RU2331883C1 - Method of angiopathy development risk diagnostics in arterial hypertension cases with metabolic syndrome - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be applied to angiopathy development risk diagnostics in arterial hypertension (AH) cases with metabolic syndrome (MS). Essentially, thrombocyte aggregative activity with collagen, antithrombin III level, euglobuline lysis time, and nitrate level in patient blood are determined, before and during temporal venous occlusion. Then vascular wall antiaggregatory activity index (VWAAI), vascular antithrombin III synthesis index (VASI), vascular wall fibrinolytic activity index (VWFAI), and nitrate index (NI) are calculated. After this, vascular antithrombotic index (VATI) by formula: VATI = (VWAAI + VASI + VWFAI + NI)/4 is calculated. If the value achieves 2.27 and more, angiopathy development risk is eliminated; if 2.26-2.10, the patients are included in angiopathy development risk group; if 2.09 and less, angiopathy develops.

EFFECT: increase in prediction accuracy of the disease development risk, possibility to administer adequate treatment in good time.

2 tbl, 3 ex, 1 dwg

Description

The invention relates to medicine, namely to cardiology and angiology.

Known methods for the study of vascular functions (Barkagan Z.S., Momot A.P. Basics for the diagnosis of hemostatic disorders. M., 1999. - 214 p.), However, a method for early detection of attenuation of antithrombotic activity of blood vessels in general has not been developed.

In clinical laboratory diagnostics, a method for studying the antiplatelet activity of the vascular wall is used, the level of antithrombin III (AT III), the activity of plasminogen before and after temporary venous occlusion, and the content of nitric oxide (NO) in plasma by the level of nitrates are estimated.

However, never before have these methods been combined into a single diagnostic complex, and the results obtained have not been statistically processed with the calculation of the antithrombotic vascular index (PTIS).

The aim of the invention is to increase the effectiveness of early diagnosis of the risk of developing vasopathy in patients with arterial hypertension with metabolic syndrome.

The essence of the proposed method lies in the fact that for the early diagnosis of the risk of developing vasopathy in patients with arterial hypertension with metabolic syndrome, the following diagnostic complex of studies is carried out: the index of antiplatelet activity of blood vessels, the activity of antithrombin III and euglobulin lysis before and after temporary venous occlusion with the calculation of the AT synthesis index are determined III in the vessels and the index of fibrinolytic activity of the vascular wall, as well as the content of nitrates in the blood before and after temporary ischemia enoznoy wall, indicating the level of NO synthesis in the vessel wall with the calculation of the index of nitrate (NO). The results are processed statistically with the calculation of the antithrombotic index of blood vessels.

The method allows to timely diagnose the risk of developing vasopathy to reduce PTIS in patients with hypertension and MS, which makes it possible to prescribe the appropriate treatment in a timely manner, translating the PTIS to a level close to that for healthy people. With regular examination in the subsequent according to the claimed method, it is possible to control the maintenance of PTIS in the optimal mode of operation, which will significantly reduce the risk of thrombotic complications, reduce the number of cases of temporary disability, exit to disability and reduce mortality among vascular patients.

The inventive method is as follows.

The anti-aggregation activity of the vascular wall was determined by the method (Baluda V.P., Lukyanova T.I., Baluda M.V. Method for determining the anti-aggregation activity of the wall of human blood vessels. // Labor. Delo, 1983. - No. 6. - p.17- twenty). The basis of this method is the calculation of the degree of inhibition of platelet aggregation in plasma obtained from blood after temporary venous occlusion.

A cuff of the sphygmomanometer is applied to the shoulder of the arm, from the blood vessels of which blood was not taken, and a pressure of 10 mm Hg is created. higher than systolic. After 3 minutes, 9 ml of blood was taken in 1 ml of a 3.8% sodium citrate solution. It was immediately centrifuged for 5 minutes at 1000 rpm to obtain platelet-rich plasma. The plasma was aspirated with a pipette and immediately placed in melting ice, where it was stored until research began. The sample was centrifuged again for 20 minutes at 3000 rpm to obtain platelet-free plasma. After counting the platelet count in platelet-rich plasma obtained before venous stasis (described in detail below), it was diluted with platelet-poor plasma (BeTP) taken before (1 test) and after (2 test) venous stasis to a concentration of 200,000 platelets / μl. The resulting mixture was shaken and incubated for 10 minutes at 20-22 ° C. Then platelets (AT) were aggregated with collagen (1: 2 dilution of the main suspension) on glass.

Assessment of AT for this study is carried out in the following way (A. Shitikova, Hemostasis. Physiological mechanisms, principles of diagnosis of the main forms of hemorrhagic diseases. Edited by N. N. Petrishchev, L. P. Papayan. St. Petersburg, 1999. P. 49- 52).

Blood is taken with sodium citrate 3.8% in a 9: 1 ratio, centrifuged for 5 minutes at 1000 rpm to obtain platelet-rich plasma (BTP). Part of the plasma is taken, and the remaining is centrifuged at 3000 rpm for 20 min, receiving a platelet-poor plasma. BTP is standardized by the number of platelets by diluting the original BTP with an autologous BeTP sample (obtained before venous stasis - 1st test or after it - 2nd test) to 200 · 10 ⁹ / L. The concentration of blood platelets in the initial BTP is calculated in the Goryaev's chamber (in 50 large squares). The volumes of mixed BTP and BeTP are determined by the formula:

V _BeTP = V _BTP · [(N / 200000) -1],

V _BeTP - volume of platelet-poor plasma,

V _BTP - the volume of platelet-rich plasma,

N is the counted platelet concentration in the original BTP (cells / μl).

From a selected volume of standardized plasma, 0.02 ml of plasma is applied to a glass slide and 0.02 ml of a collagen inducer solution at a standard concentration with another pipette (1: 2 dilution of the main suspension). Plasma is mixed with inducers with a glass rod and the stopwatch is turned on. The mixture is stirred so that the liquid occupies a circle with a diameter of about 2 cm.

Shaking the glass in a circular motion in the transmitted light of the illuminator, on a black background they monitor the occurrence of aggregates through a magnifying glass. When distinct aggregates appear, the solution becomes clear and some of the aggregates adhere to the glass, the stopwatch is turned off, fixing the time of platelet aggregation.

The reaction is repeated 2-3 times, finding the arithmetic mean value.

The degree of inhibition of platelet aggregation after temporary venous stasis is used to judge the anti-aggregation activity of blood plasma due to the entry of prostacyclin into the bloodstream from the vascular wall. The index of antiaggregatory activity of the vascular wall (IAASC) in the study of AT on glass was calculated by the formula:

The normal values for collagen can be considered IAASC 1.50-1.60.

Determination of the anticoagulant properties of the vascular wall, depending on the release of antithrombin III from it, evaluating its basal level of pre- and post-time dosed ischemia of the vein wall are given below (Barkagan Z.S., Momot A.P. Basics of the diagnosis of hemostasis disorders. M. 1999 - 214 from.).

The principle of the method is based on the detection of a decrease in activity (in the absence of heparin) in antithrombin III plasma, which is determined by the ability of the studied plasma to inactivate thrombin, for which it is pre-treated with a heparin sorbent, then subjected to thermal defibrination and mixed with a standard amount of thrombin. After incubation of the mixture, the residual coagulation activity of thrombin is determined in it. The level of decrease in thrombin activity as a percentage of the norm assesses the activity of AT III in the studied plasma.

The following reagents are used. 1. Tris-HCl buffer 0.05 M, pH 7.4. 2. Thrombin solution in buffer: 0.2 ml of the solution should coagulate with 0.3 ml of 0.4% fibrinogen solution for 15-16 seconds. The thrombin working solution is prepared in a plastic or silicone tube, stored at room temperature and used in the test on the day of the study. 3. Heparin sorbent Hepasorb-1 (company "Technology-Standard"). 4. Freshly prepared solution of fibrinogen at a concentration of 3.5-4.0 g / l, which is used to test the residual activity of thrombin. It is allowed to replace the fibrinogen solution with a normal platelet-poor citrate plasma diluted in buffer 2 times; 5. Fresh plasma platelet-poor plasma obtained from 6-8 healthy people, or proprietary lyophilized with known AT III activity, which is used to construct a calibration graph (see Figure 1).

The material for the study is a citrate platelet-poor plasma.

To prepare sorbed and defibrinated plasma in a test tube, 10 mg of heparin sorbent is mixed with 1.0 ml of the studied plasma. Constantly shaking the tube, mix its contents at room temperature for 8 minutes, excluding the formation of foam and sediment at its bottom. After that, the mixture is centrifuged for 2 minutes at 1000-1500 rpm. The plasma in the supernatant was transferred to a clean tube and defibrated in a water bath at 56 ° C for 6 min, after which it was again centrifuged at 3000 rpm for 10 min, the supernatant was removed and examined for the first 2 hours.

The course of determination. 0.4 ml of the thrombin working solution is added to the test tube and heated in a water bath at 37 ° C for 2 minutes. Then add 0.1 ml of the studied adsorbed and defibrified plasma, include a stopwatch. After 2 minutes (exactly!) 0.2 ml of the mixture was transferred to a test tube with 0.3 ml of fibrinogen solution (or diluted normal plasma), preheated to 37 ° C. Immediately turn on the stopwatch and record the clotting time.

From the calibration curve, AT III activity is found in percent.

The construction of the calibration curve is as follows. The plasma of healthy people is defibrated as described above. Then, dilutions are prepared from it for constructing a calibration curve in accordance with Table 1.

Table 1 Scheme for preparing plasma dilutions for constructing a calibration curve in determining the activity of AT III Breeding number Plasma + buffer Activity AT III,% one Buffer free plasma one hundred 2 0.2 ml + 0.2 ml fifty 3 0.1 ml + 0.3 ml 25

With each of the dilutions, the clotting time is determined by the above procedure. All measurements are carried out three times, the average result is calculated. When constructing a calibration curve along the logarithmic axis of ordinates, the clotting time in seconds is plotted, along the abscissa, the activity of AT III in% in accordance with the above dilutions. An approximate calibration curve is shown in the drawing.

A calibration curve for one series of thrombin and buffer is built once. Only the activity of the thrombin working solution is periodically monitored.

Normally, without venous occlusion, AT III activity is 85-115%. When taking blood after temporary venous occlusion, as described in the method for assessing the antiplatelet activity of blood vessels and determining AT III in it, it is possible to indirectly assess the level of excretion of AT III from the vessel wall. Against the background of temporary venous occlusion, the activity of AT III increases and is within 120-143%. The index of AT III synthesis in blood vessels (ISAS) is calculated by dividing the value of AT III activity against venous occlusion by the value of AT III without it. Normally, it is 1.20-1.45.

The synthesis activity of tissue plasminogen activators by the time of lysis of the euglobulin clot of pre- and post-temporal venous occlusion was evaluated as follows.

The principle of the method is to precipitate the euglobulin fraction of plasma, while the main inhibitors of fibrinolysis, in particular α ₂ -antiplasmin, remain in the supernatant and are removed. Therefore, euglobulin lysis reflects the activity of fibrinolysis under conditions of exclusion of the inhibitory effect of antiplasmin. Its speed mainly reflects the amount of plasminogen in the plasma and the degree of its activation. The time of spontaneous lysis of the clot obtained from the euglobulin fraction of plasma is determined by adding a solution of calcium chloride to it.

The following reagents are used: 1. 1% solution of acetic acid. 2.2.277% solution of calcium chloride. 3. Michaelis buffer or Tris-HCl buffer, 0.05M, pH 7.3-7.4.

The material for the study is a citrate platelet-poor plasma before and after temporary venous occlusion.

The course of determination. To obtain the euglobulin fraction, 0.15 ml of 1% acetic acid and 0.5 ml of the test plasma are added to 8.0 ml of distilled water. After 30 minutes of cooling the mixture in the refrigerator at + 2-8 ° C, the euglobulins are precipitated by centrifugation at 1500 rpm for 7 minutes. The supernatant is drained, the tube is dried by tipping over onto filter paper. The precipitate of euglobulins is diluted in 0.5 ml of buffer and placed in a water bath (37 ° C). Then, without removing the tube from the bath, add 0.5 ml of calcium chloride solution, mix gently by shaking the tubes (without shaking!), Turn on the stopwatch and incubate in a water bath at 37 ° C. Record the time from the moment of adding calcium chloride to the complete dissolution of the clot.

The result is expressed in minutes. Normally, the time of spontaneous lysis of the euglobulin clot is 180-240 minutes. After temporary venous occlusion, the lysis time decreases, being in the range of 126-170 minutes. When dividing the duration of spontaneous lysis of the euglobulin clot before occlusion by the value after, the index of fibrinolytic activity of the vascular wall (IFASS) was calculated, normally equal to 1.40-1.50.

Blood nitrates were determined by the method (Emchenko N.L., Tsyganenko O.I., Kovalevskaya T.V. Universal method for the determination of nitrates in the biological environment of an organism.// Clinical laboratory diagnostics. 1994. - No. 5, - p.19-20 )

The course of determination. 0.25-1.50 ml of a 50% zinc sulfate solution, 0.25-1.50 ml of a 17% iron ferricyanide solution, 1-5 ml of an analyzed sample are placed in 10 ml centrifuge tubes (a pipette for blood sampling is pre-moistened with a citrate solution sodium). Then add 0.5-2 ml of ammonium chloride buffer (pH 9.6 ± 0.05) and from 1 to 8 ml of distilled water for analysis of blood plasma.

The volumes of the analyzed sample and the reagents used for its deproteinization are presented in Table 2.

table 2 Volumes (in ml) of the analyzed samples and reagents added for their deproteinization Biosubstrate Try ZnSO ₄ K ₂ Fe (CN) ₆ Buffer Distilled water Blood 1.00 1.00 1.00 2.00 5.00

The total volume of the mixture should be 10 ml.

The resulting mixture was stirred and centrifuged for 20 minutes at 2500 rpm. In parallel, they conduct a blank experiment.

An aliquot of a 5 ml centrifugate was taken into a 50 ml flask, 2.5 ml of ammonia-chloride buffer, 2.5 ml of distilled water were added, stirred and a blank sample was passed through a sponge cadmium column, first a blank sample, then the test one. Before passing each of the samples, the column is washed sequentially with 5 ml of 1 N. HCl, 50 ml of distilled water and 25 ml of ammonium chloride buffer diluted in a ratio of 1: 9. The eluate is collected in the same volumetric flask, the column is washed with distilled water, not bringing the volume of solution in the flask to the mark of about 10 ml. Then, 5 ml of a 0.25% streptocide solution, 1 ml of HCl (1: 2) are added to the flask, and after 5 minutes, 1 ml of a 0.1% solution of N-naphthylethylenediamine dihydrochloride. The solution was adjusted to the mark with distilled water, stirred and left for 10 minutes. After this time, the samples are photometered on a photoelectric colorimeter at a wavelength of 540 nm (green filter) in a cuvette with an optical path length of 5 cm against a blank sample.

The concentration of nitrates is determined by the graded schedule, which is built in each laboratory separately and independently. To build it, 0.25, 0.5, 1, 2.5 and 5 ml of potassium nitrate solution with a concentration of nitrate ion equal to 10 μg / ml are added to volumetric flasks with a capacity of 50 ml, 10 ml of distilled water are added and 5 ml of ammonium chloride buffer and elute through a cadmium column. The color reaction and photometry is carried out as described above. The optical density is measured against a zero solution.

The nitrate content (X, mg / l) in the analyzed samples of biological media is calculated by the formula:

where C ₁ is the concentration of nitrate ions in the photometric solution, found according to the calibration graph (μg / ml); V ₁ - the total volume of protein-free extract (ml); V ₂ - the total volume of photometric solution (ml); V ₃ - the volume of the sample taken for analysis (ml); V ₄ - the volume of protein-free extract taken for analysis (in ml). Normally, in healthy people, the concentration of nitrites in the blood is 1.20-2.2 mg / L. With temporary application of a tourniquet on the shoulder and the creation of temporary ischemia of the vessel wall according to the scheme described above, increased release of nitric oxide into the blood from the vessel with an increase in the nitrate content in the blood from 6.0 to 13.0 mg / L is noted. It is possible to calculate the nitrate index by the formula:

Normally, NI is in the range from 5.0 to 5.9.

PTIS is calculated by the formula:

Normal PTIS values are values not lower than 2.27. With PTIS within 2.26-2.10, there is a risk of developing vasopathy, and with a PTIS of 2.09 or lower, clinical manifestation of vasopathy is noted.

The introduction of this risk method for the development of vasopathy in patients with hypertension with MS in the practice of medical institutions will solve a number of pressing socio-economic problems of our time. Early diagnosis in this category of patients will allow them to start treatment of vasopathy in a timely manner, which will shorten the period of temporary disability in these patients, prevent strokes and heart attacks, reduce the number of disability exits and reduce mortality among this group of patients.

Example 1. Patient B. 42 years old, suffering from arterial hypertension with metabolic syndrome for 2 years, leading a healthy lifestyle, was examined during a physical examination for arterial hypertension. Blood was taken from the patient and examined according to the above scheme, with an assessment of the antiaggregatory activity of the vascular wall, antithrombin III content, euglobulin lysis time, nitrate levels before and after venous occlusion, and the calculation of IAASC, ISAS, IFASS, NR and antithrombotic vascular index.

The patient showed no signs of inhibition of PTIS. IAASC was 1.5, ISAS, IFASS, NI were 1.3, 1.4, and 5.1, respectively. PTIS amounted to 2.32. The patient was recommended to continue to monitor blood pressure and lead a healthy lifestyle. A study of the functions of the vascular wall in her after 12 weeks did not reveal negative changes in the value of PTIS (2.34).

Example 2. Patient L. 47 years old, suffering from arterial hypertension with metabolic syndrome for 6 years, was examined during a physical examination for arterial hypertension. Blood was taken from the patient and studied according to the scheme described above with an assessment of the antiplatelet activity of the vascular wall, the content of antithrombin III, the time of euglobulin lysis, the nitrate level of pre- and post-venous occlusion and the calculation of IAASC, ISAS, IFASS, NI and PTIS calculation.

IAASC was 1.36. ISAS, IFASS and NI amounted to 1.18, 1.37 and 4.7, respectively. PTIS amounted to 2.15. The patient had a risk of developing vasopathy. The patient was recommended for adequate control of blood pressure and normalization of the state of the vascular wall to lead a healthy lifestyle, take normodipine 10 mg 1 time per day and examined monthly. So, a study of the functions of the vascular wall after 12 weeks did not reveal negative deviations in IAASC - 1.51, ISAS - 1.43, IFASS - 1.48, NI - 5.4. PTIS at her level was normal - 2.45.

Claims (1)

A method for diagnosing the risk of developing vasopathy in patients with arterial hypertension with metabolic syndrome, characterized in that the aggregation activity of platelets with collagen, the level of antithrombin III, the time of euglobulin lysis and the content of nitrates in the blood of patients before and against the background of temporary venous occlusion are determined, and then the antiaggregation activity index is calculated vascular wall (IAASS), the synthesis index of antithrombin III in the vessels (ISAS), the index of fibrinolytic activity of the vascular wall (IFASS) and the nitrate index (N I), calculated as the ratio of the values of the corresponding indicators against the background and to temporary venous occlusion, then the antithrombotic vascular index (PTIS) is calculated using the formula PTIS = (IAASS + ISAC + IFASS + NI) / 4, and with its value 2.27 and higher there is no risk of developing vasopathy; with its value in the range of 2.26-2.10, patients are at risk for the development of vasopathy; with its value of 2.09 and below, vasopathy develops.