RU2348041C1 - Method for diagnostics of aspirin resistance in patients with coronary heart disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is related to medicine, namely to cardiology. In order to realize method for diagnostics of aspirin resistance in patients with coronary heart disease prior to treatment with aspirin, in patient peripheral blood platelets activity of reactions is determined in NAD- and NADP-dependent dehydrogenases (NADP-HDH, NADPN-HDH, NADN-HDH, NAD-LDG). Then coefficient of cofactor platelets exchange (CCPE) is calculated according to the following formula: CCPE =

where

is ratio of activity levels of NADP- and NADPN-dependent reactions of glutamate dehydrogenase, and

is ratio of activity level of NADN-dependent reaction of glutamate dehydrogenase to activity level of NAD-dependent reaction of lactate dehydrogenase. If CCPE value exceeds 0.3, aspirin resistance is diagnosed, and if CCPE value is equal or less than 0.3, aspirin sensitivity is diagnosed.

EFFECT: provision of patient aspirin resistance diagnostics prior to treatment with aspirin with high accuracy of prediction.

2 tbl, 2 ex

Description

The invention relates to medicine, namely to cardiology. It can be used in the treatment of patients with coronary heart disease (CHD).

There is a method of determining aspirin resistance (AR) by means of platelet aggregometry, in which AR is detected after 2-3 days and 5 days after the start of aspirin [1]. In the case of AR, the patient is prescribed another drug. The diagnostic value of the known method is limited, since the method does not allow to diagnose AR before starting treatment with the drug.

The objective of the invention is to develop a reliable method for the diagnosis of aspirin resistance in patients with coronary artery disease before treatment with aspirin.

The problem is solved in that before the start of therapy with aspirin in the platelets of the peripheral blood of patients with coronary artery disease, the activity indicators of the reactions of NAD and NADP-dependent dehydrogenases are determined, after which the coefficient of platelet cofactor metabolism (CCOT) is calculated according to the formula CCOT = IOR / IVN. IOR is the ratio of the activity levels of NADP and NADPH-dependent glutamate dehydrogenase reactions, i.e. IOR = NADF-GDG / NADPH-GDG. IVN is the ratio of the activity level of the NADH-dependent reaction of glutamate dehydrogenase to the activity level of the NADH-dependent reaction of lactate dehydrogenase, i.e. IVN = NADN-GDG / NAD-LDG. If the value of CCOT is higher than 0.3, aspirin resistance is predicted, and if the value of CCOT is equal to or less than 0.3, the patient's sensitivity to aspirin is predicted.

A value of 0.3 was obtained by comparing the values of CCOT in patients with coronary artery disease before starting aspirin therapy with an assessment of the aspirin resistance of these patients.

It is known that in patients with coronary artery disease in the study of hemostasis, a hyperfunctional state of platelets and changes in the plasma link are revealed. These violations significantly worsen the prognosis of the disease and complicate treatment. Platelets play a key role in the hemostatic system. It is these cells that form the basis of the cellular link of the hemostatic system, and also a number of factors of the plasma link of hemostasis are synthesized in platelets, in particular fibrinogen. Platelet function is largely determined by the state of their intracellular metabolic system. It is through the metabolic system that a number of substances act on platelets, including the effects of aspirin. The activity of dehydrogenases most objectively reflects the state of intracellular metabolism [2].

LDH (EC 1.1.1.27) is a glycolysis enzyme that reversibly catalyzes the oxidation of lactate to pyruvic acid with the participation of NAD as a coenzyme. LDH occupies a key position in the regulation of the cytoplasmic level of NADH / NAD. In the case of an excess of NADH in the cytoplasm, LDH reduces pyruvate to lactate, which is then removed from the cell (anaerobic LDH reaction). At the same time, when aerobic processes are activated, LDH can oxidize lactate to pyruvate to form NADH (LDH aerobic reaction).

Glutamate dehydrogenases carry out oxidative deamination of G-glutamic acid. Two glutamate dehydrogenases are distinguished, using NAD (EC 1.4.1.2) or NADP (EC 1.4.1.4) as cofactors. The enzymatic reactions of glutamate dehydrogenases are reversible, respectively, ammonia in the presence of NAD (F) H and α-ketoglutaric acid can participate in the synthesis of glutamate. NAD- and NADP-dependent glutamate dehydrogenases are regulatory in the amino acid metabolism system.

The method is as follows.

Platelets are isolated from 9 ml of venous blood according to the method proposed by Savchenko E.A. et al. [3]. For this, venous blood is mixed with a 3.8% solution of sodium citrate in a ratio of 9: 1. Platelet-rich plasma is obtained by centrifuging stabilized blood at 140g 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 MaCl, 5 mM KCl, 36 mM sodium citrate, 10 mM EDTA, pH = 7.2). The resulting mixture was centrifuged for 15 min at 400g. The precipitate was resuspended in 10 ml of buffer No. 1 and re-centrifuged for 1 min at 400g. 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 s at 140g. For further studies, 250 μl of the supernatant was collected. To determine the activity of the reactions of NAD- and NADP-dependent dehydrogenases, a volume containing 10 ⁷ cells was selected from the collected supernatant with platelets. 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 the reactions of LDH, NADP-GDH, NADPH-GDG and NADH-GDH is determined using the bioluminescent method [4, 5]. 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 determining the activity of enzymatic reactions are presented in table 1.

Table 1 Enzymatic reactions Substrate, mm Cofactor, mm PH buffer NAD-LDH Lactate 2.0 NAD-0.50 9.0 NADF-GDG Glutamate 0.5 NADF-1.65 9.8 NADPH-GDG 2-oxoglutarate-100.0 NADPH-0.0025 7.4 NADN-GDG 2-oxoglutarate-100.0 NADN-0.0025 7.0

To determine the activity of the reactions NADPH-GDH and NADH-GDH, NH ₄ CL was additionally added to the incubation mixture at a concentration of 5.0 mM. After incubation of the test samples at 37 ° C for 30 minutes for LDH and NADP-GDH reactions or for 5 minutes for NADPH-GDH and NADH-GDG reactions, 50 μl of flavin mononucleotide (FMN) in a concentration of 1.5 is added to 200 μl of the incubation mixture × 10 ^-5 M, 50 μl of 0.0005% myristic aldehyde and 10 μl of the NADH: FMN oxidoreductase-luciferase enzyme system (all reagents of the bioluminescent system are diluted in 0.1 M K ⁺ , Na ⁺ phosphate buffer with pH 7.0). After mixing the bioluminescent reagents and the incubation sample, the luminosity is measured using a biochemiluminometer, for example, brand "BLM-8803". 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 carried out 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 biochemiluminometer, relative activity values of the studied enzymes are obtained. To obtain absolute activity values, graphs are plotted of the dependence of the bioluminescence intensity on the concentration of NADH and NADPH (calibration graph). For this, 200 μl of a standard solution of NAD (F) N in the range of 10 ^-9 -10 ^-4 M is added to the bioluminometer cuvette containing FMN, myristic aldehyde and NAD (F) N: FMN oxidoreductase-luciferase in the concentrations indicated above, after which they are produced measurement of bioluminescence intensity. Due to the wide range of pH buffers used to determine the activity of dehydrogenase reactions, 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 reaction activity of NAD- and NADP-dependent dehydrogenases is calculated by the formula:

where A is the activity of reactions of dehydrogenases, E on 2 × 10 ⁵ platelets (1E = 1 μmol / min [2]);

Δ [C] is the difference in the concentrations of NAD (F) N in the samples "enzyme" and "background enzyme", mmol;

V is the sample volume, ml;

T - incubation time, min

Then, the IOR index is calculated by the ratio of the activity levels of the reactions NADP-GDH and NADPH-GDH (IOR = NADP-GDG / NADPH-GDG). The IVN index is determined by the ratio of the activity levels of the reactions NADH-GDH and NAD-LDH (IVN = NADH-GDH / NAD-LDH). Then find the coefficient KKOT by the ratio of the indices IOR and IVN (KKOT = IOR / IVN). The value of CCOT above 0.3 indicates AR, and CCOT equal to or less than 0.3 indicates the sensitivity of platelets of patients with coronary heart disease to aspirin.

Table 2 presents the results of examinations of 38 patients with coronary heart disease conducted in the cardiac surgery department of the Krasnoyarsk Regional Clinical Hospital No. 1. Before starting treatment with aspirin (at a dose of 75-150 mg / day) according to the proposed method, the activity of the reactions of NAD- and NADP-dependent dehydrogenases was determined in the blood platelets of patients, KKOT was calculated and, depending on its value, AR or platelet sensitivity to aspirin was diagnosed. 5 days after the start of aspirin treatment, the observed patients were determined by aspirin resistance according to aggregatometry: in 12 patients, AR was diagnosed, in 28 patients the platelets were sensitive to the action of aspirin. The data presented in the table show that 36 of 38 patients had a coincidence of the prognosis for the proposed method (94.7%).

table 2 Number p / p The forecast for the proposed method IOR IVN KCOT Forecast Match one AR 0.02 0.02 1.00 Yes 2 ACh 0.00 0.19 0.00 Yes 3 AR 0.02 0.06 0.33 Yes four AR 0.02 0.06 0.33 Yes 5 ACh 0.00 0.39 0.00 Yes 6 AR 0.05 0.02 2.25 Yes 7 AR 0.02 0,03 0.67 Yes 8 ACh 0.00 0.06 0.00 Yes 9 ACh 0.00 0.06 0.00 Yes 10 ACh 0.00 0.06 0.00 Yes eleven AR 0,07 0.06 1.17 Yes 12 AR 0.02 0.02 1.00 Yes 13 ACh 0.00 0.15 0.00 Yes fourteen ACh 0.00 0.27 0.00 Yes fifteen ACh 0.00 0.08 0.00 Yes 16 ACh 0.01 0.10 0.10 No 17 ACh 0.00 0.01 0.00 Yes eighteen AR 0.55 0.01 55.00 Yes 19 ACh 0.00 0.33 0.00 Yes twenty ACh 0.00 0.25 0.00 Yes 21 ACh 0.00 0.26 0.00 Yes 22 ACh 0.00 1.17 0.00 Yes 23 AR 0.11 0.02 5.50 Yes 24 ACh 0.00 0.04 0.00 Yes 25 ACh 0.00 0,07 0.00 Yes 26 AR 0.06 0,03 2.00 No 27 AR 0.02 0.02 1.00 Yes 28 ACh 0.01 0.33 0,03 Yes 29th ACh 0.01 0.08 0.13 Yes thirty ACh 0.00 0.09 0.00 Yes 31 ACh 0.00 0.24 0.00 Yes 32 ACh 0.01 0.05 0.20 Yes 33 ACh 0,03 0.11 0.27 Yes 34 ACh 0.00 0.08 0.00 Yes 35 ACh 0.01 0.11 0.09 Yes 36 ACh 0.02 0.09 0.22 Yes 37 ACh 0.01 0.12 0.08 Yes 38 AR 0.16 0.10 1,60 Yes Note: AR - aspirin resistance; AF - aspirin sensitivity.

Clinical examples.

Example 1. Patient N., 64 years old (data No. 11 in table 2). Case history No. 4984. He was hospitalized in the cardiac surgery department of the Regional Clinical Hospital No. 1 from 02.03.2005 to 05.04.2005 with a diagnosis of IHD, angina pectoris 3 functional class, post-infarction cardiosclerosis. A survey on the proposed method. The activity of dehydrogenase reactions was determined in platelets of the patient’s peripheral blood: LDH = 947.15 μE per 2 × 10 ⁵ platelets; NADP-GDH = 3.39 μE per 2 × 10 ⁵ platelets; NADH-GDH = 54.03 μE per 2 × 10 ⁵ platelets; NADPH-GDH = 46.94 μE per 2 × 10 ⁵ platelets. The values of IOR, IVN and KKOT were calculated: IOR = 0.07; IVN = 0.06; CCOT = 0.07 / 0.06 = 1.17. According to the invention, CCFT> 0.3 indicates the patient's aspirin resistance. Upon admission to the hospital, a platelet aggregation study (laser aggregometer "Biola", Moscow) with ADP (5 μM) was conducted: light transmission was 40%. 5 days after the start of aspirin treatment, platelet aggregation with ADP was performed: light transmission was 60%. Therefore, against the background of aspirin treatment, a decrease in platelet aggregation did not occur, which confirms the patient's aspirin resistance.

Example 2. Patient A., 54 years old (data No. 17 in table 2). Case history No. 8205. He was hospitalized in the cardiac surgery department of the Regional Clinical Hospital No. 1 from 04/08/2005 to 05/12/2005 with a diagnosis of IHD, angina pectoris 2 functional class, post-infarction cardiosclerosis. A survey on the proposed method. The activity of dehydrogenase reactions was determined in platelets of the patient’s peripheral blood: LDH = 14373.42 μE per 2 × 10 ⁵ platelets; NADP-GDH = 0.00 μE per 2 × 10 ⁵ platelets; NADH-GDH = 170.48 μE per 2 × 10 ⁵ platelets; NADPH-GDH = 37.63 μE per 2 × 10 ⁵ platelets. The values of IOR, IVN and KKOT are calculated: IOR = 0,00; IVN = 0.01; CCOT = 0.0. According to the invention, CCFT <0.3 indicates an aspirin sensitivity of the patient. Upon admission to the hospital, a study was conducted of platelet aggregation with ADP (5 μM): light transmission was 41%. 5 days after the start of aspirin treatment, platelet aggregation with ADP was performed: light transmission was 27%. Therefore, against the background of aspirin treatment, a decrease in platelet aggregation occurred, which confirms the patient's sensitivity to aspirin.

The technical result from the implementation of the proposed method:

- the possibility of diagnosis in patients with coronary artery disease before treatment with aspirin;

- high level of coincidence of the forecast - 94.7%.

Thus, the proposed method allows to reliably diagnose aspirin resistance of patients with coronary artery disease before treatment and can be recommended for use in clinical practice.

Information sources

1. Momot A.P. Pathology of hemostasis. Principles and algorithms of clinical laboratory diagnostics. - St. Petersburg: FormT, 2006. - P.95.

2. Berezov T.T., Korovkin B.F. Biological chemistry. - M.: Medicine, 1998 .-- 704 p.

3. Savchenko E.A., Savchenko A.A., Gerasimchuk A.N., Grishchenko D.A. Assessment of the metabolic status of platelets in normal and coronary heart disease // Clinical laboratory diagnostics. - 2006. - No. 5. - S.33-36.

4. Savchenko A.A., Suntsova L.N. Highly sensitive determination of the activity of dehydrogenases in peripheral blood lymphocytes by the bioluminescent method // Lab. a business. - 1989. - No. 11. - S.23-25.

5. Savchenko A.A. Bioluminescent determination of the activity of NAD- and NADP-dependent glutamate hydrogenases of lymphocytes // Lab. - 1991. - No. 11. - S.22-25.

Claims (1)

A method for diagnosing aspirin resistance in patients with coronary heart disease, which consists in determining platelet resistance to aspirin, characterized in that before the start of aspirin therapy in patients' peripheral blood platelets, the activity of the NAD- and NADP-dependent dehydrogenases reactions is determined, after which the coefficient of platelet cofactor metabolism (CCOT) is calculated ) according to the formula KKOT = IOR / IVN, where IOR is the ratio of the activity levels of NADP- and NADPH-dependent reactions of glutamate dehydrogenase (i.e., IOR = NADP-GDH / NADPH- GDH), and IVN is the ratio of the activity level of the NADH-dependent reaction of glutamate dehydrogenase to the activity level of the NAD-dependent reaction of lactate dehydrogenase (i.e., IVN = NADH-GDG / NAD-LDH), and aspirin resistance is predicted with an ACC value above 0.3, and with a value of CCOT equal to or less than 0.3, sensitivity to aspirin.