RU2665139C1 - Method for determining probability of presence of unstable atherosclerotic plaques in coronary arteries in patients with coronary atherosclerosis - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to the field of medicine, namely to cardiology, and relates to a method for determining the probability of the presence of unstable atherosclerotic plaques in the coronary arteries in patients with coronary atherosclerosis. Method consists in determining the values of inflammatory biomarkers in the blood serum of a patient with coronary atherosclerosis: C-reactive protein, interleukin-6, interleukin-8, monocyte chemoattractant protein-1, and an oxidative biomarker — a FORT test, the obtained values are compared with the threshold value of the corresponding biomarker: for a C-reactive protein, a value of 10 mcg/ml, for interleukin-6, a value of 10 pg/ml, for interleukin-8, a value of 12 pg/ml, for monocyte chemoattractant protein-1, a value of 480 pg/ml, for the FORT test, a value of 2.3 mmol/l. If there are at least four biomarker values greater than the threshold value of the corresponding biomarker, there is a probability of the presence of unstable plaques in the coronary arteries in a patient with coronary atherosclerosis.EFFECT: this method will allow to determine at an early stage the possibility of development of complications of IHD, to accelerate the pace of additional examination and choice of tactics for treatment of the patient.1 cl, 2 tbl, 3 ex

Description

The invention relates to medicine, namely to cardiology, and can be used for early diagnosis of complications of coronary heart disease (CHD), such as acute coronary syndrome, and the choice of tactics for treating this disease.

The prevalence and mortality from complications of cardiovascular diseases, such as myocardial infarction, unstable angina and sudden cardiac death, combined by the general term “acute coronary syndrome” (ACS), comes first in many countries of the world, including Russia [ one].

There are many research methods that allow you to assess the condition of the vessels of the heart:

1. Echocardiography or ultrasound (ultrasound) is a popular non-invasive method for examining the heart and blood vessels using ultrasound. This study allows us to determine the elasticity and rigidity of the vascular walls, the integrity and functional state of the inner surface of blood vessels, changes in the structure and thickness of the walls of the vessel. If atherosclerotic blood clots and plaques are detected, it is possible to determine their localization, echogenicity, and surface condition.

2. Ultrasonic scanning allows you to accurately assess the condition of large vessels of the heart, which cannot be said about smaller vessels of the heart and this is a disadvantage of the method. In addition, atherosclerotic lesions impede the passage of the ultrasound wave and may distort the results. As well as an ultrasound study depends on the qualification level of the doctor researcher.

3. Coronarography (coronaroangiography) is an X-ray examination using a contrast agent that allows you to diagnose all coronary lesions, including severe multivascular lesions, with high accuracy. Such an examination is considered the standard for the diagnosis of coronary heart disease, as it allows you to accurately determine the place and degree of spasm, stenosis or obstruction of the coronary arteries of the heart.

The disadvantage of this method is its invasive nature and a high risk of various complications during the study. In addition, one should take into account the limited availability of this method in real medical practice. The presence of such equipment is possible only in large specialized cardiovascular centers.

4. Computed tomography (CT) is a modern method of studying the internal organs of a person using x-ray radiation. To increase the information content, CT is performed using a contrast medium (in particular, in the study of blood vessels and hollow organs).

The disadvantage of this method is its high cost and the availability of special equipment. As a rule, only large medical centers have such equipment. In addition, CT gives a radiation load on the tissue, so it can not be used repeatedly.

5. Magnetic resonance imaging (MRI) is based on the principle of obtaining an array of data and modeling on its basis a three-dimensional image of an organ using electromagnetic waves. MRI can detect the changes in the vessels that occur with atherosclerosis and are not available to other diagnostic methods: the presence of atherosclerotic plaques; their size, shape, stability; the degree of narrowing of the vessel at the location of the plaque; process prevalence; the presence of blood clots; abnormalities of the development of blood vessels.

The disadvantage of this method is the high cost and long research time. Similar studies are carried out in specialized clinics with the participation of highly qualified specialists.

The well-known "Method for the diagnosis of coronary lesions" (RF patent No. 2261657, АВВ 8/00), in which during stress echocardiography with physical activity determine violations of regional contractility of the left ventricular myocardium. On the basis of changes in the speeds of movement of the basal segment of the anterior wall and the median segment of the lateral wall of the left ventricle, damage to the anterior interventricular branch and the envelope of the branch of the left coronary artery is diagnosed.

The disadvantage of this method is the use of expensive equipment, the participation of highly qualified specialists to perform complex diagnostic tests, which introduces limitations on the implementation of this method in real clinical practice.

The well-known "Method for the diagnosis of coronary lesions" (RF patent No. 2180520, A61B 8/06, A61N 1/3), which consists in the fact that during stress echocardiography tests with transesophageal cardiac pacing or dobutamine record shock volume (UO). When it is reduced in the first steps of the test with transesophageal electrocardiostimulation by 30% or more compared with the initial value or decreased in the last steps of the test with dobutamine, a severe multivascular lesion of the coronary bed is diagnosed.

The disadvantage of this method is the conduct of volumetric diagnostic procedures using expensive equipment and highly qualified specialists and requires a lot of time to conduct. This technique allows you to diagnose severe multivascular damage. But during the study, complications are possible, unfavorable for patients, up to resuscitation.

It should be noted that in all cases of clinical manifestations of coronary atherosclerosis and coronary artery disease, the trigger is the violation of the integrity of the endothelium in the ulceration / destruction of the tire of unstable atherosclerotic plaque (ASB) and subsequent thrombosis, leading to occlusion of the coronary artery (CA), ischemia and necrosis , 3].

It is known that stable ASB is characterized by a thick tire, a homogeneous lipid nucleus, the absence of inflammatory changes, and unstable ASB is characterized by a thin tire (thickness <65 μm) or a thinned tire with focal endothelial destruction, inflammatory cell infiltration (more than 25 cells in the field of view of length 0 , 3 mm), a loose lipid core (> 40% of the volume of ASB) with areas of necrosis [3-5].

An important role in the development of unstable ASB is played by the inflammatory and destructive process [6-10], during which significant infiltration of unstable ASB by monocytes / macrophages (MF) and T-lymphocytes (T-LF) secreting pro-inflammatory cytokines, including interleukin 1 (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-18 (IL-18), tumor necrosis factor alpha (TNF-α) and others. IL-1β and TNF-α induce the formation of adhesion molecules by endothelial cells, leading to a decrease in the release and anti-coagulation properties of the endothelium, IL-6 - the cytotoxic factor of T-LF differentiation - stimulates the synthesis and secretion of the main proteins of the acute phase of inflammation, IL-8 stimulates the production of monocytes / MF of factors activating chemotaxis of neutrophils and T-LF into atherosclerotic foci, IL-18 secreted by T-LF activates monocyte / MF and initiates apoptosis [6-10].

Activated MFs also secrete a number of chemoattractants, including monocytic chemotactic protein (MCP-1), endothelial-monocytic activating polypeptide (EMAP-II), cell-cell adhesion molecules (sICAM-1), and endotheliocyte adhesion molecules (sVCAM-1). MCP-1, expressed by MF in response to exposure to the cytokines TNF-α, IL-1β and IL-6, is a monocyte and T-LF-specific chemoattractant [3, 11-14].

According to a number of studies, patients with ACS in the blood have elevated levels of inflammatory cytokines and biomarkers such as IL-1β, IL-6, TNF-α, C-reactive protein (CRP), their independent associations with the development of atherosclerosis and ACS have been identified [15-18].

The leading pathogenetic component of cardiovascular diseases of atherosclerotic origin, including ischemic heart disease and myocardial infarction, is oxidative stress with increased generation of active oxygen metabolites (AKM). With the development of oxidative stress, all the main classes of biomolecules, including lipids, proteins, and nucleic acids, can become critical elements of the damaging effects of AKM, which leads to their pathological oxidative modification. One of the key roles in the appearance and development of the atherosclerotic lesion belongs to the formation of oxidized modified low density lipoproteins in the blood and in the vascular wall of rich cholesterol [19-21].

The objective of the claimed invention is the creation of a non-invasive, affordable and inexpensive biochemical method for determining the likelihood of unstable atherosclerotic plaques in the coronary arteries, which can be used for early diagnosis of coronary heart disease complications in patients with coronary atherosclerosis, will accelerate the pace of additional examination and choice of treatment tactics for patients.

The essence of this method lies in the fact that to determine the likelihood of unstable atherosclerotic plaques in the coronary arteries in patients with coronary atherosclerosis in the patient's blood serum, the values of inflammatory biomarkers are determined: C-reactive protein, interleukin-6, interleukin-8, monocytic chemoattractant protein-1 , and the oxidative biomarker - FORT test, the obtained values are compared with the threshold value of the corresponding biomarker: for C-reactive protein equal to 10 μg / ml, for interleukin-6 explicit 10 pg / ml, for interleukin-8 equal 12 pg / ml, for monocytic chemoattractant protein-1 equal to 480 pg / ml, for the FORT test equal to 2.3 mmol / l, and if at least four biomarkers are greater threshold values of the corresponding biomarker indicate the likelihood of unstable plaques in the coronary arteries in a patient with coronary atherosclerosis.

The method is as follows.

In an examined patient, an oxidative biomarker - FORT test, inflammatory biomarkers: rf-CRP, IL-6, IL-8, MCP-1, are determined in laboratory blood tests by laboratory tests and compared with threshold values of these biomarkers.

Statistical analysis of previous studies [9–10] made it possible to determine the conditional threshold boundary for each indicated indicator based on the construction of distribution histograms. An increase in the incidence of patients with unstable atherosclerotic plaques for the MCP-1 indicator is observed at values above 480 pg / ml. The threshold value for IL-6 was 10 pg / ml. The distribution of patient results according to IL-8 gives us the basis for determining the threshold value of 12 pg / ml. A guideline for assessing the critical level of CRP in the blood was a concentration of 10 μg / ml. For the FORT test results, values greater than 2.3 mmol / L are considered elevated.

An increase in four or more indicators from the studied laboratory and biochemical complex indicates the likelihood of unstable atherosclerotic plaques in the coronary arteries in patients with coronary atherosclerosis.

The proposed method was developed on the basis of a study of a group of patients with coronary atherosclerosis. The group consisted of 62 patients 48-72 years old (average age 55.4 ± 3.9 years) with coronarographically verified coronary atherosclerosis, with stable angina pectoris of functional class II-III, who were admitted to the Clinic of the Federal State Research Institute for Research and Management of Rosmedical Technologies for coronary bypass surgery. In patients admitted to the operation, endarterectomy from the SC was performed during intraoperative indications during the operation.

Each endarterectomy material containing intima-media of the spacecraft was longitudinally and transversely symmetrically divided into several fragments for histological and biochemical studies. Histological analysis of fragments of the intima-media of the AC after a macroscopic description of the samples (prevalence of ASB, degree of narrowing of the lumen of the artery, hemorrhage in the structure of the ASB, calcification sites, blood clots) and standard staining with hematoxylin and eosin and according to Van Gieson was performed using an Axiostar Plus binocular microscope. After histological analysis of intima-media fragments, only stable ASB were determined in 38 patients, only unstable ASB in 24 patients. Stable and unstable ASBs were differentiated according to the described criteria [3-5].

In homogenates of intima-media fragments and in blood serum, the levels of pro-inflammatory cytokines TNF-α, IL-6, IL-8 (Bender MedSystems kits), RF-CRP, and chemoattractant MCP-1 were determined by enzyme-linked immunosorbent assay.

A direct assessment of oxidative stress (FORT test - Free Oxygen Radicals Testing) was performed on a FORM Plus CR3000 analyzer (Callegary, Italy). The severity of oxidative stress (FORT test) was evaluated by the allocation of reactive oxygen species using colorimetric registration. The method is based on the ability of metal ions to catalyze the formation of free radicals in the presence of hydroperoxides.

The research results obtained earlier [9, 10] showed that for unstable ASB, in comparison with other stages of development of atherosclerotic foci (lipid spot, stable young and fibrous ASB), the highest concentrations of IL-6 and IL-8 are characteristic (15, 9 ± 1.1 and 37.1 + 4.8 pg / mg protein, respectively), chemoattractant MCP-1 - 401.7 + 39.8 pg / ml [9, 10]. Despite the fact that the level of rf-CRP in the blood of more than 2.0 μg / ml is considered to be increased [18], in patients with unstable ASB, the concentration of rf-CRP in the blood is taken at a concentration of 10 μg / ml.

To determine significant biomarkers of blood instability of atherosclerotic foci of coronary arteries, a statistical analysis of the results was carried out in 2 stages.

First, to search for associations between indicators in the vascular wall of the spacecraft and in the blood, all patients, according to the results of histological studies, were divided into 2 groups:

Group 1 - patients in whom only stable ASB were found in fragments of the intima-media and CA;

Group 2 - patients in whom only unstable ASB were found in fragments of the intima-media and CA.

The results of studies in blood serum are shown in table 1.

The concentration of rf-CRP and IL-6 in serum in patients of the 2nd group were 1.3 times higher than in patients of the 1st group. These data correspond to [9, 10] the results of a study of the content of inflammatory cytokines at different stages of development of atherosclerotic foci. Similar, but significantly more pronounced differences between the groups were revealed for the concentration of IL-8 in the blood. So, in patients of group 2, this indicator was more than 3 times higher than in patients of group 1. The data obtained emphasize the significant place of this cytokine, which stimulates the production of monocytes / MF [7, 14], among biomarkers of destabilization of atherosclerotic foci not only in the vascular wall, but also in the blood.

Elevated values of MCP-1 in the blood were also detected in patients with unstable ASB in the coronary arteries (Table 1).

At the second stage of statistical data processing, a correlation analysis was performed between the parameters of inflammatory biomarkers in the blood and inflammatory biomarkers in the vascular wall, taking into account the nonparametric distribution of characters. The results are shown in table 2.

Note: * at p <0.05; ** - at p <0.001

When discussing the result, it is important to note that HF-CRP, an acute phase inflammatory protein, is mainly synthesized by hepatocytes under the influence of IL-6, but it can also be produced by MF and lymphocytes in ASB [7, 14]. In the present study, it was difficult to determine the source of an increased level of rf-CRP in the blood (rf-CRP from hepatocytes or rf-CRP from the vascular wall). Most likely, both sources existed: since in our study, as in others [7, 10, 14, 22], the levels of IL-6 and rf-CRP, in addition to the correlation of rf-CRP in the vascular wall and IL-6 in the blood ( table 2), correlated with each other (r = 0.521; p <0.01) in the blood and in the vascular wall (r = 0.379; p <0.01). It should be noted that only HF-CRP, IL-6 (weak bonds) and IL-8 (average bond strength) correlated in the blood with ASB instability in the blood. There were also correlations in the blood of IL-8 with the content of TNF-α and MCP-1 in the vascular wall (table 2).

The increase in oxidative processes in the body, in particular the oxidative modification of LDL, contributes to the progression of the atherosclerotic process in the vessels [20-23]. Lipid peroxidation reactions are free radical and constantly occur in the body. The activity of lipid peroxidation processes can be assessed by the concentration of MDA or determine the total degree of oxidative stress in the blood.

In the study, when determining the overall assessment of oxidative stress in the blood (FORT test), the inventors obtained the following results: in patients of group 2, the level of oxidative damage was 2.93 ± 0.2 mmol / l, and in patients of group 1 - 2.81 ± 0 3 mmol / L. Moreover, in previous studies, it was determined that in patients without coronary heart disease, the level of oxidative damage on average is 1.6 ± 0.3 mmol / l [23].

Clinical examples

Example 1. Patient K. (No. 21), man, age 57 years. Clinically, laboratory and instrumentally confirmed the diagnosis of coronary heart disease. A biochemical analysis of blood serum was performed:

IL-6 - 26.74 pg / ml

IL-8 - 2.85 pg / ml

rf-CRP - 18.35 mcg / ml

MCP-1 -490.89 pg / ml

FORT - 3.11 mmol / L

The obtained values were compared with threshold values:

IL-6 - 10 pg / ml

IL-8 - 12 pg / ml

rf-CRP - 10 μg / ml

MCP-1 - 480 pg / ml

FORT - 2.3 mmol / L

Four indicators are increased from the studied complex in the patient, which indicates a high probability of the presence of unstable ASB in the coronary arteries.

Histological examination of a sample of the intima-media fragment of the spacecraft obtained during endarterectomy according to intraoperative indications confirmed the presence of unstable atherosclerotic plaques in the coronary vessels.

Example 2. Patient M. (No. 22), man, age 63 years. Clinically, laboratory and instrumentally confirmed the diagnosis of coronary heart disease.

Conducted biochemical research methods:

IL-6 - 36.68 pg / ml

IL-8 - 9.07 pg / ml

RF-CRP - 17.24 mcg / ml

MCP-1 - 614.01 pg / ml

FORT - 2.87 mmol / L

The obtained values were compared with threshold values:

IL-6 - 10 pg / ml

IL-8 - 12 pg / ml

rf-CRP - 10 μg / ml

MCP-1 - 480 pg / ml

FORT - 2.3 mmol / L

From the studied complex, the patient has increased values of four indicators, which indicates a high probability of the presence of unstable ASB in the coronary arteries.

Histological examination of a sample of the intima-media fragment of the CA obtained during endarterectomy confirmed the presence of unstable atherosclerotic plaques in the coronary vessels.

Example 3. Patient S. (No. 73), man, age 67 years. Clinically, laboratory and instrumentally confirmed the diagnosis of coronary heart disease.

Conducted biochemical research methods:

IL-6 - 9.8 pg / ml

IL-8 - 21.42 pg / ml

rf-CRP - 0.16 mcg / ml

MCP-1 - 383.16 pg / ml

FORT - 3.28 mmol / L

The obtained values were compared with threshold values:

IL-6 - 10 pg / ml

IL-8 - 12 pg / ml

rf-CRP - 10 μg / ml

MCP-1 - 480 pg / ml

FORT - 2.3 mmol / L

From the studied complex, the patient has increased values of two indicators, which indicates a low probability of the presence of unstable ASB in the coronary arteries.

Histological examination of a sample of the intima-media fragment of the CA obtained during endarterectomy confirmed the presence of only stable atherosclerotic plaques in the coronary vessels.

Thus, key oxidative and inflammatory blood biomarkers were identified that characterize the presence of unstable atherosclerotic plaques in the coronary arteries.

The proposed method allows to determine in patients with coronary atherosclerosis the likelihood of unstable atherosclerotic plaques in the coronary arteries. This method is non-invasive, safe, not requiring large financial costs, can be performed in a clinical biochemical laboratory.

In addition, this method will allow at an early stage to determine the possibility of developing complications of coronary heart disease, to accelerate the pace of additional examination and the choice of treatment tactics for the patient.

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Claims (1)

A method for determining the likelihood of unstable atherosclerotic plaques in the coronary arteries in patients with coronary atherosclerosis, characterized in that the values of the inflammatory biomarkers are determined in the patient's blood serum: C-reactive protein, interleukin-6, interleukin-8, monocytic chemoattractant protein-1, and oxidative biomarker - FORT test, the obtained values are compared with the threshold value of the corresponding biomarker: for C-reactive protein equal to 10 μg / ml, for interleukin-6 equal to 10 pg / ml, for interleukin-8 equal to 12 pg / ml, for monocytic chemoattractant protein-1 equal to 480 pg / ml, for the FORT test equal to 2.3 mmol / l and if there are at least four biomarkers above the threshold value of the corresponding biomarker, the probability of unstable plaques in the coronary arteries in a patient with coronary atherosclerosis.