RU2792739C1 - Method for early diagnosis of alcoholic liver disease in patients with alcohol dependence syndrome - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to clinical laboratory diagnostics, and can be used for early diagnosis of alcoholic liver disease in patients with alcohol dependence syndrome. Catalase activity in the blood serum of a patient is determined photometrically, followed by recalculation of catalase activity in mkat/l according to the formula: E=(Abl - Aopt)×V×t×K×100, where E is the activity of catalase in mkat/l; A - optical density of blank and experimental samples; V is the volume of the introduced sample, 0.1 ml; t - incubation time, 10 min; K - coefficient of millimolar extinction of hydrogen peroxide, equal to 22.2×10³ mm^-1×cm^-1. If the value of catalase activity is more than 37 mkat/l, alcoholic liver disease is diagnosed in patients with alcohol dependence syndrome. With a value of E less than or equal to 37 mkat/l - the absence of alcoholic liver disease.

EFFECT: method provides the possibility of reducing trauma, no risk of complications, accessibility, ease of execution with high sensitivity, specificity and efficiency by determining the activity of catalase in blood serum by photometric method.

1 cl, 4 ex

Description

SUBSTANCE: invention relates to medicine, namely to laboratory diagnostics and internal diseases, and can be used for early diagnosis of alcoholic liver disease in patients with alcohol dependence syndrome.

Known laboratory method for the diagnosis of alcoholic liver disease, based on intravital morphological study of liver biopsy specimens (Ivashkin V.T., Maevskaya M.V., Pavlov Ch.S., Sivolap Yu.P., Lunkov V.D., Zharkova M.S. ., Maslennikov R.V. Clinical guidelines of the Russian Society for the Study of the Liver, on the management of adult patients with alcoholic liver disease. Russian Journal of Gastroenterology, Hepatology, Coloproctology. 2017; 27(6): 20-40). Disadvantages of the method: laboriousness, invasiveness, the method requires special conditions, there is a technical difficulty in obtaining material and a high risk of severe complications during and after the manipulation, covers only 0.2% of the hepatic parenchyma, low information content about the distribution of infiltrates, assessment of the histological structure of organs quite long in time, the procedure is subjectively unpleasant.

EFFECT: reduced trauma, no risk of complications, accessibility, ease of execution with high sensitivity, specificity and efficiency.

This result is achieved by determining the activity of catalase in the blood serum of the examined patient with alcohol dependence syndrome when performing early diagnosis of alcoholic liver disease.

The method is carried out as follows: in a patient on an empty stomach, venous blood is taken from the cubital vein in the amount of 5 ml into a vacuum tube with a coagulation activator. Then centrifuge for 10 min at 1500 rpm to obtain serum. The activity of the enzyme of the antioxidant system of catalase in the blood serum was determined by the method of Korolyuk M.A. photometrically on KFK-3 (photoelectric photometer, Russia) at a wavelength of 410 nm (Korolyuk M.A., Ivanova L.I., Mayorova I.G., Tokarev V.E. Method for determining catalase activity // Laboratory business. No. 1. S. 16-19).

The method for determining the activity of the catalase enzyme is based on the ability of hydrogen peroxide (H ₂ O ₂ ) to form a stable colored complex with molybdenum salts. Catalase activity was determined by the transformation of the substrate (hydrogen peroxide) by the enzyme, which is capable of forming a colored complex with ammonium molybdate salts. Extinction is recorded at 410 nm. A unit of catalase activity was taken as the amount of enzyme involved in the conversion of 1 mkat of H ₂ O ₂ in 1 second under given conditions. Catalase activity was calculated per 1 liter of blood serum.

Required reagents:

1. 0.03% hydrogen peroxide (H2O2) solution designed to react with an enzyme as a substrate.

2. 4% solution of ammonium molybdate (manufacturer LLC "Biotech", Russia).

Definition progress. Experimental sample - 2 ml of 0.03% solution of H ₂ O ₂ +0.1 ml of serum. A blank sample - instead of serum, 0.1 ml of distilled water was added. To correct the results, a control sample was prepared - instead of peroxide, 2.0 ml of distilled water was added. The samples were incubated for 10 minutes at 37°C in a thermostat. The reaction was then stopped by adding 1.0 ml of a 4% ammonium molybdate solution. After that, the samples were centrifuged for 10 minutes at 3000 rpm. The optical density of blank and experimental samples was measured against the control on KFK-3 at a wavelength of 410 nm. After that, the catalase activity was calculated according to the formula:

E \u003d (Ahol - Aopt) ^× V ^× t ^× K ^× 100, where

E - catalase activity, mkat / l;

Ahol - optical density of a blank sample;

Aopt - optical density of the test sample;

V is the volume of the introduced sample, 0.1 ml;

t - incubation time, 10 min;

K is the coefficient of millimolar extinction of hydrogen peroxide, equal to 22.2 ^× 10 ³ mm-1 ^× cm-1.

If the catalase activity value is more than 37 mkat/l, alcoholic liver disease is diagnosed in patients with alcohol dependence syndrome, if the catalase activity is less than or equal to 37 mkat/l, the absence of alcoholic liver disease is diagnosed.

Examples of specific implementation.

Example 1. Patient N., 35 years old, turned to a narcologist at the Perm Regional Clinical Narcological Dispensary for the treatment of alcohol dependence syndrome with complaints of dyspepsia, moderate weakness. In the anamnesis there was data on the systematic intake of alcohol on average 1 bottle of vodka per week. When collecting an anamnesis, it was found that the patient had been suffering from chronic alcoholism for 10 years.

An objective examination revealed: moderately pronounced sweating of the hands, face, slight tremor of the hands. Skin and visible mucous membranes of physiological color. The physique is normosthenic. The liver protrudes from under the edge of the costal arch by 1 cm, moderate density, smooth, painless. Biochemical study: ALT 30 U/l, ACT 26 U/l, total bilirubin 10 µmol/l, cholesterol 5.0 mmol/l, TG 0.9 mmol/l, HDL-C 3.56 mmol/l, cholesterol- LDL 3.9 mmol / l. We calculate the activity of catalase in blood serum:

E \u003d (0.00000073-0.00000039) * 0.1 * 600 * 22.2 ^× 10 ³ mm-1 ^× cm-1 * 100 \u003d 45.33 mkat / l

Serum catalase activity is 45.33 mkat/l.

Thus, the proposed method confirmed the diagnosis of "alcoholic liver disease".

Example 2

A 45-year-old patient has been registered at the Perm Regional Clinical Narcological Dispensary with a diagnosis of Alcohol Dependence Syndrome for 16 years. The patient came for another appointment with a narcologist. Complaints of discomfort, dull pain in the right hypochondrium, epigastrium, loss of appetite. Objective status: skin, visible mucous membranes of physiological color. Physique is asthenic. The liver protrudes from under the edge of the costal arch by 2 cm, moderate density, smooth, with a rounded edge. Biochemical study: ALT 25 U / l, ACT 29 U / l, total bilirubin 15 μmol / l, cholesterol 4, mmol / l, TG 0.9 mmol / l, HDL-C 3.1 mmol / l, LDL-C 3.8 mmol/l.

We calculate the activity of catalase in blood serum:

E \u003d (0.00000073-0.00000027) * 0.1 * 600 * 22.2 ^× 10 ³ mm-1 ^× cm-1 * 100 \u003d 61.33 mkat / l

Serum catalase activity is 61.33 mkat/l.

Thus, the proposed method confirmed the diagnosis of "alcoholic liver disease".

Example 3

A 39-year-old patient first applied for an appointment with a narcologist at the Perm Regional Clinical Narcological Dispensary. With a careful collection of alcohol history, it was found that the patient regularly, for 12 years, takes about 60 g of ethanol per day. Complaints about a feeling of moderate discomfort after eating, loss of appetite. Objective status: skin, visible mucous membranes of physiological color. The physique is normosthenic. The liver is 1 cm below the edge of the costal arch, smooth, dense. Biochemical study: ALT 31 U/l, ACT 21 U/l, total bilirubin 16 µmol/l, cholesterol 5.0 mmol/l, TG 0.9 mmol/l, HDL-C 3.56 mmol/l, cholesterol- LDL 3.9 mmol / l.

We calculate the activity of catalase in blood serum:

E \u003d (0.00000073-0.00000031) * 0.1 * 600 * 22.2 ^× 10 ³ mm-1 ^× cm-1 * 100 \u003d 55.99 mkat / l

Serum catalase activity is 55.99 mkat/l.

Thus, the proposed method confirmed the diagnosis of "alcoholic liver disease".

Example 4

Patient, 48 years old. He drinks alcohol about 4 times a month (about 20 g of 11% dry wine). No complaints. -LDL 3.1 mmol / l.

We calculate the activity of catalase in blood serum:

E \u003d (0.00000061-0.0000005) * 0.1 * 600 * 22.2 ^× 10 ³ mm-1 ^× cm-1 * 100 \u003d 14.66 mkat / l.

Catalase activity 14.66 mkat/l. Alcoholic liver disease in a woman is excluded. This example illustrates the result of the proposed diagnostic method.

The positive effect of using this method is the timely detection of alcoholic liver disease at an early stage, a significant reduction in the economic costs of treatment, for paying sick leave.

Claims (3)

A method for early diagnosis of alcoholic liver disease in patients with alcohol dependence syndrome, which consists in the fact that catalase activity is photometrically determined in the patient's blood serum, followed by recalculation of catalase activity in mkat/l according to the formula: E \u003d (Ahol - Aopt) × V × t × K × 100, where E is the activity of catalase in mkat/l; A - optical density of blank and experimental samples; V is the volume of the introduced sample, 0.1 ml; t - incubation time, 10 min; K is the coefficient of millimolar extinction of hydrogen peroxide, equal to 22.2×10 ³ mm ^-1 × cm ^-1 , and with a catalase activity value of more than 37 mkat/l, alcoholic liver disease is diagnosed in patients with alcohol dependence syndrome, with a value of E less than or equal to 37 mkat/l - the absence of alcoholic liver disease.