RU2425640C1 - Method of predicting recurrent cerebrovascular complications in patients with hypertension in combination with ischemic heart disease in acute period of ischemic stroke - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: invention relates to medicine, namely to neurology and cardiology. In patients with hypertension (H) in combination with ischemic heart disease (IHD) after ischemic stroke (IS) by means of bioluminescent method levels of activity of G6DG, NADP MDG, GR and NADPH-GDG platelets. Value of coefficient of ratio of reactions of NADP reduction and NADPH oxidation is used to make conclusion about possibility of development or absence of recurrent cerebro-vascular complications. ^ EFFECT: method makes it possible to predict on the first day of development of IS in patients with H in combination with IHD development of recurrent cerebro-vascular complications during 12 months after IS by single venous blood sampling. ^ 2 tbl, 4 ex

Description

The invention relates to medicine, namely to neurology and cardiology, can be used for the effective prevention of cerebrovascular complications in patients with hypertension (GB) in combination with coronary heart disease (CHD) after an ischemic stroke (II).

A known method for predicting the course of AI, consisting in the first day of acute cerebrovascular accident analysis of blood serum and cerebrospinal fluid for uric acid content. When the content of uric acid in blood serum is higher than 390 μmol / L, and in cerebrospinal fluid above 140 μmol / L, an adverse course (lethal outcome) of ischemic stroke is predicted [3]. The disadvantage of this method is the mandatory conduct of a patient with AI spinal puncture, which is not always an easily performed and necessary manipulation.

A known method for predicting the course of AI, which consists in conducting immunophenotyping of lymphocytes. The essence of the method consists in the fact that on the 1st, 10th, 21st day from the moment of AI development, the average level of CD95 ⁺ lymphocytes is determined. If the level of CD95 ⁺ lymphocytes on the 1st day is more than 7.2 ± 2.8% and return to normal on the 21st day, a favorable outcome is predicted, when the level of CD95 ⁺ lymphocytes is above 7.2 ± 2.8% and maintaining the average level of the indicator on the 21st day, a favorable prognosis for life and unfavorable for the restoration of neurological deficit are predicted, and with an average value of CD95 lymphocytes above 29.12 ± 3.1%, a poor prognosis for life is predicted

[2]. A significant disadvantage of this method is the complexity of the calculations and repeated blood sampling in patients with AI.

A known method for predicting the clinical course of AI by determining hemostatic activation parameters such as degradation products of fibrinogen D-dimer, fibrin-monomeric soluble complexes (RKFM), as well as platelet aggregation activity. Measurements are taken throughout the acute and acute periods of ischemic stroke. In the case of even a one-time increase in the concentration of the D-dimer over 1 μg / ml and / or the concentration of RKFM more than 50 mg% in combination with a significant decrease in platelet aggregation to 10-15% compared with the values detected at the beginning of the acute period, the probability of death is predicted course of ischemic stroke [1]. The disadvantage of this method is the need for repeated determination of the studied parameters of hemostasis in patients with AI.

In literary sources, no prognosis of the development of cerebrovascular complications was found for patients undergoing AI and suffering from combined cardiovascular pathology (GB and IHD).

The objective of the proposed method is to prevent the occurrence of repeated cerebrovascular complications of a thrombotic nature: acute cerebrovascular accident, transient ischemic attack, acute myocardial infarction, peripheral vascular thrombosis in patients with hypertension combined with coronary heart disease after the development of ischemic stroke.

The task is carried out due to the fact that, using the bioluminescent method, the activity levels of G6FDG, NADPMDG, GR and NADPH-GDH of platelets are determined and the possibility of the occurrence or absence of complications is judged by the ratio of the reaction ratios of NADP ⁺ reduction and NADPH oxidation, with a coefficient value <1 , 00 predict the development of complications, with a value of ≥1.00 - the absence of complications.

The method is as follows. In patients, on the first day after the development of AI in the morning, on an empty stomach, using venipuncture with a sharp needle, a free flow of venous blood from the ulnar vein is taken in a volume of 10 ml into a plastic tube. As an anticoagulant, a 3.8% aqueous solution of sodium citrate is used. The study is carried out within one hour after blood sampling. Platelets are isolated from venous blood according to the method proposed by Savchenko E.A. et al. [four]. For this, venous blood is mixed with a 3.8% aqueous solution of sodium citrate in a ratio of 9: 1. Platelet-rich plasma is obtained by centrifuging stabilized blood at 140 g for 10 minutes. The supernatant was carefully taken from the tube, transferred to a clean tube and adjusted to 10 ml with buffer No. 1 (90 mM NaCl, 5 mM KCl, 36 mM sodium citrate, 10 mM EDTA, pH = 7.2). The resulting mixture was centrifuged for 15 min at 400 g. The precipitate was resuspended in 10 ml of buffer No. 1 and centrifuged again for 1 min at 400 g. 9 ml of the supernatant are collected and centrifuged again at 400 g for 15 minutes. The supernatant is carefully drained and 10 ml of buffer No. 2 (0.13 M NaCl, 0.02 M Tris-HCl buffer, 0.03 M EDTA, 0.015 M glucose, pH = 7.4) is added to the precipitate and centrifuged in the same mode. Then the precipitate was diluted in 400 μl of buffer No. 2 and centrifuged for 50 seconds at 140 g. For further studies, 250 μl of the supernatant was collected. To determine the activity of NADPH and NADP-dependent dehydrogenases, a volume containing 10 ⁷ cells was selected from the collected platelet supernatant. Platelets are destroyed by osmotic lysis with a total volume of 2.5 ml (the final cell concentration is 4 × 10 ⁶ / ml). The activity of G6FDH, NADFMDG, GR and NADPH-GDH is determined using the bioluminescent method. For this, in 50 μl of the incubation mixture containing the appropriate substrate and cofactor, add 50 μl of a suspension of destroyed platelets. The specific values of the concentrations of substrates and cofactors, as well as the pH of the medium for the determined enzymes are presented in table 1.

Table 1 The concentration of substrates, cofactors and pH indicators for determining the activity of NAD (F) -dependent dehydrogenases in blood platelets by a bioluminescent method Enzyme Substrate, mm Cofactor, mm PH buffer G6FDG G6F - 1.5 NADPH - 0.025 9.8 NADFMDG Malat - 7.5 NADF - 0.375 9.8 GR GSH - 0.5 NADPH - 0.0025 7.4 NADPH-GDG Oxoglutarate - 100 NADPH - 0.0025 7.4 Note: pH 9.8 medium is prepared in Tris-HCl buffer (ICN Biomedicals Inc., USA); with a pH of 7.4 - on K ⁺ , Na ⁺ -phosphate buffer (buffer prepared from K ₂ HPO ₄ and NaH ₂ PO ₄ (Reakhim, Russia)).

After incubation of the test samples at 37 ° C for 30 minutes for G6FDG and NADPMDG or 5 minutes for GR and NADPH-GDG, 50 μl of flavin mononucleotide (FMN) at a concentration of 1.5 × 10 ^-5 M, 50 μl are added to 200 μl of the incubation mixture 0.0005% myristic aldehyde and 10 μl of the NADH: FMN oxidoreductase-luciferase enzyme system (all bioluminescent system reagents are diluted in 0.1 M K ⁺ , Na ⁺ phosphate buffer with pH 7.0). After mixing the bioluminescent reagents and the incubation sample using a biochemiluminometer, for example, the brand "BLM-8803" measure the glow. Given that the cells have a certain number of substrates for the course of various metabolic reactions, including those catalyzed by the studied enzymes, the parameters conventionally called “substrate background of enzymes” are determined. The determination is made under the same conditions as for the above dehydrogenases, but a buffer is added to the incubation mixture instead of the corresponding substrate. As a result of measuring the glow on a bioluminometer, relative values of the activity of the studied enzymes are obtained. To obtain the absolute values of activity, build graphs of the dependence of the intensity of bioluminescence on the concentration of NADPH (calibration graph). For this, 200 μl of a standard NADPH solution in the range of 10 ^-9 -10 ^-4 M is added to the bioluminometer cuvette containing FMN, myristin aldehyde and NADPH: FMN oxidoreductase-luciferase at the concentrations indicated above, after which the intensity of bioluminescence is measured. Due to the wide range of pH buffers used to determine dehydrogenase activity, as well as the pH dependence of the bioluminescence of the enzymatic system from luminous bacteria, calibration plots are plotted for each pH buffer. The activity of NADP and NADPH-dependent dehydrogenases is calculated by the formula

A = Δ [C] × V / T

Where

A - dehydrogenase activity, E (unit of enzyme activity) per 2 × 10 ⁵ platelets (1E = 1 μmol / min [1]);

Δ [C] is the difference in the concentrations of NADPH in the samples “enzyme” and “background enzyme”, mmol;

V is the sample volume, ml;

T is the incubation time, min

Then calculate the ratio of the reactions of reduction of NADP ⁺ and oxidation of NADPH

With a coefficient value <1.00, the development of complications is predicted, with a value of ≥1.00 - the absence of complications. A ratio of less than 1.00 for the reactions of NADP ⁺ reduction and NADPH oxidation indicates the prevalence of NADPH oxidation reactions over NADP ⁺ reduction reactions.

This method is confirmed in 21 patients in the acute period of ischemic stroke. All patients with acute cerebrovascular accident upon admission to the hospital underwent generally accepted laboratory blood tests: general analysis, biochemical analysis with determination of cholesterol and lipid spectrum. The parameters of the hemostatic system and the levels of enzyme activity in platelets were determined in the first 24 hours after the development of AI. The coincidence of the prognosis was evaluated by the fact of the occurrence of cerebrovascular complications in patients with hypertension in combination with coronary heart disease after the development of ischemic stroke (repeated stroke, TIA, AMI, peripheral vascular thrombosis).

table 2 The prognosis of the occurrence of repeated combination with coronary heart disease after the development of cerebrovascular complications in patients with hypertension in AI by the value of the NADP ⁺ / NADPH coefficient No. Coefficient Cerebrovascular complication there is no one 2 3 four one NADF ⁺ / NADPH - 23,381 2 NADF ⁺ / NADPH - 44,669 3 NADF ⁺ / NADPH - 624,019 four NADF ⁺ / NADPH - 58,408 5 NADF ⁺ / NADPH 0.130 - 6 NADF ⁺ / NADPH 0.205 - one 2 3 four 7 NADF ⁺ / NADPH 0.859 - 8 NADF ⁺ / NADPH - 21,695 9 NADF ⁺ / NADPH - 6,999 10 NADF ⁺ / NADPH 0.363 - eleven NADF ⁺ / NADPH - 11,887 12 NADF ⁺ / NADPH - 5,030 13 NADF ⁺ / NADPH 0.174 - fourteen NADF ⁺ / NADPH - 16,822 fifteen NADF ⁺ / NADPH 0.989 - 16 NADF ⁺ / NADPH - 291,719 17 NADF ⁺ / NADPH 0.696 - eighteen NADF ⁺ / NADPH - 156,258 19 NADF ⁺ / NADPH - 12,763 twenty NADF ⁺ / NADPH - 2,980 21 NADF ⁺ / NADPH - 19,272

According to the results of the examination (cardiological and neurological examinations, cardiac echocardiography, recording of an electrocardiogram, Holter monitoring), it was found that within 12 months after the development of AI, 14 patients had no cerebrovascular complications. In 7 - in the range from 1 week to 12 months, one of the ischemic cerebrovascular complications of a thrombotic nature developed.

Thus, a coincidence of the prognosis of the occurrence of repeated cerebrovascular complications in patients with hypertension in combination with coronary heart disease, obtained by determining the ratio of the reactions of recovery of NADP ⁺ and NADPH oxidation in the acute period of II, with the results of examination of patients after 12 months (100%), was found.

Clinical example 1.

Patient Ch., 55 years old (medical history No. 19577), was admitted as an emergency to the neurological department of the Municipal Health Institution City Clinical Hospital (Municipal Clinical Hospital) No. 6 named after N.S. Karpovich of Krasnoyarsk with a diagnosis of Ischemic stroke in the left cerebral artery basin (LSMA) with right-sided hemihyposthesia. Cardiological diagnosis: Hypertension 3 degrees, stage III, risk 4, dyslipidemia. Ischemic heart disease. Angina pectoris I FC.

Upon admission to the neurological department of the Municipal Health Institution, City Clinical Hospital No. 6 named after N.S. Karpovich, Krasnoyarsk, the patient complained of a headache, a violation of sensitivity in the right extremities. The diagnosis of AI was confirmed by neurological examination and computed tomography (CT) of the brain.

From the anamnesis it is known that there was no earlier acute cerebrovascular accident (ONMK); since 2003, blood pressure rises to 240/130 mm Hg. Since 2006, the clinic of angina pectoris at the level I functional class (FC), acute myocardial infarction (AMI) did not tolerate. I did not receive drug therapy.

The results of the study of the hemostatic system on the 1st day: hypercoagulation, thrombinemia, weakening of the anticoagulation potential of the vascular wall, a decrease in the induced platelet aggregation, an increase in the prothrombotic activity of the endothelium.

When studying the activity of NADPH- and NADP-dependent platelet dehydrogenases, it was found that the ratio of the reactions of NADP ⁺ reduction and NADPH oxidation was 5.03, which indicates the absence of cerebrovascular complications within a year after the onset of AI.

When examined after 12 months in the neurological department of the Municipal Clinical Hospital № 6 named after N.S. Karpovich in the patient did not reveal cerebrovascular complications. Did not take drug therapy.

Clinical example 2.

Patient S., 57 years old (medical history No. 6718), was urgently admitted to the neurological department of the Municipal Healthcare Institution City Clinical Hospital No. 6 named after N.S. Karpovich of Krasnoyarsk with a diagnosis of repeated ischemic stroke in the pool of the right middle cerebral artery (PSMA). Cardiological diagnosis: Hypertension 3 degrees, stage III, risk 4. IHD. Postinfarction cardiosclerosis (PEAKS). Angina III functional class (FC). Heart failure stage 2A - III functional class.

Upon admission to the hospital, the patient complained of weakness in the left leg and left arm, limping when walking. The diagnosis of AI is confirmed by neurological examination, computed tomography of the brain.

In the history of 2000, blood pressure rises to 190/120 mm Hg. In 2001 and 2005 she suffered an acute violation of cerebral circulation. In 2003, she suffered an acute myocardial infarction, followed by a clinic of exertional angina at the level of functional class III. Phenomenon of heart failure with stagnation of a large circle of blood circulation. I did not regularly take antihypertensive drugs.

The results of the study of the hemostatic system on the 1st day: hypercoagulation, thrombinemia, fibrinogenemia, weakening of the anticoagulation potential of the endothelium, platelet aggregation indicators within the normal range, weakening of the antiplatelet activity of the vascular wall against the background of an increase in prothrombotic.

When studying the activity of NADPH and NADP-dependent platelet dehydrogenases, it was found that the ratio of the reactions of NADP ⁺ reduction and NADPH oxidation was 291.72, which indicates the absence of exacerbation.

When examining the patient after 12 months in the neurological department of the Municipal Health Institution City Clinical Hospital No. 6 named after N.S. Karpovich was not found cerebrovascular complications. Antihypertensive and antianginal therapy was not taken regularly.

Clinical example 3.

Patient G., 67 years old (medical history No. 10349), was urgently admitted to the neurological department of the Municipal Healthcare Institution City Clinical Hospital No. 6 named after N.S. Karpovich of Krasnoyarsk with a diagnosis of Acute ischemic disturbance of cerebral circulation in the pool of the left midbrain artery. Cardiological diagnosis: IHD. Postinfarction cardiosclerosis. Angina III functional class. Paroxysmal form of atrial fibrillation. Hypertension 3 degrees, stage III, risk 4, dyslipidemia. Heart failure stage 2A - III functional class.

Complaints upon receipt of speech impairment, weakness, dizziness, headache, vomiting once. The diagnosis of AI is confirmed by neurological examination, computed tomography of the brain.

From the anamnesis it is known that in 2006 suffered a transient ischemic attack (TIA). Since 1998, blood pressure rises to 180/90 mm Hg. In 2004, he suffered an acute myocardial infarction, followed by a clinic of exertional angina at the level of functional class III. Since that time, paroxysms of atrial fibrillation. Phenomenon of heart failure with congestion in the pulmonary circulation. I did not regularly take antiarrhythmics.

The results of the study of the hemostatic system on the 1st day: thrombinemia, hypercoagulation, weakening of the anticoagulant activity of the endothelium, induced and spontaneous platelet aggregation within the normal range, increased prothrombotic activity of the vascular wall against the background of a significant weakening of antiplatelet.

When studying the activity of NADPH and NADP-dependent platelet dehydrogenases, it was found that the ratio of the reactions of NADP ⁺ reduction and NADPH oxidation was 0.76, which indicates the development of cerebrovascular complications within a year after the onset of AI.

When examining a patient after 12 months in the neurological department of the Municipal Clinical Hospital № 6 named after N.S. Karpovich revealed that the patient developed a repeated ischemic stroke within a year. Antihypertensive and antianginal therapy was not taken regularly.

Clinical example 4.

Patient P., 60 years old (medical history No. 4561), was urgently admitted to the neurological department of the Municipal Healthcare Institution City Clinical Hospital No. 6 named after N.S. Karpovich of Krasnoyarsk with a diagnosis of Recurrent acute cerebrovascular accident according to ischemic type in the left cerebral artery basin. Cardiological diagnosis: Hypertension 3 degrees, stage III, risk 4. IHD. Angina II functional class. Heart failure II A stage - II functional class.

Upon admission to the hospital, the patient complained of speech impairment, weakness, headache. The diagnosis of AI was confirmed by neurological examination, computed tomography of the brain.

From the anamnesis it is known that the patient suffered 2 ischemic strokes in 2004. Over 10 years, blood pressure rises to 180/100 mm Hg. Myocardial infarction did not suffer, since 2000 the clinic of exertional angina pectoris at the level of II functional class. Mild stagnation in the pulmonary circulation.

The results of the study of the hemostatic system on the 1st day: hypercoagulation, thrombinemia, a marked decrease in the anticoagulant activity of the vascular wall, a significant increase in the spontaneous and induced platelet activity, a marked weakening of the antiplatelet activity of the endothelium against the background of an increase in prothrombotic.

When studying the activity of NADPH and NADP-dependent platelet dehydrogenases, it was found that the ratio of the reactions of NADP ⁺ reduction and NADPH oxidation was 0.99, which indicates the development of cerebrovascular complications within a year after the onset of AI.

When examined after 12 months in the neurological department of the Municipal Clinical Hospital № 6 named after N.S. Karpovich revealed that within a year the patient against the background of atherosclerosis developed peripheral vascular thrombosis. I did not take antihypertensive and antianginal therapy.

The above examples indicate that, despite the unidirectional changes in the parameters of the hemostatic system, the prognosis of the development of cerebrovascular complications significantly differs in patients.

The advantages of the proposed method are:

1) the ability to predict on the first day of the development of AI in patients with hypertension in combination with coronary heart disease the occurrence of repeated cerebrovascular complications within 12 months after stroke;

2) a single sampling of venous blood only;

3) a high level of forecast coincidence - 100%.

The proposed method can be recommended for use in clinical practice by neurologists, cardiologists, therapists.

Information sources

1. RF patent No. 2217754 C1, G01N 33/49, G01N 33/68, BIPM No. 33, 11/27/2003.

2. RF patent No. 2236685 C1, G01N 33/577, BIPM No. 26, 09/20/2004.

3. RF patent №2324941 C1, G01N 33/50, BIPM No. 14, 05.20.2008.

4. Savchenko A.A. Bioluminescent determination of the activity of NAD- and NADP-dependent glutamate dehydrogenases of lymphocytes // Lab. a business. - 1991. - No. 11. - S.22-25.

Claims (1)

A method for predicting repeated cerebrovascular complications in patients with hypertension in combination with coronary heart disease in the acute period of ischemic stroke by a blood test, characterized in that using the bioluminescent method, the levels of G6PDH, NADPMDH, GR and NADPH-GDG platelets are determined and the possibility of or absence of complications is judged by the value of the recovery ratio of NADP ⁺ and NADPH oxidation at largest coefficient <1,00 reactions predict the development of complication in the under-largest ≥1,00 - the absence of complications.