RU2507518C1 - Method for prediction of clinical course of ischemic heart disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for prediction of the clinical course of ischemic heart disease consists in analysing pre- and post-therapeutic modified lipoproteins (a) by processing blood serum 0.5 ml in 0.1% Triton X-100 0.2 ml, incubating for 15 min at 20°C, agitating the mixture 120 times a minute and adding 7% polyethylene glycol 6000 and performing electrophoretic separation in agarose gel in a well 4-20 mm; if observing a decrease of modified lipoproteins (a) by 30% and more, cholesterol by 18% and more and an increase of A to A-1 by 25% and more as compared to the reference, the prognosis of the clinical course of ischemic heart disease is considered to be favourable and facilitating the transfer of functional class III-IV exertional angina into class I-II exertional angina, while a decrease of modified lipoproteins (a) by less than 30%, and total cholesterol less than 18% and an increase of A to A-1 by less than 25% as compared to the reference, the prognosis is considered to be unfavourable.

EFFECT: higher prediction accuracy.

2 ex, 1 tbl, 6 dwg

Description

The invention relates to medicine and can be used in cardiology or therapy.

Known methods of separation into fractions of blood lipoproteins by the method of analytical ultracentrifugation (A. N. Klimov, N. G. Nikulcheva. Lipids, lipoproteins and atherosclerosis. - Peter. Press., Pp. 98-102).

A known method of separation into LP fractions in a polyacrylamide gel (NN Shatskaya. Biochemical studies in assessing the state of the cardiovascular system. In the book "Research Methods in Occupational Pathology", Moscow, 1988, p.95-97).

There are also known methods of separation into LP fractions by agarose gel electrophoresis (Lab. Research Methods / Edited by V.V. Menshikov. M: Medicine, 1987, p.248-249), SU 1720015 A, 03/15/1992, RU 2063040 C1, 06/27/1996, RU 2115121 C1, 07/10/1998, RU 2097038 C1, 11/27/1997, RU 2060034 C1, 05/20/1996, EP 0074610 A, 03/23/1983.

The disadvantage of these methods is that they do not allow to detect all fractions of blood LP, including chylomicrons (ChM), high density lipoproteins (HDL), low density lipoproteins (LDL), very low density lipoproteins (VLDL) and LP (a), and serve to separate into fractions of ChM, HDL, LDL, VLDL from the complex of albumin with unesterified fatty acids, and also do not allow to detect minor fractions of modified blood lipoproteins, namely modified Lipoprotein abnormal (Lp (a) or LP (a)).

A known method for determining the fractions of blood lipoproteins of the authors Kanskoy N.V., Fedorova N.A., Perova N.V., Garganeeva N.P., Kozhanova A.A., Baykova A.N., Kansky A.V., Pohryaeva E.N. RU 2210079 C2 G01 N 33/92, 08/10/2003. Bull. Number 22.

This method is the closest to the proposed technical essence and the achieved result and is selected as a prototype, however, it does not allow to identify minor fractions of modified drugs (a). The minor fraction of modified LP (a) is the most atherogenic. Therefore, for the early diagnosis of the disease, it is especially important to detect LP (a) along with the most complete determination of fractions of other blood LPs. Modified LP (a) are a predictor of atherosclerosis and a genetically determined factor leading to myocardial infarction or ischemic stroke.

The task of the invention is to improve the accuracy of the method. This task is achieved by the fact that, in addition to blood lipids, determined LPs are determined before and after treatment by treatment with 0.5 ml of blood serum 0.2 ml of 0.1% Triton X-100 solution, incubation for 15 minutes at 20 ° C, stirring shaking the mixture 120 times per 1 min, followed by the addition of a 7% solution of polyethylene glycol 6000 and electrophoretic separation in agarose gel in a 4 × 20 mm well and with a decrease in the level of PL (a) by 30% or more, and the level of total cholesterol by 18% and more and an increase in blood Apo A-1 by 25% or more compared with the initial level, the prognosis of the diseases are considered favorable, contributing to the transition of angina pectoris of FC III-IV to FC I-II, and with a decrease in the level of modified LP (a) less than 30%, total cholesterol less than 18% and an increase in blood Apo 1 less than 25% compared with baseline prognosis of the disease is considered unfavorable.

New in this method is that the patient’s blood serum is treated with Triton X-100 detergent, incubated for 15 minutes at 20 ° C, the mixture is stirred by shaking 120 times in 1 minute, followed by the addition of 7% solution of polyethylene glycol 6000, which allows additional minor fractions of modified blood lipoproteins and simultaneously determine the total cholesterol and Apo A-1 blood before and after treatment of coronary heart disease.

Therefore, only a comprehensive modernization of the prototype method allows to obtain the desired result.

The study of drugs of different classes in the diagnosis of coronary heart disease (CHD) is recommended by the All-Russian Scientific Society of Cardiology according to the provisions of the recommendations of the European Society for the Study of Atherosclerosis - "Diagnosis and correction of lipid metabolism disorders for the prevention and treatment of atherosclerosis" (Moscow, 2005; Clinical laboratory diagnostics, No. 10, 2008, p.21-32).

At present, lipid studies with detergents are promising / Triton X-100 et al. / In laboratory practice, direct or homogeneous methods for determining lipoproteins (LPs) and their lipids are increasingly being used. Such methods are based on the use of various detergents that can block or solubilize drug classes for the specific isolation of APO A-1, LDL and other drug fractions.

Using these methods, the isolation of other classes of drugs does not require additional operations and the concentration of cholesterol (cholesterol) in classes of drugs can be determined directly in the blood serum by conventional enzyme methods in the same cuvette (Clinical Laboratory Diagnostics, No. 10, 2008, p.21- 32).

An increase in the concentration of abnormal LP or LP (a) in the blood is considered an independent risk factor for atherosclerosis. With a blood content of LP (a) of more than 300 μg / ml at a rate of 0-300 μg / ml, the risk of coronary atherosclerosis is doubled, and with a simultaneous increase in the level of LP (a) of cholesterol and LDL cholesterol - by 5 times (JAMA - 2001. - Vol. 285. - p 2486-2497, Eur. Heart. J. - 2003. - Vol.24, - p. 1601-1610).

To obtain PEG-6000 precipitate of blood serum, a 7% solution of PEG-6000 was used. It is known that 7% PEG-6000 is the limiting concentration for obtaining serum precipitates containing immune complexes and not containing coarse proteins of blood serum. The use of a 6% PEG-6000 solution does not completely precipitate the immune complexes of blood serum. Therefore, to increase the accuracy of the method, a 7% concentration of PEG-6000 was chosen experimentally, which allows, on the one hand, to completely precipitate immune complexes from blood serum with a guarantee of the absence of coarse (high molecular weight) proteins of blood serum, and on the other hand, to completely precipitate circulating immune complexes (CECs) present in blood serum. So, in the case of a serum CEC concentration of 2.5 g / l, the same CEC concentration was detected in 7% serum precipitates (with a result of + 3%), which is associated with an increase in the number of manipulations, and not a change in concentration CEC in serum precipitates.

Incubation of a 7% PEG-6000 precipitate of the test blood serum with Sudan B dye increases the affinity of the dye for the drug precipitates in the blood, and the large volume of the blood sample taken for the study reveals the modified drugs (a) contained in the blood of patients with coronary heart disease (CHD) in low concentrations. Additional processing of blood serum with triton X-100 allows to increase the accuracy of the method and obtain the desired result. The proposed method allows to identify the following minor fractions of modified drugs: HDL, LDL, VLDL and LP (a).

Each feature newly introduced into the claims fulfills the function of increasing the accuracy and efficiency of the method: treating 0.5 ml of a patient’s blood serum sample with 0.2 ml of a 0.1% Triton X-100 solution, incubating the sample at 20 ° C for 15 minutes, mixing shaking the mixture 120 times per 1 min followed by the addition of a 7% solution of polyethylene glycol 6000 and additional detection of minor fractions of modified lipoproteins simultaneously with determination of total cholesterol and Apo A-1 blood before and after treatment of coronary heart disease.

Currently, special attention is paid to the study of minor fractions of modified drugs (a), in connection with which laboratory diagnostic methods are being developed to study this blood lipoprotein. It refers to apo-B-containing lipoproteins rich in cholesterol (cholesterol). LP (a) is identical to sinking pre-β-Lp (sinking pre-β-Lp), which has the mobility of pre-β-LP during electrophoresis. PL (a) contains 27% protein, 8% carbohydrates and 65% lipids, of which ECS make up 59%, NEHS 14%, and PL 14%.

The protein component of LP (a) is the highly glycosylated apo (a) polypeptide, which has a close structural affinity for plasminogen - one of the factors of the coagulation system - anticoagulation of blood. With an increase in the concentration of both LP (a) and its modified forms in the blood, the microcirculation processes in the blood arteries are disrupted with the possible formation of microthrombi.

Due to the presence in the structure of apo (a) sialic acids, PL (a) is more negatively charged compared to B-PL in an electric field, it is better soluble in water, and can interact with metal ions (calcium). This lipoprotein and its modified forms are heterogeneous. All this indicates the special role of LP (a) and modified LP (a) in atherogenesis.

LP (a) can interact with LDL receptors, exerting a weak effect on the activity of GMK-CoA reductase, on the esterification of cholesterol. The half-life of the drug (a) is longer than that of LDL and is 3.3 days. The content of LP (a) in the blood normally does not exceed 30 mg / L. At a high concentration in the blood, PL (a) is detected at the sites of vascular damage in the area of accumulation of fibrinogen. An increased concentration of LP (a) is often combined with II ^a , II ⁶ types of hyperlipoproteinemia, in which modified LPs are often detected. Therefore, in clinical practice it is extremely important to determine the LP (a) simultaneously with the determination of proteins of the acute phase of inflammation. It was found that most lipid-lowering drugs do not affect elevated levels of LP (a).

The LP (a) fraction is heterogeneous. It was established that during electrophoresis, the drugs (a) are in the region of β-globulins, but up to 5% of the drugs (a) can be detected in the region of α-globulins. Due to such pronounced heterogeneity, it is quite difficult to evaluate the whole LP fraction (a) during electrophoresis, and even less its minor fractions, which can remain on the start line if their particle size is large enough. Therefore, the use of the triton X-100 detergent commonly used in studies of the last fifty years for the treatment of blood serum, namely blood lipoproteins, leading to partial delipidation of drugs and an increase in their mobility during electrophoresis, allows the detection of minor drug fractions (a). In the chemical industry, various detergents are used (in the manufacture of washing powder, detergents, etc.), but when working with biological material, Triton X-100 is used predominantly. The processing mode of the sample by Triton X-100 was selected on the basis of experimental studies empirically. For this, various dilutions of Triton X-100, a different temperature and a different exposure in minutes were used. When using different concentrations of the Triton X-100 solution, small concentrations (below 0.1%) did not increase the release of minor drug fractions, including LP (a), and high concentrations (more than 0.1%) led to complete delipidization and destruction of the structure LP. The result of the study is presented in table No. 1.

Processing 0.5 ml of blood serum to study lipoproteins with different concentrations of Triton X-100 solution for different time of incubation of the sample at different temperatures.

Therefore, the optimal conditions for treating blood serum with Triton X-100 solution is a concentration of 0.1% in a volume of 0.1 ml, at a temperature of 20 degrees Celsius for 15 minutes.

Since LP (a) is the most atherogenic, it is very important in the early stages of the disease to identify the maximum content of LP (a) in the blood of each patient. This allows you to diagnose coronary heart disease before the stage of significant changes in other clinical and laboratory parameters, improves the accuracy of the diagnosis of the disease. In turn, pathogenetically substantiated therapy is prescribed to such patients early. Equally important is the identification of minor LP fractions (a) to assess the effectiveness of treatment of the disease and predict the course of coronary heart disease.

All of the above indicates the extreme importance of the development of laboratory diagnostic methods that allow the most complete identification of LP (a), its modified forms and their minor fractions.

Currently, in a wide clinical laboratory practice, the methods for determining LP (a) are not sufficiently used. At the same time, the popularity of the method of electrophoretic separation into blood LP fractions in agarose gel is due to its high sensitivity, ease of implementation, and sufficient adequacy of the results obtained (Clinical Laboratory Diagnostics, No. 10, 2008, P.21-32).

The essential features characterizing the invention, showed in the claimed combination of new properties that are not explicitly derived from the prior art in this field, and are not obvious to a specialist.

An identical set of features was not found in the study of patent and medical literature.

This invention can be used in medical practice to improve the accuracy of diagnosing the degree of atherosclerosis in coronary heart disease.

Thus, the proposed invention should be considered relevant to the conditions of patentability: "novelty", "inventive step", "industrial applicability".

The method is based on the electrophoretic mobility of modified lipoproteins and the simultaneous determination of total blood cholesterol and Apo A-1.

The invention will be clear from the following description of the attached drawings.

Figure 1 presents the results of electrophoretic separation into fractions of LP serum: a - in the control group by the prototype method; b - in the control group of the proposed method.

On figa, b presents the results of electrophoretic separation into fractions of LP serum in the group of patients with coronary artery disease before treatment ;.

Figure 3 (a and b) presents the results of electrophoretic separation into fractions of LP serum of patients after treatment.

Figure 4 (a, b) presents the results of electrophoretic separation into blood LP fractions of patients with coronary heart disease with hypercholesterolemia.

Figure 5 (a, b) presents the results of electrophoretic separation into LP fractions of the blood serum of a patient with coronary heart disease according to the prototype method and the proposed method.

Figure 6 (a, b, c) presents the results of electrophoretic separation into LP fractions of the blood serum of patients according to the proposed method in a patient with coronary heart disease after treatment, as well as before and after treatment in another patient with coronary heart disease.

The method is as follows in stages:

1) preparation of a solution of Sudan B: 400 mg of Sudan B is dissolved in 20 mg of ethylene glycol in a boiling water bath for 50 minutes, filtered, stored in a glass dish;

2) preparation of agarose gel: 320 mg of Sigma A agarose A is dissolved in 20 ml of water by boiling, then placed in a thermostat at 55 ° C; add 20 ml of albumin solution (1 g of albumin in 200 ml of veronal-medial buffer, pH 8.6), 3 ml of agarose gel is applied to a fat-free horizontally mounted glass slide, a metal steel rod-bar 20 × 4 mm (10 mm high) is placed which, after solidification of the gel is removed with a magnet;

3) for the study, 1 ml of blood serum was taken, which is divided into 2 servings. 0.2 ml of a 0.1% Triton X-100 solution are added to 0.5 ml of a patient’s blood serum sample, incubated for 15 minutes at 20 ° C, the mixture is stirred by shaking 120 times every 1 minute, then 20 ml of 7% polyethylene glycol is added ( PEG) -6000 and incubated for 1 h at 20 ° C, centrifuged for 40 min at 25000 g. The precipitate is washed twice.

4) the precipitate solution is used for electrophoretic research. 0.15 ml of Sudan B solution is added to 0.25 ml of blood serum precipitate, placed for 1 h in a dark thermostat at 40 ° C, then 0.2 ml of a hot agarose gel solution is added, mixed, heated at 55 ° C and added with warm in the groove of agarose gel;

5) a glass slide is placed in the electrophoresis chamber with the agarose layer down, electrophoresis is carried out for an hour in a refrigerator at a temperature of 4 ° C at a voltage of 100 V and a current of 40-45 mA;

6) the electrophoregram is fixed in a 5% solution of acetic acid for one hour, then dried between sheets of filter paper, continuously wetting with 96% ethyl alcohol;

7) densitometry is carried out on an MF-4 microphotometer;

8) carry out electrophoresis of blood lipoproteins.

The improvement of the method relates to mechanical shaking of the sample with a frequency of 120 times per 1 min, followed by disintegration of the sample on a laboratory shaker for preparing reagents of the company "Immunotech a.s.", Czech Republic. More frequent or prolonged shaking followed by disintegration leads to an increase in the temperature of the sample and the appearance of recombinant forms of blood lipoproteins, which prevents further research and distorts the result of electrophoretic analysis of the sample. A shorter shaking period followed by disintegration of 30 seconds does not improve the result of the study.

The densities are quantified by densitometry as follows: the area of each peak in the chromatogram is determined by the formula S = h · B1 / 2h, where S is the peak area, h is the peak height, B1 / 2h is the peak width at half its height.

The calculation is carried out automatically according to the appropriate program and the final result is the percentage of each fraction of lipoproteins, if any, in relation to the total amount of fractions taken as 100%.

Total blood cholesterol is determined by a standardized method in dynamics. Pretreatment of blood serum with Triton X-100 solution does not affect the analysis.

The method is based on measuring the optical density of cholesterol and its esters with the Lieberman-Burchard reagent. Most of the cholesterol in the blood serum is esterified, the reaction product of cholesterol with the indicated reagent absorbs light in the same region of the spectrum and has a slightly higher extinction coefficient than the reaction product of free cholesterol. To account for this difference, as well as to partially account for matrix effects (the effects of proteins and bilirubin on the results of the analysis), the method is calibrated using calibration serum, the cholesterol content of which is determined by the Abel-Kendal reference method.

Preparation of the Lieberman-Burchard reagent:

600 ml of acetic anhydride and 300 ml of acetic acid are poured into a two-liter flask, placed in an ice bath and stirred on a magnetic stirrer, then 100 ml of chilled sulfuric acid are added. After 30 minutes, the flask was removed from the ice bath, put on a stirrer and 20 g of sodium sulfate was added, stirred until the salt was completely dissolved for 4-5 hours. The reagent is stable for 2 weeks.

Analysis progress:

1. 5 ml of Lieberman-Burchard reagent is poured into dry tubes, placed in an ice bath.

2. 0.2 ml of distilled water is poured into the first test tube (along the wall, slowly over 10 minutes). Shake gently and place in a water bath.

3. At intervals of 2 minutes, 0.2 ml of the test serum is poured into the remaining tube, shaken, placed in a water bath.

4. 25 minutes after adding water in the first test tube, measure the optical density of its content against water. Next, test samples are measured at a wavelength of (625 ± 10) nm.

5. Calculate the concentration of cholesterol in blood serum compared with the calibration.

For solutions of the Lieberman-Burchard reaction products, the Bouger-Lambert-Beer law is observed in the range of 0-400 mg / dl of cholesterol.

When conducting a second study after treatment, the magnitude of the LP fraction (a) before treatment is taken as 100% and the percentage value of the decrease in the fraction of modified LP (a) after the treatment of coronary heart disease is calculated.

A-1 was determined on a Konelab analyzer (code 981662). Apolipoproteins A form the majority of the main proteins found in high density lipoproteins and are also found in chylomicrons. Approximately 50% of the mass of high density lipoproteins belongs to proteins, while Apo A-I and A-II comprise approximately 90% of this mass. The amounts of A-I and A-II are approximately equal to 3: 1. Apo A-I and A-II are formed in the intestines and liver. When it enters the blood after isolation from the intestine in the form of fragments of chylomicones Apo A-I and A-II, according to the data obtained, accumulate in HDL. Studies have shown that some particles of lipoproteins contain only apolipoprotein A-I, while other particles contain both A-I and A-II. Apolipoprotein A-II plays a role in the activation of LHAT (lecithin cholesterol acyltransferase) and the excretion of free cholesterol from extrahepatic tissues. High Apo B content and low Apo A-II content indicate an increased risk of coronary heart disease.

Determination method

The method is based on measuring immunoprecipitation enhanced with polyethylene glycol (PEG) at 340 nm. An excess of specific antiserum is added to samples with buffer solution. Then, an increase in light absorption caused by immunoprecipitation is recorded when the reaction reaches its endpoint. The change in light absorption is proportional to the amount of antigen I (apolipoprotein A-1) contained in the solution.

Results are calculated automatically using a calibration curve. The normal content of Apo A-1 in blood serum is 1.0-2.3 g / l.

In the control group in the case of using the prototype method (n = 10) and the proposed method (n = 10), lipoproteins were not detected (figa and b). The level of total blood cholesterol was estimated, which was 4.7 ± 0.5 mmol / L in the first 10 patients and 5.4 ± 0.5 mmol / L in the second group of patients and the level of Apo A-1, which is in group I amounted to 2.0 ± 0.2 g / l, and in the second, 2.2 ± 0.2 g / l. We examined 2 groups of patients with coronary heart disease before and after treatment, 10 patients each for the prototype method of group I and 30 patients of group II for the transplantable method.

The diagnosis of patients: CHD, angina pectoris, FC III-IV.

In patients of group I, prior to treatment, mLDPE and mLPONP were revealed (Fig. 2a, b). The level of serum cholesterol was 8.0 ± 0.7 mmol / L, and the level of Apo A-1 was 1.4 ± 0.1 g / L. After treatment with electrophoresis, the mLDPE and mLPONP fractions became less intense (Figs. 3a and b).

The level of total serum cholesterol after treatment was 6.3 ± 0.5 mmol / L, and the level of Apo A-1 was 1.6 ± 0.1 g / L.

Diagnosis after treatment: ischemic heart disease, exertional angina, FC II-III. Conclusion: the prognosis of the course of coronary heart disease is favorable, but not accurate enough. In patients of group II before treatment, modified mLDPE and mLPONP were detected, the cholesterol level was 7.8 ± 0.7 mmol / L, and ApoA-1 1.3 ± 0.2 g / L.

After treatment of coronary artery disease in 6 patients, a trace hard-to-detect fraction of modified LP (a) was detected (Figs. 4a and b). In other patients, this fraction was not detected.

After treatment, the level of total serum cholesterol in the whole group was 5.2 ± 0.5 mmol / L, Apo A-1 1.9 ± 0.2 g / L. The diagnosis after treatment in patients of this group (29 people): ischemic heart disease, exertional angina, FC II. Conclusion: the prognosis of CHD is favorable. Patients in this group are presented in clinical examples. In one patient of the second group, with a decrease in total blood cholesterol after treatment, the level of LP (a) in the blood serum remained quite high. No dynamics after treatment were detected. The prognosis of the course of coronary heart disease in this patient was regarded as unfavorable.

We did not identify false positive and false negative cases, because only objective criteria for the effectiveness of the treatment of coronary heart disease were evaluated: the results of bicycle ergometry, the frequency of angina attacks and, accordingly, the dose of glycerol taken, as well as the results of laboratory research methods, expressed quantitatively.

Example 1. Patient P., 66 years old, medical history No. 189, was admitted to the department of IHD and atherosclerosis of the Research Institute of Cardiology of Tomsk. Complaints upon receipt of pain in the region of the heart stitching, radiating to the left subscapular region. The pains occurred during physical and emotional stress, were relieved by prolonged action nitrates. Bicycle ergometry was discontinued at a load of 50 watts due to increased chest pain. HELL 70/110 mm Hg, pulse at rest 60 beats. in minutes Pain is stopped by nitroglycerin.

Laboratory results: total cholesterol 8.8 mmol / l, triacylglycerides 1.8 mmol / l, HDL cholesterol 1.1 mmol / l, blood HDL 16%, Apo A-1 1.4 g / l.

The results of an electrophoretic study of a drug of 7% PEG-6000 serum precipitate are presented in Fig. 5a, and according to the proposed method in Fig. 56. The fractions of MLPNP, MLPONP and MLP (a) were revealed.

Diagnosis at admission: coronary heart disease, angina pectoris, FC III-IV.

After treatment, the results of a laboratory test: total cholesterol 5.6 mmol / l, triacylglycerides 1.4 mmol / l, HDL cholesterol 1.2 mmol / l, HDL 34%, Apo A-1 2.1 g / l.

When electrophoretic study of the drug 7% PEG-6000 precipitated blood serum according to the proposed method, the fraction of the drug (a) were not detected. Figa. contains a start line and a weakened fraction of mLDPE. Conclusion: the absence of MLP (a), a significant increase in the level of HDL blood to 34% and a decrease in blood cholesterol by 65% and an increase in Apo A-1 by 50% indicates a significant improvement in the patient's condition. The LDL fraction is weakened.

Diagnosis at discharge: coronary heart disease, exertional angina FC II. Conclusion: the prognosis of the course of coronary heart disease is favorable, as evidenced by a decrease in the functional class of angina pectoris from III-IV to II.

Example 2. Patient M., 58 years old, medical history No. 192, was admitted to the department of coronary heart disease and atherosclerosis of the Research Institute of Cardiology of Tomsk with complaints of pain in the region of the heart, radiating to the shoulder blade, arising from physical exertion. Bicycle ergometry was stopped at a load of 50 watts due to chest pain and shortness of breath. HELL 160/100 mm Hg, pulse at rest 66 beats. in minutes Pain is stopped by nitroglycerin.

Laboratory results: total cholesterol 7.8 mmol / l, triacylglycerides 1.8 m mol / l, HDL cholesterol 0.9 m mol / l, blood HDL 13%, Apo A-1 1.4 g / l.

The results of electrophoresis of 7% PEG-6000 precipitate of blood serum revealed the presence of mLPNP, mLPONP and mLP (a) (Fig.6). Densitometry results of mLP: mLDPE - 50%, mLPONP - 42%, m PL (a) - 8%.

Diagnosis at admission: coronary heart disease, angina pectoris, FC III-IV.

After treatment, the results of a laboratory test: total cholesterol 5.4 mmol / l, triacylglycerides 1.8 mmol / l, HDL cholesterol 1.3 mmol / l, HDL blood 31%, Apo A-1 2.2 g / l.

The results of electrophoretic studies of blood MLP according to the proposed method after treatment are presented in figv. Densitometry results for mLP: mLDPE - 55%, VLDL - 43%, mLP (a) - 2%.

Diagnosis at discharge: coronary heart disease, angina pectoris, FC II. A decrease in the level of LHT (a) by 72%, and total cholesterol by 44% compared with the initial one and an increase in Apo A-1 by 57% compared with the initial one testified to the effectiveness of the treatment of IHD. Conclusion: the treatment and prognosis of the course of coronary heart disease is favorable, as evidenced by a decrease in the functional class of angina pectoris from III-IV to II.

So, when applying the prototype method, the prognosis of the course of coronary heart disease was based on the level of total serum cholesterol and clinical data, therefore it was not accurate enough. The method was applied in 40 patients with coronary heart disease and 20 patients of the comparison group (control) with neurocirculatory dystonia. With a decrease in the level of MLP (a) by 30% or more of total serum cholesterol by 18% or more and an increase in Apo A-1 of blood by 25% or more, the prognosis of the course of IHD was assessed as favorable, as evidenced by a change in the functional class (FC) angina of exertion from III-IV to I-II, and when changing only one of the indicators, an unfavorable prognosis of the course of IHD was established (the transition of a stable course to an unstable form of angina).

False positive cases of predicting the course of coronary heart disease when using our proposed method was not observed. Moreover, the proposed method is simple in the execution and interpretation of the results.

Table 1 Detergent Concentration Incubation time Temperature, ºС Detection of the LP fraction (a) Triton X-100 0.1 ml 0.01% 10 minutes fifteen - Triton X-100 0.1 ml 0.01% 15 minutes twenty - Triton X-100 0.1 ml 0.01% 20 minutes 25 - Triton X-100 0.1 ml 0.1% 10 minutes fifteen traces Triton X-100 0.1 ml 0.1% 15 minutes twenty + Triton X-100 0.1 ml 0.1% 20 minutes 25 traces Triton X-100 0.1 ml 0.15% 10 minutes fifteen - Triton X-100 0.1 ml 0.15% 15 minutes twenty - Triton X-100 0.1 ml 0.15% 20 minutes 25 -

application

Table 1. Study of the effect of the Triton X-100 detergent on the detection of lipoprotein fractions at different incubation times and different temperatures, that is, determination of optimal conditions.

Figure 1. The results of electrophoretic separation into blood serum LP fractions are presented: a - in the control group by the prototype method; b - in the control group of the proposed method.

Figure 2 (a, b). The results of electrophoretic separation into blood serum LP fractions in the group of patients with coronary artery disease before treatment are presented.

Figure 3 (a and b). The results of electrophoretic separation into the LP fractions of the blood serum of patients after treatment are presented.

Figure 4 (a, b). The results of electrophoretic separation into blood LP fractions of patients with coronary heart disease with hypercholesterolemia are presented.

Figure 5 (a, b). The results of electrophoretic separation into LP fractions of the blood serum of a patient with coronary heart disease by the prototype method and the proposed method are presented.

Fig.6 (a, b, c). The results of electrophoretic separation into the LP fractions of the blood serum of patients according to the proposed method in a patient with coronary heart disease after treatment, as well as before and after treatment in another patient with coronary heart disease are presented.

Claims (1)

A method for predicting the course of coronary heart disease by examining blood serum lipids, characterized in that before and after treatment, modified drugs (a) are examined by treating 0.5 ml of blood serum with 0.2 ml of 0.1% Triton X-100 solution, by incubation 15 min at 20 ° C, stirring the mixture by shaking 120 times in 1 min, followed by adding 7% solution of polyethylene glycol 6000 and electrophoretic separation in agarose gel in a 4 × 20 mm well and with a decrease in the level of PL (a) by 30% or more, and level of total cholesterol by 18% or more and increase Ap A-1 blood count of 25% or more compared with the initial level, the prognosis of the course of the disease is considered favorable, contributing to the transition of angina pectoris FC III-IV to FC I-II, and with a decrease in the level of modified LP (a) less than 30%, total cholesterol less 18% and an increase in blood Apo A-1 of less than 25% compared with the initial level, the prognosis of the disease is considered unfavorable.

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