RU2402016C1 - Method of predicting course of coronary heart disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: in order to predict course of coronary heart disease before and after treatment in blood serum general cholesterol and Lp(a) are determined by additional processing of 0.3 ml 0.1% solution of Triton X-100 and incubation for 15 minutes at 20°C. Further incubation of patient's blood serum is carried out with Sudan B solution for 1 hour in dark thermostat at 40°C. Samples are introduced into hollow in agarose gel with base area 4×20 mm for electrophoresis with further fixation of electrophoregrams, their drying and densitometry. In case of simultaneous 20% and more reduction of general cholesterol level and 40% and more reduction of Lp(a)level in comparison with initial level prognosis of disease course is considered to be favourable, contributing to transition of exertional angina from functional class III-IV into functional class I-II. In case of lower than 20% reduction of general cholesterol and less than 40% reduction of Lp(a) level in comparison with initial level prediction is considered unfavourable.

EFFECT: method increases sensitivity and accuracy of predicting coronary heart disease course.

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Description

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

Known methods of separation into blood lipoprotein (LP) fractions by analytical ultracentrifugation of blood (A. N. Klimova, N. G. Nikulcheva. "Lipids, lipoproteins and atherosclerosis", 1995, Peter, press, pp. 98-102) and separation into LP fractions in a polyacrylamide gel (N.N. Shatskaya. Biochemical studies in assessing the state of the cardiovascular system. In the book: Research Methods in Occupational Pathology. M., 1988, pp. 95-97).

There are also known methods of separation into LP fractions by agarose gel electrophoresis (Phenotyping of hyperlipoproteinemia based on agarose gel lipoprotein electrophoresis, Lab. Delo, 1980, No. 5, p. 287-290);

RU 2099693 C1, 12.20.1997. RU 2102767 C1, 01.20.1998. SU 1334087 A1, 08.30.1987. US 5547873 A, 08.20.1996.

The disadvantage of these methods is that they do not allow to identify all fractions of blood LP, including HM, HDL, LDL, VLDL and LP (a), but are used to separate HM, HDL, LDL, VLDL from the complex of albumin with unesterified fatty acids .

There is also a method of determining the fractions of blood lipoproteins in patients with coronary heart disease (RU No. 2200950 C2 G01N 33/49, 03.20.2003 Bull. No. 8.)

This method is the closest to the proposed technical essence and the achieved result and is selected as a prototype.

The disadvantage of the prototype method is the low accuracy, since it does not allow to identify minor fractions of the drug. This is due to the fact that the LP (a) fraction is the most atherogenic and for the early diagnosis of the disease, it is especially important to identify the minor fraction of the LP (a), along with the most complete determination of the fractions of other blood LPs.

The task of the invention is to increase the accuracy and sensitivity of the method.

This problem is solved by separation into blood lipoprotein fractions in patients with coronary heart disease (IHD) by agarose gel electrophoresis by additional processing of 0.3 ml of 0.1% Triton X-100 solution and incubation for 15 min at 20 ° C, followed by incubation of the patient's blood serum with Sudan B solution for 1 h in a dark thermostat at 40 ° C and then making a sample in a well in an agarose gel with a base area of 4 × 20 mm for electrophoresis, followed by fixation of electrophoregrams, their drying, densitometry, and at the same time lowering the level LP (a) by 40% or more, and total cholesterol by 20% or more compared with the initial level, the prognosis of the course of the disease is considered favorable, contributing to the transition of angina from functional class III-IV to functional class I-II, and with a decrease in the level LP (a) less than 40%, and total cholesterol less than 20% compared with the initial prognosis is considered unfavorable.

New in the invention is the additional incubation of 0.3 ml of a patient’s blood serum sample with 0.1 ml of a 0.1% solution of Triton X-100 detergent at 20 ° C for 15 minutes, which makes it possible to identify minor fractions of blood LP at the same time as determining the total blood cholesterol before and after the treatment of coronary heart disease.

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).

Currently, promising are methods for the study of lipids with detergents / Triton X-100 and others /. 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 classes of drugs for the specific isolation of HDL, LDL and other fractions of the drug.

When using these methods, 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).

Currently, special attention is paid to the study of LP (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 a highly glycosylated polypeptide - apo (a), which has a close structural affinity for plasminogen, one of the factors of the coagulation-anticoagulation system. With an increase in the concentration of LP (a) in the blood, the microcirculation processes in the blood arteries are disrupted with the possible formation of microtrombi.

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

LP (a) can interact with LDL receptors, having 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 ^b types of hyperlipoproteinemia. Therefore, in clinical practice, the determination of LP (a) is extremely important, 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). World population studies have shown that LP (a) is an independent and independent risk factor for coronary artery disease - the most severe manifestation of atherosclerosis.

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 subfractions, 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 delipidization of drugs and an increase in their mobility during electrophoresis makes it possible to identify minor fractions of drugs (a). 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 indications, 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 the treatment of the disease and to predict the course of IHD.

All of the above indicates the extreme importance of the development of laboratory diagnostic methods that allow the most complete detection of drugs (a), including minor fractions of drugs (a) (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 diagnosis in patients with coronary artery disease.

Thus, this technical solution 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.

The invention will be clear from the following description and 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.

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

Figure 3 (a, b) presents the results of electrophoretic separation into the LP fractions of the blood serum of patients with coronary artery disease after treatment.

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

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

Figure 6 (a, b, c) presents the results of electrophoretic separation into blood serum LP fractions according to the proposed method in a patient with coronary artery disease after treatment, as well as before and after treatment in another patient with coronary artery 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 boiling water, then placed in a thermostat at 55 ° C; add 20 ml of albumin solution (1 g of albumin in 200 ml of veronalmedinal buffer, pH 8.6). 3 ml of agarose gel is applied to a fat-free horizontally mounted microscope slide, a metal steel rod is placed - a bar 4 × 20 mm (10 mm high), which, after gel setting, is removed with a magnet;

3) 0.1 ml of a 0.1% solution of Triton X-100 is added to 0.3 ml of a patient’s blood serum sample, incubated for 15 min at 20 ° С, 0.15 ml of Sudan B solution is added, placed for 1 hour in a dark thermostat at 40 ° С, then 0.2 ml of a hot agarose gel solution is added, mixed, heated at 55 ° С and 4 × 20 × 10 mm in size are added to a well of agarose gel;

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

5) 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;

6) densitometry is carried out on an MF-4 microphotometer.

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 spectral region and has a slightly higher extinction coefficient than the reaction product of free cholesterol. To take into account this difference, as well as to partially take into account the marin effects (the effects of proteins and bilirubin on the results of the analysis), the method is calibrated using a calibration serum, in which the cholesterol content was 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 narya 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. With an interval of 2 minutes, 0.2 ml of the test serum is poured into the remaining test 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.

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 LDL, VLDL, HDL and LP (a), if it is in relation to the total amount of fractions, taken as 100%.

When conducting a repeated 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 LP fraction (a) after the treatment of coronary heart disease is calculated.

In the control group, in the case of using the prototype method (n = 10) (Fig. 1a) and the proposed method (n = 12) (Fig. 1b), CI, LDL, VLDL, HDL were detected - this is a normal lipid profile. The level of total blood cholesterol was estimated, which was in the first 10 patients (4.6 ± 0.2) mmol / L and in the second group of patients, n = 12 (4.7 ± 0.2) mmol / L. We examined 2 groups of patients with coronary heart disease before and after treatment, 10 patients each for the prototype method — group I and 30 patients — group II for the proposed method.

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

Before the treatment, patients of group I revealed CI, LDL, VLDL, HDL (Fig. 2a, b). Their serum cholesterol level was (7.4 ± 0.5) mmol / L. After treatment with electrophoresis of LP, the LDL and VLDL fractions became less intense (figa and b).

The level of total serum cholesterol after treatment was (5.7 ± 0.4) mmol / 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 22 patients of group II with hypercholesterolemia, LDL, VLDL, HDL, LP (a), ChM were detected during electrophoresis (Figs. 4a and b).

In the remaining 8 patients of the group, the LP fraction (a) was not detected. The level of total cholesterol in patients of the entire second group was (8.7 ± 0.6) mmol / L.

After treatment of coronary artery disease in 10 patients, a trace hard-to-detect fraction of LP (a) was detected; in 20 patients, LP (a) was not detected.

The level of total serum cholesterol after treatment was in the whole group (6.0 ± 0.5) mmol / 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 V., 54 years old, medical history No. 187, was admitted to the Department of IHD and Atherosclerosis, Research Institute of Cardiology, Tomsk. Complaints upon receipt of pain in the region of the heart and behind the sternum of a stitching nature with irradiation under the scapula. The pains occurred during physical exertion and were relieved by nitroglycerin. HELL 160/90 mm Hg Pulse at rest 66 beats. in minutes Bicycle ergometry was stopped at a load of 50 watts for objective reasons.

Laboratory results: total cholesterol 7.8 mmol / l, triacylglycerides 1.8 mmol / l, HDL cholesterol 1.0 mmol / l.

The results of electrophoretic studies of blood LP according to the prototype method are presented in figa, and according to the proposed method in fig.5b. LP fraction (a) is present.

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

After the treatment, the results of a laboratory study: total cholesterol 5.6 mmol / l, triacylglycerides 1.6 mmol / l, HDL cholesterol 1.3 mmol / l.

The results of electrophoretic studies of blood LP according to the proposed method are presented in figa.

The LP fraction (a) is absent, the LDL fraction is weakly expressed.

Diagnosis at discharge: coronary heart disease, angina pectoris, FCII. Conclusion: the treatment is effective. The prognosis of CHD is favorable.

Example 2. Patient P., 65 years old, medical history No. 148, was admitted to the department of coronary heart disease and atherosclerosis of the Research Institute of Cardiology of the city of Tomsk with complaints of pressing pains in the heart and behind the sternum, radiating under the scapula and in the cervical region. No significant changes were detected on the ECG. Bicycle ergometry was stopped at a load of 50 watts due to pain behind the sternum. Pain is stopped by nitroglycerin. An oblique depression of the ST segment in leads V4-V6 appeared on the ECG. HELL 150/110 mm Hg, pulse at rest 68 beats. in minutes

Laboratory results: total cholesterol 8.3 mmol / l, triacylglycerides 1.8 m mol / l, HDL cholesterol 1.0 m mol / l.

The results of electrophoretic studies of blood LP before treatment by the proposed method are presented in Fig.66. The results of densitometry of drugs: LDL - 56%, VLDL - 20%, HDL - 15%, PL (a) - 7%.

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

After treatment, the results of a laboratory study: total cholesterol 6.0 mmol / l, triacylglycerides 1.5 mmol / l, HDL cholesterol 1.3 mmol / l.

The results of electrophoretic studies of blood LP according to the proposed method after treatment are presented in figv.

The results of densitometry of drugs: LDL - 56%, VLDL - 25%, HDL - 17%, PL (a) - 2%.

Diagnosis at discharge: coronary heart disease, angina pectoris, FCII. A decrease in the level of LP (a) by 58%, and total cholesterol by 25% compared with the original testified to the effectiveness of the treatment of coronary artery disease. Conclusion: the treatment is effective, the prognosis of the course of IHD is favorable.

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 and 22 patients of the comparison group with a diagnosis of neurocirculatory dystonia. With a decrease in the level of LP (a) by 40% or more and total serum cholesterol by 20% or more, the prognosis of IHD was assessed as favorable, as evidenced by a change in the functional class (FC) of angina pectoris, and when only one of the indicators changed An unfavorable prognosis of the course of coronary heart disease (transition of a stable course to an unstable form of angina pectoris) has been established.

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.

Claims (1)

A method for predicting the course of coronary heart disease by examining blood serum, characterized in that before and after treatment, total cholesterol and LP (a) are determined in blood serum by additional processing of 0.3 ml of a 0.1% solution of Triton X-100 and incubation for 15 min at 20 ° C, followed by incubation of the patient's blood serum with Sudan B solution for 1 h in a dark thermostat at 40 ° C, and then making a sample in a well in an agarose gel with a base area of 4 × 20 mm for electrophoresis and subsequent fixation electrophoregrams, drying them, By sensitometry and a simultaneous decrease in the level of total cholesterol by 20% or more and PL (a) by 40% or more compared with the initial level, the prognosis of the course of the disease is considered favorable, contributing to the transition of angina from functional class III-IV to functional class I-II and with a decrease in total cholesterol less than 20%, and the level of LP (a) less than 40% compared with the initial prognosis is considered unfavorable.

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