pain in the right hypochondrium, vomiting, fever up to 38 ° C.

On examination: the patient is pale, lethargic, low nutrition. The tongue is dry, coated with a white coating. Heart rate 120 beats per minute, respiratory rate 22 per minute. The abdomen is symmetrical, does not participate in breathing, is sharply painful in the right hypochondrium during palpation, is tense, the symptoms of peritoneal irritation are sharply positive, intestinal motility is sharply weakened. After additional instrumental studies, the diagnosis was made: Acute calculous obstructive cholecystitis. Perivesical abscess. Endogenous intoxication was determined by the level of substances of low and medium weight by method No. 2 as follows. 5 ml of blood was taken from the cubital vein, centrifuged at 6000 rpm for 5 minutes to separate the serum from the shaped elements. Then 1 ml of serum was placed in a test tube and heated in a water bath to 100 ° for 3 minutes. After that, 4 ml of distilled water was added to the tube with the denatured protein and the glass rod was thoroughly mixed, then the tube for sedimentation was centrifuged at 6000 rpm for 5 minutes. After that, 0.5 ml of clarified supernatant was taken and 7 ml of distilled water was added. Then, the optical density of the solution was determined on a spectrophotometer in the range of 200-230 nm, with a measurement step of 2 nm. To determine the level of endogenous intoxication, an estimated indicator of the content of substances of low and medium molecular weight was calculated by the formula

где

Where

Thus, the level of endogenous intoxication in this patient was 89%, which indicated the moderate severity of endogenous intoxication.

Standard clinical and laboratory examination data confirmed the presence of severe endogenous intoxication. Blood test: red blood cells 4.7 × 10 ¹² , hemoglobin 98 g / l, white blood cells 21.1 × 10 ⁹ , eosinophils - 1, stab neutrophils - 41, segmented neutrophils - 40, lymphocytes - 10, monocytes - 8; ESR - 28 mm / h.

Example 3

Patient O-va M.M., 67 years old, was treated in the intensive care unit of Clinical Hospital No. 13 for: Mixed pancreatic necrosis with retroperitoneal tissue damage on both sides. Diffuse enzymatic peritonitis.

The patient, upon admission, carried out, along with a general examination, the determination of endogenous intoxication by the claimed method as described in Example 2. The level of endogenous intoxication, which was determined by the claimed method, was 241%.

Given the degree of endogenous intoxication of the patient, it was decided to add extracorporeal detoxification to the ongoing complex therapy.

On the second day, the patient had a plasma sorption session. The initial level of endogenous intoxication, determined by the claimed method, decreased from 241% to 133%.

As can be seen from the example, the proposed method allows not only to assess the severity of endogenous intoxication, but also to control the effectiveness of extracorporeal detoxification methods.

Example 4

30 healthy donors were examined, the results are compared with the survey data of 30 patients with necrotic pancreatitis.

The determination of the degree of endogenous intoxication was determined by the claimed method as described in example 2.

For each sample, an estimated indicator of the content of substances of low and medium molecular weight was calculated by the formula:

где

Where

The results obtained in table 2 indicate that the difference between the minimum value of the estimated endogenous intoxication Δ for patients with necrotic pancreatitis and the maximum value of the estimated endogenous intoxication Δ for a healthy person is 14 times greater than the measurement error of the present method.

A comparison of the minimum Δ value for patients and the maximum Δ value for donors to measurement error indicates a high diagnostic reliability and sensitivity of the proposed method for determining endogenous intoxication by the content of low and medium molecular weight substances in blood serum.

The inventive method is of great socio-economic importance, since it significantly improves the diagnosis of endogenous intoxication in acute surgical diseases and increases the reliability of the results, which is very important in the treatment of this category of patients.

Table 1
Comparison of student criteria for developed and known methods The inventive method Known method No. healthy sick healthy sick healthy sick Δ _healthy ,% Δ _patients ,% D is _healthy (254 nm) D _sick (254 nm) D is _healthy (280 nm) D _sick (280 nm) one 12.0 93.9 0.20 0.42 0.39 0.61 2 6.1 79.6 0.15 0.32 0.33 0.51 3 11,2 113.6 0.24 0.39 0.31 0.61 four -11.6 68.6 0.17 0.32 0.30 0.46 5 2.6 75,2 0.15 0.39 0.33 0.50 6 -13.9 94.4 0.13 0.38 0.25 0.54 7 -5.1 64.8 0.13 0.20 0.27 0.37 8 -0.9 61.2 0.22 0.28 0.38 0.36 9 -6.4 53.8 0.27 0.27 0.28 0.44 10 9,4 80.1 0.24 0.24 0.33 0.42 eleven -6.7 57.8 0.18 0.40 0.29 0.47 12 13,2 48.6 0.35 0.24 0.39 0.38 13 -16.3 91.8 0.11 0.48 0.28 0.56 fourteen 7.6 63.9 0.22 0.38 0.40 0.46 fifteen -1.1 34.2 0.19 0.18 0.34 0.32 0.01 ± 9.8% 72.1 ± 20.7% 0.2 ± 0.06 units 0.33 ± 0.09 units 0.32 ± 0.05 units 0.48 ± 0.09 units

p≤0.0001 p = 0.24 p = 0.17

table 2
Comparison of the minimum value of Δ for patients and the maximum value of Δ for donors to measurement error Sample number Healthy donors Sample number Sick NP Δ _healthy ± δΔ _healthy % Δ _sick. ± δΔ _sick. % one -11 ± 3.0 one 109 ± 2.1 2 -12 ± 3.0 2 152 ± 2.0 3 -14 ± 3.0 3 90 ± 2.1 four -11 ± 3.0 four 110 ± 2.1 5 -7 ± 2.9 5 247 ± 1.8 6 -1 ± 2.8 6 63 ± 2,3 7 -7 ± 2.9 7 75 ± 2.2 8 9 ± 2.7 8 135 ± 2.0 9 0 ± 2.8 9 206 ± 1.9 10 1 ± 2.8 10 142 ± 2.0 eleven 6 ± 2.7 eleven 134 ± 2.0 12 -7 ± 2.9 12 101 ± 2.1 13 -7 ± 2.9 13 75 ± 2.2 fourteen 25 ± 2.5 fourteen 132 ± 2.0 fifteen 6 ± 2.7 fifteen 148 ± 2.0 16 7 ± 2.7 16 113 ± 2.1 17 -3 ± 2.8 17 74 ± 2.2 eighteen 21 ± 2.6 eighteen 132 ± 2.0 19 22 ± 2.6 19 97 ± 2.1 twenty 22 ± 2.6 twenty 114 ± 2.1 21 4 ± 2.8 21 199 ± 1.9 22 1 ± 2.8 22 134 ± 2.0 23 -5 ± 2.9 23 97 ± 2.1 24 -4 ± 2.9 24 110 ± 2.1 25 2 ± 2.8 25 60 ± 2.5 26 -11 ± 3.0 26 195 ± 1.9 27 -5 ± 2.9 27 184 ± 1.9 28 -5 ± 2.9 28 141 ± 2.0 29th 2 ± 2.8 29th 129 ± 2.0 thirty -11 ± 3.0 thirty 103 ± 2.1

Claims (2)

1. A method for determining the presence of endogenous intoxication by the content of low and medium molecular weight substances in the blood, characterized in that the blood serum is freed from high molecular weight proteins by boiling, followed by centrifugation, then spectrophotometry of the aqueous supernatant solution is performed in the wavelength range from 200 to 330 nm, after what is the calculation of the estimated indicator of the content of substances of low and medium molecular weight according to the formula:

where D _x (λ) is the optical density spectrum of the supernatant solution obtained from a blood serum sample;

- the average spectrum of optical density obtained by averaging individual absorption spectra of supernatant solutions from the blood serum of healthy donors; λ is the wavelength of light, and with a Δ value of more than 50%, the presence of endogenous intoxication is determined. 2. The method according to claim 1, characterized in that the boiling of blood serum is carried out for 3 minutes at a temperature of 100 ° C and centrifuged for 5 minutes at 6000 rpm, then the supernatant is diluted with distilled water.