RU2659145C1 - Method for determining systemic metabolic disorders - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to biochemistry. Method for determining systemic metabolic disturbances is as follows: take a blood sample, centrifuge, precipitate high-molecular compounds with acetonitrile, determine the optical density of metabolic products in the supernatant at wavelengths of 210, 220 and 230 nm, effective concentration of albumin is determined by fluorometric method, after which the value of albumin index is established according to the formula:

, where AI is albumin index; ECA – effective concentration of albumin; ε_pool – total content of the indices of optical densities of metabolic products at wavelengths of 210, 220 and 230 nm, and with albumin index of 9.01±0.5, establish the absence of systemic metabolic disturbances.

EFFECT: method for determining systemic metabolic disorders is proposed.

1 cl, 3 ex, 1 tbl

Description

The present invention relates to medicine, namely to biochemistry, and can be used to determine the presence or absence of a systemic metabolic disorder - a systemic inflammatory response syndrome, the basis of which is the accumulation in biological fluids of the body above physical norms as end products of metabolism, due to the delay in their elimination , and intermediate - in connection with the violation of metabolic processes.

A known method for determining the presence or absence of systemic metabolic disorders by establishing the integral coefficient of endogenous intoxication, which is calculated by the formula:

, где:

where:

CEI - coefficient of endogenous intoxication

- оптическая плотность веществ средней молекулярной массы при 254 нм у больных;

- optical density of substances of average molecular weight at 254 nm in patients;

- оптическая плотность веществ средней молекулярной массы при 254 нм у клинически здоровых людей;

- optical density of substances of medium molecular weight at 254 nm in clinically healthy people;

- оптическая плотность веществ средней молекулярной массы при 280 у больных;

- optical density of substances of average molecular weight at 280 in patients;

- оптическая плотность веществ средней молекулярной массы при 280 нм у клинически здоровых людей;

- optical density of substances of medium molecular weight at 280 nm in clinically healthy people;

MDA ^P - content of malondialdehyde in patients;

MDA ^N is the content of malondialdehyde in patients with clinically healthy people.

In this case, the optical density of medium molecular weight substances is preliminarily determined at 254 nm and at 280 nm, and the content of malondialdehyde in blood serum is determined by spectrophotometric method (Criterion for evaluating endogenous intoxication in patients with pelvic peritonitis. S.M. Matveev, B.V. Davydov, Yu.O. Teselkin, E. V. Klychnikova, O. B. Shakhova, Clinical Laboratory Diagnostics. - 2009. - No. 4. - p. 15-16).

A value of CEI greater than 1.14 indicates the presence of a metabolic disorder, and a value of CEI less than 1.14 indicates the normalization of metabolic processes.

A significant disadvantage of this method should include its lack of information, because the determination of medium-molecular compounds is carried out only at two wavelengths - 254 nm and 280 nm, which does not allow to assess the presence of other substances contained in the metabolic pool and reflecting the clinical picture of the disease.

At the same time, the determination of malondialdehyde gives a general idea of the state of peroxidation without taking into account the more reactive products formed during this process - diene, ketodiene and other peroxidation products.

Therefore, the indicators used by the authors of the known method do not fully evaluate the systemic metabolic disorders.

The closest in technical essence to the proposed is a method for determining systemic metabolic disorders by establishing the value of the index of intoxication, which reflects the content of toxic products of low and medium molecular weight in the blood serum (Malakhova M.Ya. Method for recording endogenous intoxication: A manual for doctors. St. Petersburg: MAPO, 1995.- p. 7-12).

The known method is as follows. A blood sample is taken from the ulnar vein on an empty stomach, the plasma from the formed elements is separated by centrifugation. High molecular weight plasma compounds are precipitated with a 15% solution of trichloroacetic acid. Determination of the optical density of the products of metabolism of low and medium molecular weight in the supernatant is carried out in the ultraviolet region in the wavelength range from 210 to 300 nm. The calculation of the amount of low and medium molecular weight substances is carried out according to the formula:

,

,

Where:

- сумма оптических плотностей в условных единицах (у.е.),

- the sum of optical densities in arbitrary units (cu),

ε ₂₁₀ -ε ₃₀₀ - the value of optical density at a given wavelength.

The known method also determines the optical density of the catabolic pool in the wavelength range from 238 to 258 nm, the value of which is: ε _cat . _Pool = ε ₂₃₈ + ε ₂₄₂ + ε ₂₄₆ + ε ₂₅₀ + ε ₂₅₄ + ε ₂₅₈ .

The authors of the known method found that normally the catabolic pool does not exceed 10-15% of the total metabolic pool and is determined by them as an intoxication index (II). In pathological conditions, AI exceeds 20% or more, which characterizes the severity of the clinical condition of the patient.

The disadvantages of this method, the authors of the proposed technical solution include the use of a 15% solution of TCA, because this precipitator distorts the spectral characteristics of the supernatant due to the absorption properties of TCA in the ultraviolet region.

The disadvantages of this method include the fact that when determining the index of intoxication toxic products in the wavelength range of 210, 220 and 230 nm are not taken into account, which, as you know, include peptide-like compounds and compounds with unsaturated saturated bonds, which are the main fractions catabolic pool.

The objective of the proposed technical solution is to develop a method for determining systemic metabolic disorders associated with the main fraction of substances absorbing in the region of 210-230 nm, and constituting a catabolic pool.

The technical result of the proposed method is to increase the accuracy of determining systemic metabolic disorders by determining the toxic substances of the catabolic pool in the ultraviolet region of the spectrum.

The technical result is achieved by the fact that the method for determining systemic metabolic disorders includes taking a blood sample, centrifuging it, precipitating high molecular weight compounds from blood plasma and determining in the supernatant the optical density of low and medium molecular weight metabolic products. The total content of these products determines the presence or absence of metabolic disorders.

The differences of the proposed method from the known one is that high molecular weight compounds are precipitated with acetonitrile, the optical density of metabolic products is determined at wavelengths of 210, 220 and 230 nm, and the effective concentration of albumin is determined by the fluorimetric method. Then set the value of the albumin index according to the formula:

, где

where

AI - albumin index;

ECA is the effective concentration of albumin;

ε _pool - the total content of indicators of optical densities of metabolic products at wavelengths of 210, 220 and 230 nm.

When the albumin index is 9.01 ± 0.5, the absence of systemic metabolic disorders is established, a decrease in the value of this indicator indicates their presence.

A comparative analysis with the prototype showed that the proposed method differs from the known above methods and, therefore, meets the criteria of the invention of "novelty."

From the analysis of patent and specialized literature it was found that the proposed method has features that distinguish it not only from the prototype, but also from other technical solutions in this and related fields of biochemistry and medicine. In the available literature, the authors of the proposed technical solution have not identified a method for determining systemic metabolic disorders by the above methods using indicators that reflect both the accumulation of toxic metabolic products in the blood and their disposal.

A distinctive technique of the proposed method is the precipitation of high molecular weight compounds with acetonitrile, which is an aprotic neutral reagent, which allowed the removal of spectra in the wavelength range of 210-230 nm. The main compounds that determine the composition of the pool in the wavelength range of 210-230 nm, identified by the authors of the proposed method by chemical methods - membrane ultrafiltration; extraction; chromatography: thin layer, liquid, high performance thin layer; IR spectroscopy They made it possible to establish the predominant content of highly reactive compounds — peptide-like substances, polyunsaturated fatty acids (PUFAs), arachidonic acid, and cholesterol.

It is known that, in many pathological conditions, increased binding of PUFA, phospholipids, unesterified fatty acids, and cholesterol to albumin is noted (Serum albumin in clinical practice. Edited by Yu.A. Gryzunov, G.E. Dobretsova. M: IRIUS, - 1994 - S. 123-125). Therefore, the main compounds of this pool bind to the active centers of albumin and undergo further changes. Therefore, the authors determine the definition of ECA as an important factor involved in the provision and maintenance of physiological metabolic processes.

The authors of the proposed method, based on the studies, it was found that in clinically healthy people the albumin index is 9.01 ± 0.5 and indicates the absence of systemic metabolic disorders. The authors also found that a decrease in the AI index indicates the presence of systemic metabolic disorders and characterizes the severity of the patient's clinical condition.

The above allows us to conclude that the technical solution meets the criterion of "inventive step".

The method comprising the claimed invention is intended for use in medicine, namely in biochemistry and physiology. The possibility of its implementation is confirmed by the methods and means described in the application, therefore, the proposed solution meets the criterion of "industrial applicability".

The inventive method is as follows.

In the morning (on an empty stomach) the patient takes blood from the cubital vein into a test tube containing a 3.8% solution of sodium citrate. Blood cells are separated by centrifugation. To 1 ml of plasma add 1 ml of acetonitrile (grade OCH), mix, centrifuged for 30 minutes at 3000 rpm. 0.5 ml of plasma supernatant (9: 1 ratio) is added to 4.5 ml of distilled water and the spectrogram is taken on an SF-2000 spectrophotometer at wavelengths of 210 (λ ₂₁₀ ), 220 (λ ₂₂₀ ) and 230 (λ ₂₃₀ ) nm. The result is expressed in units of optical density (cu). The total content of the metabolic pool is determined by the formula:

.

.

The effective concentration of albumin (ECA) is determined by the fluorimetric method on an AKL-01 Zond device (Russia) with a set of reagents for it (Yu.A. Gryzunov. Measurement of the parameters of ECA and OCA on the AKL-01 analyzer. // Serum albumin in clinical medicine.Edited by Yu.A. Gryzunov, G.E. Dobretsov. - M .: GEOTAR, 1998. - pp. 104-107). After determining the value of the total metabolic pool and ECA, the albumin index value is established by the formula:

, где

where

AI - albumin index;

ECA is the effective concentration of albumin;

ε _pool - the total content of indicators of optical densities of metabolic products at wavelengths of 210, 220 and 230 nm.

When the albumin index is 9.01 ± 0.5, the absence of systemic metabolic disorders is established.

The proposed method is illustrated by examples of specific performance.

Example No. 1. Patient A., diagnosis: disseminated purulent peritonitis. In the morning, this patient (on an empty stomach) had a blood sample taken from the ulnar vein into a test tube containing a 3.8% sodium citrate solution. Blood plasma from the formed elements was separated by centrifugation at 15,000 rpm. To 1 ml of the obtained plasma, 1 ml of acetonitrile (grade OCh) was added, mixed and centrifuged at 3000 rpm for 30 minutes. Then, 0.5 ml of plasma supernatant (9: 1 ratio) was added to 4.5 ml of distilled water and the spectrogram was recorded on a spectrophotometer at wavelengths of 210 (λ ₂₁₀ ), 220 (λ ₂₂₀ ), 230 (λ ₂₃₀ ) nm. The result is expressed in units of optical density (cu)

UV spectra of patient A. upon admission:

at a wavelength of 210 nm (λ ₂₁₀ ) - 2.8 cu;

at a wavelength of 220 nm (λ ₂₂₀ ) - 2.2 cu;

at a wavelength of 230 nm (λ ₂₃₀ ) - 1.9 cu;

The determination of the total content of the metabolic pool was carried out according to the formula:

The determination of the effective concentration of albumin (ECA) was carried out on an AKL-01 Zond device (Russia) with a set of reagents for it. ECA = 22.

был рассчитан альбуминовый индекс:Then according to the formula

the albumin index was calculated:

.

.

The AI in patient A at the time of admission was 2.8 times less than in clinically healthy people, which indicated systemic metabolic disorders and characterized the severity of the clinical condition of this patient.

The severity of peritonitis in patient A. was confirmed by a modified Manheimer index (MPI), which was 15 points and confirmed the extremely serious condition of this patient (A. V. Shumov. Staged abdominal sanitation in the treatment of postoperative disseminated purulent peritonitis. Abstract. Cand. Medical Sciences. - Irkutsk. - 1991. - S. 12-13).

Surgery was performed, which included an inspection of the abdomen, relaparotomy, radical elimination of the source of peritonitis, debridement and drainage of the abdominal cavity. On day 5, the patient's condition improved, AI was 4.20; MPI - 9 points. Those. determination of the albumin index made it possible to quickly conduct laboratory monitoring of the severity of the clinical condition of the patient and confirm the effectiveness of the therapy.

Example No. 2. Patient K. Diagnosis: iron deficiency anemia. The total content of the optical density indices of metabolic products at wavelengths of 210, 220, and 230 nm was 5.02 cu; ECA - 29.4; AI - 5.85. The value of the albumin index in this patient was reduced by 1.5 times compared with clinically healthy people, which indicated the severity of the clinical condition and indicated an excessive accumulation of toxic products, i.e. about systemic metabolic disorders.

Example No. 3. Patient V., diagnosis: CHD, angina pectoris of tension and rest, 3rd functional class. Hospitalized in the cardiology department of OKB.

On admission ε _210-230 was 5.94 cu .; ECA - 34.2; AI - 5.75.

When the extract was 4.96 ε _210-230 cu .; ECA - 38.1; AI - 7.68.

Using the proposed method showed that the products of perverted metabolism accumulated in the blood serum of patient B. The albumin index was reduced by 1.56 times compared with clinically healthy people, which indicated the severity of the clinical condition and required not only medical treatment, but also the use of efferent methods.

In total, the authors of the proposed method examined 120 patients undergoing treatment in different departments of the OKB, of which 40 patients with advanced purulent peritonitis, with pancreatic necrosis - 20, with coronary disease - 40 and 20 - hematological patients. A group of clinically healthy people were 20 people.

The obtained data on the values of the albumin index, the effective concentration of albumin and the total content of the metabolic pool in the examined patients are presented in the table below.

The implementation of the proposed method allowed to evaluate the clinical condition of the examined patients.

The authors of the proposed method, based on the studies, it was found that in clinically healthy people the albumin index is 9.01 ± 0.5 and indicates the absence of systemic metabolic disorders. The authors also found that a decrease in the AI index indicates the presence of systemic metabolic disorders and characterizes the severity of the patient's clinical condition.

Thus, the proposed method for determining systemic metabolic disorders by the size of the albumin index allows us to assess the severity of the clinical condition of the patient, the effectiveness of the treatment and to predict the outcome of the disease. The albumin index is a simple, affordable and objective diagnostic laboratory criterion.

Claims (5)

A method for determining systemic metabolic disorders, including blood sampling, centrifugation, precipitation of high molecular weight compounds from blood plasma, determination in the supernatant of the optical density of low and medium molecular weight metabolic products, the total content of which determines the presence or absence of metabolic disorders, characterized in that high molecular weight compounds precipitated with acetonitrile, the determination of the optical density of metabolic products is carried out at wavelengths 210, 220 and 230 nm, and the effective concentration of albumin is determined by the fluorimetric method, after which the value of the albumin index is determined by the formula:

AI - albumin index; ECA is the effective concentration of albumin; ε _pool - the total content of the optical density indices of metabolic products at wavelengths of 210, 220, and 230 nm, and when the albumin index is 9.01 ± 0.5, the absence of systemic metabolic disorders is established.