RU2592237C1 - Method for diagnosis of clinical course of "asymptomatic" carotid atherosclerosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be used for diagnosis of clinical course of "asymptomatic" carotid atherosclerosis. Diagnosis of clinical course of "asymptomatic" carotid atherosclerosis is carried out by enzyme immunoassay of blood plasma biomarkers. Enzyme immunoassay of blood plasma is used to determine level of tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), adiponectin, asymmetrical dimethylarginine (ADMA), and biochemical method is used to determine nitrate, nitrite and nitrogen oxide (NO) levels. Then each biomarker is assigned 0 or 1 point depending on decrease or increase thereof relative to standard. Particularly, if levels of t-PA, adiponectin and nitrogen oxide are higher than standard said biomarkers are assigned 0 points, and if lower than standard - 1 point, if values of PAI-1, ADMA, nitrate and nitrite levels are higher than standard, biomarkers are assigned 1 point, and if lower relative to standard - 0 points. Method then includes calculating total score, and if its value is less than 4, favourable clinical course of "asymptomatic" carotid atherosclerosis is diagnosed, and if value is equal to or higher than 4, unfavourable course is diagnosed.

EFFECT: method provides higher accuracy for diagnosis of clinical course of "asymptomatic" carotid atherosclerosis due to specificity and sensitivity of method of simultaneous determination of biomarkers - t-PA, PAI-1, adiponectin, ADMA, nitrate, nitrite and NO.

1 cl, 3 tbl, 2 ex

Description

The invention relates to medicine, in particular to neurology, and can be used to diagnose the course of "asymptomatic" carotid atherosclerosis.

The most important and clinically significant localization of atherosclerotic plaques is the area of bifurcation of the common carotid arteries. The prevalence of carotid atherosclerosis is 25.4% in men and 26.4% in women (aged 18 to 99 years) (Prati Ρ, Vanuzzo D, Casaroli M, Di Chiara A, De Biasi F, Feruglio GA, Touboul PJ. Prevalence and determinants of carotid atherosclerosis in a general population. Stroke. 1992 Dec; 23 (12): 1705-11). Carotid atherosclerosis is one of the main causes of ischemic stroke. The development of ischemic disorders of cerebral circulation (NMC) in patients with stenotic lesions of the arteries supplying the brain can be realized by all known mechanisms. Thus, in the general structure of all verified subtypes of ischemic stroke, the atherothrombotic subtype of stroke (according to the mechanism of arterial-arterial embolism from unstable ASB) occurs in 34% of cases, and hemodynamic stroke (as a result of reduced perfusion pressure against the background of a wall occlusion process) - in 15% of cases (Suslina ZA. Vascular pathology of the brain: results and prospects. Annals of clinical and experimental neurology, 2007, 1; 1: 10-16). At the same time, the process of atherosclerotic lesions of the arteries is the dominant vascular process in all heterogeneous subtypes of NMC.

Based on the fact of the presence or absence in the history of data for ischemic stroke in the interested zone of blood supply, stenosis of the internal carotid artery (ICA) is divided into "symptomatic" and "asymptomatic".

The “asymptomatic” ICA stenosis includes narrowing of the lumen of the carotid artery ≥50% in the absence of a history of retinal ischemia (amaurosis ftigax) and / or brain during the previous 6 months. In the general population, the prevalence of “asymptomatic” ICA stenosis is: from 0% to 7.5% for moderate stenosis (ie 50-70%) and 0-3.1% for severe stenosis (≥70%) (den Hartog AG, Achterberg S, Moll FL, Kappelle LJ, Visseren FLJ, van der Graaf Y, et al. Asymptomatic carotid artery stenosis and the risk of ischemic stroke according to subtype in patients with clinically manifest arterial disease. Stroke. 2013; 44: 1002 -1007).

The amount of scientific information available today on the factors of initiation, development and progression of the atherosclerotic process indicates the extreme complexity and ambiguity of the detected clinical and molecular genetic interactions.

A known method for diagnosing and predicting the course of the atherosclerotic process, which consists in obtaining a tissue sample from an artery wall as a result of a biopsy or during surgery, lyophilization of a sample, recording the EPR spectrum by high-frequency electron paramagnetic resonance (EPR), measuring relaxation times of paramagnetic Mn ² complexes ⁺ on a certified W-band electron paramagnetic resonance spectrometer. When detecting the time of transverse relaxation of Mn ^{2+ ions of} less than 100, a weak degree of calcification of the atherosclerotic plaque is not determined (RU 2468368 C1, 11.27.2012). However, this method does not provide an assessment of the course of “asymptomatic” carotid atherosclerosis and is used only to diagnose the presence of diseases caused by atherosclerosis. In addition, this technique is invasive and can be used for a narrow category of patients who are indicated for surgery. The disadvantage of this method is that it can only be used in specialized centers.

A known method for the study of patients with "asymptomatic" ICA stenosis (50-79%) by MP-research in TOF, Τ1, T2, PDw modes in the area of bifurcation of the common carotid artery at the beginning of observation and then every 18 months. The observation period was 54 months. A group of patients was identified in whom plaque hemorrhage was detected during this period and the rate of progression of the atherosclerotic process (as an increase in ICA wall volume by MRI) was calculated before and after hemorrhage. It turned out that before the development of hemorrhage it was - 20.5 ± 13.1 mm ³ / year (p = 0.129), and after - 18.3 ± 6.5 mm ³ / year (p = 0.008); in patients who did not develop hemorrhage in the plaque, this parameter was 5.3 ± 7.2 mm ³ / year (p = 0.466) (Sun J, Underhill HR, Hippe DS, Xue Y, Yuan C, Hatsukami TS. Sustained acceleration in carotid atherosclerotic plaque progression with intraplaque hemorrhage. JACC: Cardiovascular imaging. 2012; 5 (8): 798-804). Thus, the presence of hemorrhage in the plaque according to the MP study can be considered a suboptimal predictor of the increase in the degree of progression of the atherosclerotic process in the carotid pool. However, in routine practice, the use of the aforementioned relatively expensive technique is not always justified, therefore, in order to clarify the group of “high risk” patients, the search for biomarkers and genetic changes that determine the susceptibility to destabilization of atherosclerotic plaque has been intensively carried out in the last decade. A special place in these studies is occupied by biochemical predictors of endothelial dysfunction. Endothelium plays a leading role in maintaining adequate blood flow through the vascular bed. Endothelial dysfunction is characterized by the formation of a tendency to a decrease in the ability to vasodilation, a pro-inflammatory state, accompanied by prothrombotic activity.

The closest technical solution to the claimed one is a method for diagnosing the course of “asymptomatic” carotid atherosclerosis by enzyme-linked immunosorbent assay in the blood plasma of adiponectin content (Iglseder B, Mackevics V, Stadlmayer A, Tasch G, Ladumer G, Paulweber B. Plasma adiponectin levels and sonographic phenotypes of sub carotid artery atherosclerosis: data from the SAPHIR Study. Stroke. 2005 Dec; 36 (12): 2577-82). The disadvantages of the method is its low diagnostic accuracy, since this biomarker reflects only one of the factors of the nature of atherosclerosis.

The authors of the present invention for the diagnosis of the course of “asymptomatic” carotid atherosclerosis as biomarkers were used tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), adiponectin, asymmetric dimethylarginine (ADMA), nitrate, nitrite and nitric oxide (NO ) This set of biomarkers is due to the following. In particular, it was found that nitric oxide (NO), formed in the endothelium with the participation of the enzyme endothelial nitric oxide synthase (eNOS), is considered by many authors as a powerful vasodilating agent, which is of great importance in the emergence of such a phenomenon as flow-dependent vasodilation. In addition, NO reduces platelet aggregation, slows down the adhesion of monocytes and leukocytes to the endothelium, inhibits the proliferation of smooth muscle cells, and also inhibits the oxidation of low density lipoprotein cholesterol. The determination of the final metabolites, nitrites and nitrates, which are regarded as stable end products of NO, has been widely used in experimental and clinical studies to evaluate the overall synthesis of NO. Increased levels of NO metabolites in hypercholesterolemia are associated with the inhibitory effect of NO during LDL oxidation. The antithrombotic effect of NO may also be associated with a negative effect on the expression of plasminogen activator inhibitor 1 (PAI-1), a prothrombotic protein. Tissue plasminogen activator (t-PA) and PAI-1 directly affect and interfere with the formation and degradation of a blood clot and, therefore, largely determine the risk of developing arterial thrombosis. In addition, an endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginine (ADMA), is synthesized in the human body, the level of which is inversely proportional to flow-dependent dilatation. Adiponectin also contributes to the development of obesity-related diseases, which inhibits the various mechanisms underlying atherogenesis: the expression of adhesion molecules in endothelial cells, proliferation of smooth muscle cells and the formation of foam cells. Changes in the biomarkers described above, which are specific for endothelial dysfunction, lead to a change in the parameters determined in blood samples, which, when used together, are suitable for diagnosing the course of "asymptomatic" carotid atherosclerosis with high accuracy.

The technical result of the claimed invention consists in high accuracy in the diagnosis of the course of “asymptomatic” carotid atherosclerosis due to the specificity and sensitivity of the method for the joint determination of biomarkers - tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), adiponectin, asymmetric dimethylarginine (ADMA) nitrate, nitrite and nitric oxide (NO).

The technical result is achieved in that the diagnosis of the course of “asymptomatic” carotid atherosclerosis is carried out by enzyme-linked immunosorbent assay in the blood of biomarkers, characterized in that in the blood plasma the levels of tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1) are determined, adiponectin, asymmetric dimethylarginine (ADMA) and a biochemical method in the blood serum levels of nitrate, nitrite and nitric oxide (NO), with each biomarker assigned 0 or 1 point depending on its increase or decrease relative to the norm, namely, when the values of t-PA, adiponectin and NO are higher than the norm, these biomarkers are assigned 0 points, and when they decrease relative to the norm, 1 point, when the values of PAI-1, ADMA, nitrate and nitrite are above the norm these biomarkers are assigned 1 point, and when they are reduced relative to the norm - 0 points, then the sum of the points is calculated and, if it is less than 4 points, a favorable course of asymptomatic carotid atherosclerosis is diagnosed, and if it is 4 or more, it is unfavorable.

The method is as follows.

Assays for the activity of t-PA, PAI-1, adiponectin and ADMA biomarkers were performed at the hemorheology and hemostasis laboratory of the FSBI NCH. The study was conducted using a Victor2 microplate reader, Perkin-Elmer (USA). In all studies, calibrators of reagent manufacturers were used. Enzyme-linked immunosorbent assays were monitored in duplicates using lyophilized control sera / plasma with a low and high content of the studied parameters.

Blood samples were taken on an empty stomach, from the ulnar vein with a disposable needle into a vacuum tube (containing EDTA). Further, within 30 minutes from the time of sampling, blood samples were centrifuged for 15 minutes at a speed of 2500 g. The studied plasma was further aliquoted and frozen at a temperature of - 80 ° C. Thawing of the samples was carried out immediately before the ELISA.

Analyzes of biomarkers of NO, nitrite and nitrate were carried out in blood serum samples by the biochemical method in a microplate format at a wavelength of 540 nm and a measurement range of 0.78-200 μmol / L.

The most common complaints of patients with “asymptomatic” atherosclerosis were: headache and / or less tinnitus, as well as instability of mood and memory. Numbness in the distal arms and legs occurred in a number of patients, but it was most likely associated with diabetic polyneuropathy, which was present in these patients.

When analyzing the neurological status of the indicated cohort of patients, the main syndrome was cephalgic, cognitive impairment was less frequent. Headaches were encountered quite often, in 56% of cases (n = 65), the overwhelming majority had a “pressing”, “dull” character, localized in the frontotemporal and occipital areas.

Cognitive impairment was represented mainly by complaints of impaired memory (mainly on current events). To objectify the revealed changes, the MosA questionnaire was used. The average score on this scale in the study group was 24.9 ± 2.6 points, which indicates the presence of mild-moderate cognitive dysfunction in patients with asymptomatic atherosclerosis.

In these patients with “asymptomatic” carotid atherosclerosis, enzyme-linked immunosorbent assays for plasma biomarkers are carried out, namely, levels of t-PA, PAI-1, adiponectin, ADMA are determined using a biochemical method to determine the levels of nitrate, nitrite and NO in blood serum. At the same time, each biomarker is assigned 0 or 1 point depending on the decrease or increase in its level relative to the norm. Moreover, when t-PA, adiponectin and NO are higher than normal, these biomarkers are assigned 0 points, and when they are lower than normal, 1 point, when PAI-1, ADMA, nitrate and nitrite are higher than normal, these biomarkers are assigned 1 point, and with their decrease relative to the norm - 0 points. After that, the sum of the points is calculated and, with its value less than 4 points, a favorable course of asymptomatic ”carotid atherosclerosis is diagnosed, and with a value of 4 or more, it is adverse.

The determination of adiponectin concentration was carried out as follows. During the BioVendor Human Adiponectin ELISA assay, standards, samples and controls were incubated in microplate wells coated with horseradish peroxidase-labeled human anti-adiponectin monoclonal antibodies. Then, other monoclonal human sTfR antibodies labeled with horseradish peroxidase enzyme were added to the wells with the immobilized antibody-sTfR complex. After the second incubation and washing procedure, tetramethylbenzidine substrate and hydrogen peroxide were added to the wells, with which the conjugated conjugated antibodies reacted. The latter was stopped by the addition of an acid solution, and the absorption of the resulting yellow solution was determined spectrophotometrically at a wavelength of 450 nm. The absorption indicators are proportional to the concentration of adiponectin in the test sample, the numerical value of which was determined by the calibration curve. The specified technique is a variant of the "sandwich" ELISA.

The determination of the concentration of the plasminogen activation inhibitor of the first type and the tissue plasminogen activator was carried out by the same method as the determination of adiponectin, only using the Technoclone kit.

The determination of the concentration of asymmetric dimethylarginine was based on a competitive ELISA, and Immundiagnostik reagents were used. Prior to the analysis, a derivative reagent was added to blood samples. Then, such samples prepared together with polyclonal anti-ADMA serum were incubated in the wells of a microplate coated with an ADMA derivative. During the incubation period, ADMA in the test plasma competes for binding to polyclonal antibodies with the ADMA derivative originally present in the onion. The following stages of this test are carried out according to the method described by the definition of adiponectin.

The determination of nitric oxide was carried out in samples of blood serum by a biochemical method in a microplate format. Wavelength: 540 nm. Measurement range: 0.78-200 μmol / L. Analytical sensitivity: 0.78-200 μmol / L. This method is based on the enzymatic conversion of nitrate to nitrite under the action of the nitrate reductase enzyme. A specific nitrite-ion reaction based on the synthesis of an azo compound (Griss reaction) is recorded colorimetrically. The Griss reaction is a two-stage diazotitration reaction in which acidic NO ₂ (a nitrosating agent reacts with sulfanilic acid to form a diazonium ion. This ion binds to N- (1-naphthyl) ethylenediamine to form a colored azo derivative that absorbs light at a wavelength of 540- 570 nm The Total NO / Nitrite / Nitrate Assay kit of R&D Systems (USA), which has 2 analytical options, was used for determination, measurement of endogenous nitrite is the first option, conversion of nitrate to nitrite using nitrate reductase and measurement of total nit Rita is the second, to obtain the concentration of nitrate, the concentration of endogenous nitrite is calculated from the total nitrite.

The study included 117 patients with “asymptomatic” stenosis of one of the internal carotid arteries (at least 50%) and 105 volunteers without signs of atherosclerosis in the carotid artery system (verified by duplex scanning of the main arteries of the head and neck, carried out no later than 6 months before the patient is included in the study). The criterion for the "asymptomaticity" of stenosis, in accordance with current recommendations, was the absence of ischemic stroke in the pool of the affected internal carotid artery for 6 months from the time stenosis was detected. However, in this study, it was decided to narrow down the criteria for inclusion of patients in the study group - only patients were selected who had no previous history of cerebrovascular accident (both ipsi and contralateral to the stenotic artery) of any etiology (as ischemic and hemorrhagic genesis). The average age of the examined was 65.7 ± 9.3 years; men predominated somewhat (59%) (see table 1). All patients in the study group were interviewed on the Montreal Cognitive Assessment test (MOCA), which included questions on identifying visual-spatial disorders, reducing auditory-speech memory, concentration and attention.

The examination of the patients included both instrumental techniques and a comprehensive assessment of the somatic and neurological status, routine clinical and laboratory tests and determination of biomarkers by enzyme-linked immunosorbent assay, namely, blood plasma levels of t-PA, PAI-1, adiponectin, ADMA, nitrate were determined nitrite and NO.

Given the multifactorial nature of atherosclerosis, an analysis was made of the biomarker “burden” of patients with “asymptomatic” stenoses of the brachiocephalic arteries (BCA). For this purpose, reference values of the studied parameters were determined and the level of a biomarker was evaluated in relation to the control group (see table 2).

Reference levels for all markers were selected based on their average values in the control group, which was the norm for the study group of patients with asymptomatic carotid atherosclerosis. Moreover, with t-PA, adiponectin and nitric oxide levels above the norm, these biomarkers were assigned 0 points, and when they are lower than the norm, 1 point, with PAI-1, ADMA, nitrate and nitrite levels above the norm, these biomarkers were assigned 1 point, and when they are reduced relative to the norm - 0 points.

It turned out that the median of the average score on the scale in patients with "asymptomatic" carotid atherosclerosis was 4 points or more, and in patients in the control group it was below 4 points.

That is, the trend towards a more “favorable” biomarker profile was in 75% of patients in the control group. At the same time, in patients with asymptomatic carotid atherosclerosis with the involvement of 4 or more biomarkers, there was a tendency to an “unfavorable” biomarker profile in 85.8% of cases (see Table 3).

Thus, the claimed method provides high accuracy in the diagnosis of the course of "asymptomatic" carotid atherosclerosis.

Examples of the method

Example 1

Patient E., 60 years old, turned to the scientific advisory department of the Scientific Center for Neurology with complaints of headaches and a slight decrease in memory for current events. From the anamnesis it is known that for a long time suffers from arterial hypertension with a moderate increase in blood pressure figures (maximum - up to 160/100 mm Hg). He did not systematically take antihypertensive therapy; in 2011, impaired glucose tolerance was diagnosed.

On examination, the constitution is hypersthenic (BMI - 29 kg / m ² ). Blood pressure - 140/90 mm Hg In neurological status, finely divided nystagmus is observed when looking to the left. The face is symmetrical at rest and when performing facial tests. Swallowing and phonation are not disturbed. The soft palate is symmetrical. Reflexes from the soft palate and posterior pharyngeal wall are live. Tongue in the midline. There are no paresis. Tendon reflexes are symmetrically reduced. There are no pathological reflexes. There is a decrease in pain sensitivity in the distal arms and legs. During the coordination tests, some uncertainty is noted. In the trial, Romberg staggers.

When conducting complex neuropsychological testing, the patient revealed mild cognitive impairment, mainly involving short-term memory. When examined on the Montreal scale of assessment of cognitive functions (MoCA), the patient scored 22 points (normal - 28 and above).

When conducting a biochemical blood test, the following were revealed: fasting glycemia - 6.3 mmol / l, glycated hemoglobin - 6.2%. Plasma glucose level 2 hours after loading during OHTT - 9.2 mmol / L. Noteworthy is a violation of lipid metabolism in the form of an increase in triglycerides to 2.24 mmol / L, a decrease in HDL cholesterol to 0.78 mmol / L and an increase in cholesterol to 6.7 mmol / L.

When assessing hemostasis, an increase in platelet aggregation under the influence of adrenaline to 54 (normal 37-43%) and ADP to 60 (normal 40-46%), an increase in fibrinogen level to 4.3 g / l, as well as a decrease in the index, were found fibrinolytic activity up to 5%.

When assessing the biomarkers of endothelial dysfunction, an increase in the concentration of nitrate and nitrite (157 and 155 μmol / L, respectively) was noted relative to the norm, with a pronounced decrease in the plasma level of NO to 2 μmol / L. A marked increase in PAI-1 (up to 8.4 U / ml) was accompanied by a decrease in the activity of tissue plasminogen activator to 1.2 ng / ml. The level of ADMA was sharply increased - 0.78 μmol / l, and adiponectin - reduced (6.5 μg / ml). According to the biomarker scale (see section 3.5), the patient scored a maximum of 7 points, since all indicators were outside the reference values (normal values). The diagnosis of an unfavorable course of "asymptomatic" carotid atherosclerosis was made.

Given such pronounced changes in the marker molecules of the atherosclerotic process, the patient underwent DS MAG, where 60% stenosis of the right ICA and 55% of the stenosis of the left ICA were detected. MRI revealed signs of dyscirculatory encephalopathy, however, no data were obtained for acute NMC.

Thus, in this patient changes are detected that affect almost all links of atherogenesis - inflammation, fibrinolytic activity, increased hemostatic activation, lipid metabolism. Despite the fact that the patient is essentially “asymptomatic,” such a large set of different molecular markers is a high risk factor for the possible destabilization of atherosclerotic plaque and the further development of acute NMC by the type of atherothrombosis, arterial-arterial embolism, or by hemodynamic subtype. All of the above was taken into account when choosing the "aggressive" therapeutic tactics for this patient.

Against the background of the appointment of adequate antiplatelet, antihypertensive and hypolipidemic therapy, as well as drugs that affect carbohydrate metabolism, an improvement in the clinical picture of this patient was noted - cognitive impairment regressed somewhat, cephalgic syndrome almost disappeared, and blood pressure numbers stabilized. 12 months after the initial examination, the biomarker indices were reanalyzed: a positive trend was revealed in the form of a decrease in the score on the biomarker scale to 5 (due to a decrease in ADMA and an increase in t-PA concentration). There was no clinical “voicing” of carotid atherosclerosis for the indicated observation period. Progression of the degree of stenosis was within acceptable values — by 5% in the left ICA.

Example 2

Patient K., 57 years old, went to the clinic with complaints of episodic headache, memory loss (mainly on current events). From the anamnesis it is known that cephalgic syndrome has been disturbing for about 3 years, is associated with psychoemotional stress. Memory impairment has been disturbing for a year now, but it doesn’t “interfere” with the patient’s daily life. Previously not examined.

An examination in the scientific advisory department revealed some staggering in the Romberg test, the value of the score on the MoCA scale is 28, which corresponds to the lower limit of the norm. Blood pressure during examination was 130/80 mm Hg, heart rate - 78 per minute. There were no signs of another somatic pathology during examination.

In general clinical and biochemical blood tests, a slight increase in total cholesterol and a decrease in high-density lipoprotein cholesterol (6.3 mmol / L and 0.8 mmol / L, respectively) are detected. When assessing hemostasis, an increase in platelet aggregation under the influence of adrenaline to 50 (normal 37-43%) and ADP to 53 (normal 40-46%) was found.

The patient was examined using the indicated biomarker model. Normal levels of t-PA (2.76 ng / ml), PAI-1 (2.4 U / ml), an increase in adiponectin to 18 μg / ml (which also corresponds to the norm) were noted. Nitric oxide and nitrate values were also within the reference values (25.9 μmol / L and 43 μmol / L, respectively), except for the level of nitrite, which was above the norm (23 μmol / L). Deviations were also observed in the level of ADMA, which amounted to 0.53 μmol / L. In total, the rating on the scale was 2 points, which corresponds to the "favorable" profile of "asymptomatic" carotid atherosclerosis.

Given the patient's age and complaints, it was decided to conduct an ultrasound examination of the brachiocephalic arteries, where only a small (up to 1.1 mm) thickening of the intima-media complex of the right internal carotid artery was revealed.

Against the background of the appointment of some sedative therapy and recommendations for lifestyle changes, the patient's condition improved markedly, cephalgic syndrome regressed, memory impairments significantly decreased.

Thus, the joint use of biomarkers - t-PA, PAI-1, adiponectin, ADMA, nitrate, nitrite and NO for the diagnosis of the course of “asymptomatic” carotid atherosclerosis allows with a high accuracy and reliability to diagnose its favorable (minimal signs of severity, thickening of the intima complex media of the carotid arteries, a small percentage of stenosis) and an unfavorable course (severe [> 50%] stenosis of the carotid arteries, unstable atherosclerotic plaque).

Claims (1)

A method for diagnosing the course of “asymptomatic” carotid atherosclerosis, including enzyme-linked immunosorbent assay in the blood plasma of biomarkers, characterized in that plasma levels of tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), adiponectin, asymmetric dimer are determined by plasma immunoassay (ADMA) and biochemical methods in the blood serum levels of nitrate, nitrite and nitric oxide (NO), and each biomarker is assigned 0 or 1 point depending on its decrease or increase relative about the norm, namely, when the levels of t-PA, adiponectin and nitric oxide are higher than the norm, these biomarkers are assigned 0 points, and when they decrease relative to the norm - 1 point, when the values of PAI-1, ADMA, nitrate and nitrite are higher than the norm for these biomarkers 1 point is assigned, and when they are reduced relative to the norm - 0 points, then the sum of the points is calculated and, if it is less than 4 points, a favorable course of “asymptomatic” carotid atherosclerosis is diagnosed, and with a value of 4 or more, it is unfavorable.